How Tony Melcher advanced our understanding of periodontal biology and regeneration.

Tony Melcher, a highly influential and forward-thinking scientist and teacher, focussed on the origins, behaviour and regulation of cells in periodontal tissues. His recent death in April 2020, has motivated us to highlight his multi-level contributions to research in biology and the dental sciences. Tony was particularly adept at recognizing the inherent instructive power of the periodontium, most notably as a model system for studying the inter-relationships between the structure, development and functions of connective tissues. Further, his mentoring of dozens of students who subsequently went on to develop their own careers in research, and his leadership in promoting collaborations in dental sciences world-wide, engendered important advances in the importance and utility of research relating to oral tissues. Here, we reflect upon his development of a large, multi-disciplinary research enterprise, the MRC Group in Periodontal Physiology at the University of Toronto and brief commentaries of those who worked with him there. We examine his early career development and then go on to consider some of his most highly cited publications and their impact on subsequent research trends.

Comparative response of epithelial cells and osteoblasts to microfabricated tapered pit topographies in vitro and in vivo.

Microfabricated tapered pits in vivo can stimulate connective tissue and bone attachment to percutaneous devices, secondarily preventing epithelial migration, and promoting long-term implant survival. Epithelial cells, which form a seal with a dental implant, acting as a barrier, and osteoblasts, which form bone, can come into contact with the same implant topography. To investigate whether the phenotypic characteristics of each cell type influenced cell response to micro-topography, we compared the response of the two cell types to the same dimensions of tapered pits, in vitro, and in vivo. Increased spreading, mature FAs, and restricted migration characterized individual PLE cell response to tapered pits. In contrast, osteoblasts were highly migratory, formed smaller, punctate adhesions and mineralized. Epithelial sheets formed from high-density PLE cultures demonstrated that tapered pits did not inhibit migration of the PLE sheets in vitro, similar to in vivo observations. In vitro, PLE sheet migration correlated with increases in vinculin, tyrosine phosphorylation, cytokeratin and ERK 1/2 phosphorylation. The findings of this study show that tapered pits stimulate osteoblast mineral deposition in vitro and in vivo, but do not prevent epithelial sheet migration. In vitro results suggest that epithelial sheet migration could involve altered FA mediated signal transduction.

Triton X-100 pretreatment of LR-white thin sections improves immunofluorescence specificity and intensity.

The staining of intracellular antigenic sites in postembedded samples is a challenging problem. Deterioration of antigenicity and limited antibody accessibility to the antigen are commonly encountered on account of processing steps. In this study preservation of the antigen was achieved by fixing the tissues with mild fixatives, performing partial dehydration, and embedding in a low crosslinked hydrophilic acrylic resin, LR-White. Permeabilization of cell membranes with Triton X-100 is well documented but can affect some antigen conformations. We tested the effect of Triton X-100 on the ED1 antigen present in the lysosomal membrane of the macrophage in cell culture. The ED1 antigen in the lysosome was resistant to extraction by Triton X-100. Interestingly pretreating the LR-White sections of macrophage pellets with Triton X-100 improved the staining intensity of ED1. The most intense and clear specific fluorescent staining was observed when sections were pretreated with 0.2% Triton X-100 for 2 min. Longer exposure of sections to 0.2% Triton or 2 min exposure to 2% Triton lead to reduced ED1 labeling. SEM observations indicated that the detergent extracted a component from the cells and not the resin and was determined to be lipid. This novel technique could be applied in many research areas where postembedding fluorescent immunolabeling with higher labeling intensity is desired.

Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: a cell morphology study.

