Differential behaviour and gene expression in 3D cultures of femoral- and calvarial-derived human osteoblasts under a cyclic compressive mechanical load.

The aim of the study was to compare the response of calvarial and femoral osteoblasts cultured in a 3D hydrogel environment to cyclic compressive mechanical loading. Human foetal femoral and calvarial osteoblasts were encapsulated in a semi-synthetic thiol-modified hyaluronan gelatin polyethylene glycol diacrylate (PEGDA) cross-linked HyStemC hydrogel. Constructs were subjected to a cyclic compressive strain of 33.4 kPa force every second for 5 s every hour for 6 h per day using FlexCell BioPress culture plates and compared to non-compressed constructs. Cell viability, mineralisation, and morphological changes were observed over 21 days. BMP2, ALP, COL1A1, COL2A1, and OCN gene expression levels were quantified. Encapsulated osteoblast numbers increased and formed hydroxyapatite over a 21-day period. Cell viability decreased under a cyclical strain when compared to cells under no strain. Femoral osteoblasts under strain expressed increased levels of BMP2 (53.9-fold) and COL1A1 (5.1-fold) mRNA compared to no strain constructs. Surprisingly, no BMP2 mRNA was detected in calvarial osteoblasts. Osteoblasts derived from endochondral (femoral) and intra-membranous (calvarial) processes behaved differently in 3D-constructs. We therefore recommend that site-specific osteoblasts be used for future bone engineering and bone replacement materials and further research undertaken to elucidate how site-specific osteoblasts respond to cyclic compressive loads.

Appliance-induced osteopenia of dentoalveolar bone in the rat: effect of reduced bone strains on serum bone markers and the multifunctional hormone leptin.

To understand, in greater detail, the molecular mechanisms regulating the complex relationship between mechanical strain and alveolar bone metabolism during orthodontic treatment, passive cross-arch palatal springs were bonded to the maxillary molars of 6-wk-old rats, which were killed after 4 and 8 d. Outcome measures included serum assays for markers of bone formation and resorption and for the multifunctional hormone leptin, and histomorphometry of the inter-radicular bone. The concentration of the bone-formation marker alkaline phosphatase (ALP) was significantly reduced at both time points in the appliance group, accompanied by a 50% reduction in inter-radicular bone volume; however, osteocalcin (bone Gla protein) levels remained unaffected. Bone collagen deoxypyridinoline (DPD) crosslinks increased 2.3-fold at 4 d only, indicating a transient increase in bone resorption; in contrast, the level of the osteoclast-specific marker, tartrate-resistant acid phosphatase 5b (TRACP 5b), was unchanged. Leptin levels closely paralleled ALP reductions at both time points, suggesting an important role in the mechanostat negative-feedback loop required to normalize bone mass. These data suggest that an orthodontic appliance, in addition to remodeling the periodontal ligament (PDL)-bone interface, may exert unexpected side-effects on the tooth-supporting alveolar bone, and highlights the importance of recognizing that bone strains can have negative, as well as positive, effects on bone mass.

Re-examination of 'Einige Beitrage zur Theorie der Zahnregulierung' (Some contributions to the theory of the regulation of teeth) published in 1904-1905 by Carl Sandstedt.

The original histological investigation that forms the basis of our present understanding of tooth movement was carried out on dogs by the Swedish dentist Carl Sandstedt at the Karolinska Institute, Stockholm. His findings were published in 1901 as a monograph in Swedish, and shortly after his death in 1904, as three articles in German entitled 'Einige Beiträge zur Theorie der Zahnregulierung'. Sandstedt observed that the bone was deposited on the alveolar wall of the tension side with both heavy and light forces, new bone spicules following the orientation of the periodontal fibres. On the pressure side, with light forces, osteoclasts resorbed the surface of the alveolar bone, but heavy forces compressed the periodontal ligament resulting in hyalinization--the formation of localized cell-free areas. At these sites, bone removal resulted from undermining resorption by osteoclasts from adjacent marrow spaces. He also observed root resorption and commented on the centre of rotation of the teeth. No English version of Sandstedt's research has ever been published, and in view of its importance, one of us (DB) has translated his three articles from the original German. The aim was to persuade an orthodontic journal to publish the articles in full--however, weighing-in at 21,000 words, the impracticality of this plan soon became clear. We concluded that excerpts from the text plus commentary would be the most practical solution. Where possible and without materially changing the intended meaning, we have translated the German text into something resembling contemporary English, accompanied by the original 16 figures.

Altered serum levels of the osteoclast-specific TRACP 5b isoform in Chinese children undergoing orthodontic treatment.