Surface topography and (bio)chemistry are key factors in determining cell response to an implant. We investigated cell adhesion and spreading patterns of epithelial cells, fibroblasts and osteoblasts on biomimetically modified, smooth and rough titanium surfaces. The RGD bioactive peptide sequence was immobilized via a non-fouling poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system, which allowed exploitation of specific cell-peptide interactions even in the presence of serum. As control surfaces, bare titanium and bio-inactive surfaces (scrambled RDG and unfunctionalized PLL-g-PEG) were used. Our findings demonstrated that surface topography and chemistry directly influenced the attachment and morphology of all cell types tested. In general, an increase in cell number and more spread cells were observed on bioactive substrates (containing RGD) compared to bio-inactive surfaces. More fibroblasts were present on smooth than on rough topographies, whereas for osteoblasts the opposite tendency was observed. Epithelial cell attachment did not follow any regular pattern. Footprint areas for all cell types were significantly reduced on rough compared to smooth surfaces. Osteoblast attachment and footprint areas increased with increasing RGD-peptide surface density. However, no synergy (interaction) between RGD-peptide surface density and surface topography was observed for osteoblasts neither in terms of attachment nor footprint area.

Effects of surface topography on the connective tissue attachment to subcutaneous implants.

PURPOSE: A major concern for implants that penetrate stratified epithelia is aggressive epithelial proliferation and migration. This epithelial downgrowth on the implant can be inhibited by a firm attachment between the underlying connective tissue and the implant. This study evaluates the connective tissue attachment to titanium implants with various well-defined surface topographies. MATERIALS AND METHODS: Titanium-coated epoxy replicas of polished (PO; R(a) = 0.06 microm), finely blasted (FB; R(a) = 1.36 microm), coarsely blasted (CB; R(a) = 5.09 microm), acid-etched (AE; R(a) = 0.59 microm), coarsely blasted and acid-etched (SLA; R(a) = 4.39 microm), titanium plasma-sprayed (TPS; R(a) = 5.85 microm), machined-like (ML; R(a) = 2.15 microm), and micromachined grooved (GR; V-shaped grooves 30 microm deep) surfaces were implanted subcutaneously in 74 rats for 1 to 11 weeks. Animals were sacrificed weekly. Surfaces were processed for histomorphometric evaluation of connective tissue attachment, capsule thickness, and where applicable, the degree of separation between the tissue and implant. RESULTS: A total of 153 test surfaces were analyzed. Statistical analysis revealed that textured and rough substrata, namely the GR, TPS, AE, CB, and SLA surfaces, exhibited significantly greater (P < .05) connective tissue attachment and thinner fibrous encapsulation when compared to the PO surface. Tissue separation from the implant interface was of significantly lower magnitude and frequency with the rough surfaces than with the PO surface. CONCLUSIONS: The results indicate that rough implant surfaces are associated with stable connective tissue attachment, which has implications for their use in percutaneous and permucosal applications. In addition, data from the AE surface may indicate that the geometry of the surface irregularities can also be a significant determinant of the connective tissue response.

Effect of titanium surface topography on macrophage activation and secretion of proinflammatory cytokines and chemokines.

The macrophage has a major role in normal wound healing and the reparative process around implants. Murine macrophage-like cells RAW 264.7 were used to investigate the effect of titanium surfaces on macrophage activation and secretion of proinflammatory cytokines [interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha] and chemokines (monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha). Four topographies were used: those produced by mechanically polishing, coarse sand blasting, acid etching, and sandblasting and acid etching (SLA). Macrophages were plated on the four titanium surfaces at a population density of 5 x 10(5) cells/mL/well. Tissue culture plastic and tissue culture plastic plus lipopolysaccharide (LPS) served as negative and positive control, respectively. In addition, all surfaces were tested for their effects on macrophages in the presence of LPS. Supernatants were collected for assays after 6, 24, and 48 h and the numbers of macrophages attached to the surfaces were quantified using the DAPI (4,6-di-amidino-2-phenylindole) assay. Cytokine and chemokine levels were measured with sandwich enzyme-linked immunosorbent assays. Statistical comparison between the surfaces and the controls was determined by using the two-way analysis of variance including interaction effect (two tailed and p < or = 0.05). Unstimulated macrophages increased their secretion of the proinflammatory cytokine (TNF-alpha) when attached to rough surfaces (acid etching and SLA, p < or = 0.05). In macrophages stimulated with LPS, the roughest surface SLA produced higher levels of IL-1 beta, IL-6, and TNF-alpha at 24 and 48 h than all other surfaces (p < or = 0.05). Surface topography also modulated the secretion of the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha by macrophages. Unstimulated macrophages attached to the SLA surface down-regulated their production of chemokines (p < or = 0.05) whereas LPS-stimulated macrophages attached to the SLA surface up-regulated their production (p < or = 0.05). Moreover, the SLA surface was found to act synergistically with LPS as well as the combination of blasting and etching features of the SLA surface resulted in significant release of proinflammatory cytokines and chemokines by stimulated macrophages at 24 and 48 h (p < or = 0.05). This in vitro study has demonstrated that surface topography, in particular the SLA surface, modulated expression of proinflammatory cytokines and chemokines by macrophages in a time-dependent manner.

The effect of surface roughness on RAW 264.7 macrophage phenotype.

Monocyte-derived cells, including macrophages and foreign body giant cells, can determine the performance of implanted devices. Upon contact with biomaterials, macrophages can be activated into a classic inflammatory (M1) or wound-healing (M2) phenotype. Previously, we showed that high macrophage density on rough SLA implants was associated with early bone formation. This study examined a possible mechanism, namely, surface roughness activation of macrophages to the M2 phenotype to enhance bone formation on the SLA surface. RAW 264.7 macrophages were seeded on SLA or smooth (Po) epoxy substrates and the expression of the M1 and M2 specific markers, NOS2 and Arg-1 measured by qPCR on days 1, 3, and 5. Additionally, secretion of inflammation-associated cytokines and chemokines was studied by antibody arrays and ELISAs. Controls included RAW 264.7 macrophages primed into the M1 or M2 phenotypes by LPS/IFN-γ and IL-4, respectively. Rough SLA surfaces did not activate Arg-1 and NOS2 expression, but relative to Po surfaces MCP-1 and MIP-1α were upregulated after 5 days, whereas the secretion of the M1-associated chemokine IP-10 was lowered. RAW 264.7 macrophages on the SLA surface thus adopted elements of an M2-like phenotype, suggesting that when implanted the SLA surfaces may enhance wound repair.

Standardization of clinical protocols in oral malodor research.

The objective of this study is to standardize protocols for clinical research into oral malodor caused by volatile sulfur compounds (VSCs). To detect VSCs, a gas chromatograph (GC) using a flame photometric detector equipped with a bandpass filter (at 393 nm) is the gold standard (sensitivity: 5 × 10(-11) gS s(-1)). The baselines of VSC concentrations in mouth air varied considerably over a week. When the subjects refrained from eating, drinking and oral hygiene including mouth rinsing, the VSC concentrations remained constant until eating. Over a 6 h period after a meal, VSC concentrations decreased dramatically (p < 0.01). These results point to optimal times and conditions for sampling subjects. Several portable devices were compared with the measurements by the GCs. Portable GCs demonstrated capabilities similar to those of the GCs. We also applied the recommended protocols described below to clinical research testing the efficacy of ZnCl(2) products, and confirmed that using the recommended protocols in a randomized crossover design would provide very clear results. Proposed protocols include: (a) a short-term study rather than a long-term study is strongly recommended, since the VSC concentrations are constant in the short term; (b) a crossover study would be the best design to avoid the effects of individual specificities on each clinical intervention; (c) measurements of VSCs should preferably be carried out using either a GC or portable GCs.

Drug intercalation in layered double hydroxide clay: application in the development of a nanocomposite film for guided tissue regeneration.