Orthodontic tooth movement is dependent upon the ability of mechanical forces to induce remodelling activity within the tooth-supporting alveolar bone. In view of the importance of bone resorption in mediating tooth movement, the aim of this study was to establish if alterations in the osteoclast-specific bone marker tartrate-resistant acid phosphatase (TRACP) 5b could be detected in the sera of patients undergoing orthodontic treatment. The sample consisted of 14 subjects (10 girls and 4 boys) aged 10.5-16.5 years (mean 12.6 years) being treated with fixed appliances and a distalizing headgear. Venous blood samples (3 ml) were collected from the cubital vein pre-treatment (T0) and 2, 4, and 6 months into treatment (T1-T3); serum TRACP 5b levels were quantified using a solid-phase immunofixed enzyme activity assay. When the data were pooled and treated cross-sectionally, a significant increase in immunoreactive TRACP 5b was detected at 2 months (T1) indicating increased bone resorptive activity. However, when the serum profiles of individual patients were recorded longitudinally, a very different pattern emerged, not all patients following the same trend. This is not surprising given normal anatomical variation and differences between the patients in age, gender, and mechanotherapy. Designed as a pilot to demonstrate 'proof of principle', this study is the first to show that the TRACP 5b isoform can be detected in the sera of patients undergoing orthodontic treatment. It further suggests that serum bone marker measurements offer a simple and minimally invasive method for correlating the findings of laboratory and animal experimentation with clinical data.

Micro-vibrations at 30 Hz on bone cells cultivated in vitro produce soluble factors for osteoclast inhibition and osteoblast activity.

OBJETIVE: It has been claimed that micro-pulse vibration can accelerate the rate of tooth movement during orthodontic treatment; however, the underlying cellular mechanism has yet to be elucidated. The purpose of this study was to understand the mechanisms underlying tooth movement acceleration by measuring alterations in a panel of intercellular signalling molecules and markers of osteoblast/osteoclast function following micro-pulse vibration for 20 min at 30 Hz. DESIGN: Primary BALB/c mouse calvarial osteoblasts were cultivatedin vitro and subjected to micro-pulse vibration (0.25 N; 30 Hz) with the AcceleDent® Aura appliance for 20 min and assayed for IL-4, IL-13, IL-17, OPG, soluble RANKL and TGF-ß protein by ELISA; for PCNA in osteoblasts and caspase 3/7 in osteoclasts by immunohistochemistry; for IL-4, IL-13, and Il-17 in osteoclasts by ELISA; and for cathepsin K by flow cytometry. RESULTS: After micro-pulse vibration, the murine osteoblast culture supernatant showed increased IL-4, IL-13, IL-17, OPG and TGF-ß levels and decreased RANKL levels; PCNA in osteoblasts and caspase 3/7 in osteoclasts were also upregulated. The osteoclast culture supernatant had increased levels of IL-4, IL-13 and IL-17, and cathepsin K was upregulated in the treatment group compared with the control group. CONCLUSIONS: Micro-pulse vibration promotes the production of soluble factors that inhibit osteoclasts, promote apoptosis and activate osteoblasts in vitro, which could increase bone mineral density. Further studies should be conducted in order to understand the biological mechanism of how micro-vibration might influence tooth movement during orthodontic treatment.

Induction of osteopenia during experimental tooth movement in the rat: alveolar bone remodelling and the mechanostat theory.

Increases in bone strains above a certain threshold have a positive effect on bone mass, whereas reductions in strain magnitude lead to bone loss and osteopenia; the term 'mechanostat' has been introduced to describe this tissue-level negative feedback mechanism. The mechanobiology of bone and particularly alveolar bone is poorly understood, and whether the mechanostat theory has any relevance to explaining the osseous changes that occur during orthodontic tooth movement remains unclear. To investigate the relationship further, an expansile force of 0.2 N was applied to the maxillary molars of 36, 6-week-old Wistar rats by helical coil springs. The animals were sacrificed at 1, 2, 4, and 8 days and the tissue response analyzed by histological, biochemical, and finite element (FE) methods. Differences between groups were determined by Student's t-test (two-tailed). The appliance produced an increase in the intermolar width averaging 0.5 mm after 8 days. Tetracycline uptake in the control rats suggested a rapid turnover of bone in both the interradicular domain and the bone-periodontal ligament interface. In the experimental group, however, incorporation of tetracycline into the interradicular domain was reduced and conventional histology revealed evidence of bone loss and osteopenia, in both the experimental and a group of sham-treated positive controls (with inactive, annealed springs). Serum alkaline phosphatase declined significantly in both experimental and sham-treated groups over the 8-day time course, indicating decreased bone formation. Serum acid phosphatase also declined, suggesting a concomitant decrease in bone resorption. Three-dimensional FE analysis of the stresses generated in the bone following occlusal (2 N) and orthodontic loading showed that the orthodontic force created a constant loading condition shielding some areas of bone from mechanical stress. Areas of low mechanical stimulation were coincident with sites of bone loss observed histologically, while bone mass was preserved in areas with higher levels of loading. These findings suggest that (1) the osteopenia resulted from stress shielding of the interradicular bone by the appliance, and a consequent reduction in occlusal loading below the critical threshold required for maintaining normal osseous architecture and (2) the mechanostat model can be employed to explain, at least in part, the response of the bone to orthodontic loading.