It has been proposed that localized and controlled delivery of alendronate and tetracycline to periodontal pocket fluids via guided tissue regeneration (GTR) membranes may be a valuable adjunctive treatment for advanced periodontitis. The objectives of this work were to develop a co-loaded, controlled release tetracycline and alendronate nanocomposite plasticized poly(lactic-co-glycolic acid) (PLGA) film that would form a suitable matrix supporting osteoblast proliferation and differentiation. Alendronate release was successfully controlled, with complete suppression of the burst phase of release by intercalation of alendronate anions in magnesium/aluminum layered double hydroxide (LDH) clay nanoparticles and dispersed in the PLGA film matrix. Tetracycline, loaded as free drug into the film together with alendronate-LDH clay complex released more rapidly than alendronate, but showed evidence of intercalation in the LDH clay particles. The dual drug loaded nanocomposite films were biocompatible with osteoblasts and after 5 week incubations, significant increase in alkaline phosphatase activity and bone nodule formation were observed.

An in vitro study of plasticized poly(lactic-co-glycolic acid) films as possible guided tissue regeneration membranes: material properties and drug release kinetics.

Guided tissue regeneration (GTR), in periodontal therapy, involves the placement of a barrier membrane, to ensure the detached root surface becomes repopulated with periodontal ligament cells capable of regenerating this attachment. GTR procedures exhibit large variability in surgical outcome as a consequence of poor membrane performance. The objective of this study was to evaluate the suitability of plasticized poly(lactic-co-glycolic acid) (PLGA) as a material for GTR membranes. The material was also investigated as a localized controlled release system for the antibiotic, anti-inflammatory agent tetracycline. Films made from PLGA (85:15), plasticized with either 10% w/v methoxypoly(ethyleneglycol) (MePEG) or a diblock copolymer [poly(D,L-lactic acid)-block-methoxypoly(ethyleneglycol)] were loaded with tetracycline base (or hydrochloride salt) and cast by solvent evaporation. Drug release was measured using high performance liquid chromatography (HPLC). The time-course of elasticity changes and swelling were determined using a stress-strain apparatus or gravimetric/dimensional determinations, respectively. Cells extracted from periodontal ligament cell explants were used to evaluate the effect of material and drug loading on cell morphology. Tetracycline·HCl released more rapidly than tetracycline from PLGA films. The addition of either MePEG or diblock caused a concentration dependent increase in release rates for both drugs. Release profiles ranged from a small initial burst phase followed by slow sustained release to almost full drug release after 1 day. After incubation in PBS, the films stiffened and swelled within 30 min. Periodontal ligament cell morphology was not affected by the inclusion of tetracycline. Plasticized PLGA films displayed desired features for possible use as GTR membranes.

Modulation of human gingival fibroblast adhesion, morphology, tyrosine phosphorylation, and ERK 1/2 localization on polished, grooved and SLA substratum topographies.

Attachment of connective tissue to dental implants, which is influenced by surface topography, is an important determinant of implant success. Approaches employed to alter topography include acid etching or blasting to produce roughened surfaces, and production of precisely defined topographies using microfabrication techniques. The aim of this study was to assess the influence of polished, microgrooved, and sand-blasted, large grit, acid-etched (SLA) topographies on fibroblast adhesion, morphology, activation, and ERK 1/2 phosphorylation and localization. Human gingival fibroblasts (HGFs) spread on all tested surfaces within 2 h, and topography influenced the pattern of phosphotyrosine localization. Fibrillar adhesion formation was prominent in HGFs cultured on microgrooves and SLA at 24 h compared with smooth. No significant difference in ERK 1/2 phosphorylation was observed at 2 or 24 h, but nuclear localization depended on culture time and substratum topography. Nuclear localization of ERK 1/2 occurred at 2 h on polished surfaces, but was not evident at 1 week. In contrast, cells on SLA and grooved surfaces did not exhibit nuclear localization of ERK 1/2 at early times, but did at 1 week. The results of this study suggest that rough and microfabricated topographies influence fibroblast adhesion and intracellular signaling through focal adhesion/integrin-dependent mechanisms in a time-dependent manner.

Controlled release of alendronate from polymeric films.

Bisphosphonate drugs alter the balance of bone resorption and formation, leading to a net increase in bone density. Therefore, these drugs are commonly used to treat osteoporosis or as an adjunct to cancer chemotherapy. Local delivery of bisphosphonates, such as alendronate, from polymeric films has the potential to improve efficacy and decrease side-effects common to oral bisphosphonate therapy. Alendronate was effectively encapsulated in film formulations composed of poly(lactic-co-glycolic acid) (PLGA) blended with poly(DL-lactic acid)-block-methoxy poly(ethylene glycol) (diblock co-polymer) and the films were characterized for elasticity, swelling, thermal and drug-release properties. Increasing the proportion of diblock co-polymer in the formulation decreased the glass transition temperature of PLGA, allowing for improved handling via increases in film elasticity. Immersion in aqueous media caused a rapid stiffening and swelling of the films. The inclusion of diblock co-polymer increased the rate of drug release from the films over a 3-week period. Drug-loaded polymeric films containing 0.25% alendronate increased osteoblast viability after 4 days compared to polymer alone. After 5 weeks, there was a significant increase in alkaline phosphatase activity and calcium nodule formation in osteoblasts grown on films containing 1.25% alendronate in 5% diblock co-polymer in PLGA.

Comparison of the response of cultured osteoblasts and osteoblasts outgrown from rat calvarial bone chips to nonfouling KRSR and FHRRIKA-peptide modified rough titanium surfaces.

Mimicking proteins found in the extracellular matrix (ECM) using specific peptide sequences is a well-known strategy for the design of biomimetic surfaces, but has not yet been widely exploited in the field of biomedical implants. This study investigated osteoblast and, as a control, fibroblast proliferation to novel consensus heparin-binding peptides sequences KRSR and FHRIKKA that were immobilized onto rough (particle-blasted and chemically etched) commercially pure titanium surfaces using a poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system. This platform enabled a detailed study of specific cell-peptide interactions even in the presence of serum in the culture medium; thanks to the excellent nonfouling properties of the PLL-g-PEG surface. Cell-binding peptide sequence RGD in combination with KRSR or FHRRIKA was used to examine a potentially-enhanced or synergistic effect on osteoblast proliferation. Bare titanium and bioinactive surfaces (i.e., unfunctionalized PLL-g-PEG and scrambled KSSR, RFHARIK, and RDG) were used as control substrates. Additionally, in a newly developed experimental setup, freshly harvested bone chips from newborn rat calvariae were placed onto the same type of surfaces investigating size and pattern of osteoblast outgrowths. The findings of the current study demonstrated that the difference in osteoblast and fibroblast proliferation was influenced by surface topography more so than by the presence of surface-bound KRSR and FHRRIKA. On the other hand, in comparison with the control surfaces, osteoblast outgrowths from rat calvarial bone chips covered a significantly larger area on RGD, KRSR, and FHRRIKA surfaces after 8 days and also migrated in an isotropic way unlike cells on the bioinactive substrates. Furthermore, the stimulatory effect of 0.75 pmol cm(-2) RGD on osteoblast migration pattern could be enhanced when applied in combination with 2.25 pmol cm(-2) KRSR.

Fabrication of TiO2-coated epoxy replicas with identical dual-type surface topographies used in cell culture assays.

The goal of this study was to reproducibly generate samples with complex surface topographies and chemistries identical to a "master surface" and to test their response in cell culture using rat calvarial cells. Negative replicas of dual-type topography were fabricated using dental impression material with half of the surface exhibiting smooth and rough topography, respectively. Positive epoxy resin replicas were cast from the same negative replica eight times consecutively and coated with a 60-nm thin film of titanium dioxide using a vapor deposition technique. Atomic force microscopy, scanning electron microscopy, confocal white light microscopy, and X-ray photoelectron spectroscopy indicated that TiO(2)-coated epoxy replicas had surface topographical features and surface compositions nearly indistinguishable from the original titanium master surfaces. The described technique showed high reproducibility over at least eight generations of replication using the same negative replica. Rat calvarial osteoblasts proliferated just as well on dual topography surfaces as on single topography surfaces. The advantage of the dual-type substrates is that they facilitate comparison within a single culture dish, thus eliminating dish-to-dish variation as well as saving material, time and costs compared to the usual method of evaluating surfaces in separate dishes.