Cultured human periodontal ligament cells constitutively express multiple osteotropic cytokines and growth factors, several of which are responsive to mechanical deformation.

BACKGROUND AND OBJECTIVE: A role for cytokines and growth factors in mediating the cellular and molecular events involved in orthodontic tooth movement is well established. The focus to date, however, has been largely on individual mediators, rather than to study cytokines in terms of complex interacting networks. Our objective was to expand our knowledge of the cytokines and growth factors expressed by human periodontal ligament (PDL) cells and to identify new genes that are responsive to mechanical deformation. MATERIAL AND METHODS: Human PDL cells were strained with a cyclic deformation of 12% for 6-24 h, and the differential expression of 79 cytokine and growth factor genes was quantified using real-time RT-PCR arrays. For statistical comparison, t-tests were used with mean critical threshold (CT) values derived from triplicate samples. RESULTS: Forty-one genes were detected at CT values < 35 and, of these, 15 showed a significant change in relative expression. These included seven interleukins (IL): IL1A, IL1F7, IL6 and IL7 (down), IL8, IL11 and IL12A (up). Eight genes representing other cytokine and growth factor families showed comparable mechanical sensitivity, including VEGFD and OPG (down) and PDGFA, INHBA, GDF8 and two transforming growth factor beta genes, TGFB1 and TGFB3 (up). The genes CSF2/GMCSF and IL11 were found to be consistently stimulated across all three time points. Genes that were not expressed included: (1) the immunoregulatory lymphokines (IL2-IL5), IL17 and IL17B; (2) IL10 and other members of the IL-10 family of anti-inflammatory cytokines (IL19, IL20, IL22 and IL24); and (3) TNF and RANKL. CONCLUSION: Human PDL cells constitutively express numerous osteotropic cytokines and growth factors, many of which are mechanoresponsive.

Engineering the periodontal ligament in hyaluronan-gelatin-type I collagen constructs: upregulation of apoptosis and alterations in gene expression by cyclic compressive strain.

To engineer constructs of the periodontal ligament (PDL), human PDL cells were incorporated into a matrix of hyaluronan, gelatin, and type I collagen (COLI) in sample holders (13×1 mm) of six-well Biopress culture plates. The loading dynamics of the PDL were mimicked by applying a cyclic compressive strain of 33.4 kPa (340.6 gm/cm(2)) to the constructs for 1.0 s every 60 s, for 6, 12, and 24 h in a Flexercell FX-4000C Strain Unit. Compression significantly increased the number of nonviable cells and increased the expression of several apoptosis-related genes, including initiator and executioner caspases. Of the 15 extracellular matrix genes screened, most were upregulated at some point after 6-12 h deformation, but all were downregulated at 24 h, except for MMPs1-3 and CTGF. In culture supernatants, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) protein levels were upregulated at 24 h; receptor activator of nuclear kappa factor B (RANKL), osteoprotegerin (OPG) and fibroblast growth factor-2 (FGF-2) were unchanged; and connective tissue growth factor (CTGF) not detected. The low modulus of elasticity of the constructs was a disadvantage-future mechanobiology studies and tissue engineering applications will require constructs with much higher stiffness. Since the major structural protein of the PDL is COLI, a more rational approach would be to permeabilize preformed COLI scaffolds with PDL-populated matrices.

Alterations in the Synthesis of IL-1ß, TNF-α, IL-6, and Their Downstream Targets RANKL and OPG by Mouse Calvarial Osteoblasts In vitro: Inhibition of Bone Resorption by Cyclic Mechanical Strain.

Mechanical strain is an important determinant of bone mass and architecture, and the aim of this investigation was to further understand the role of the cell-cell signaling molecules, IL-1ß, TNF-α, and IL-6 in the mechanobiology of bone. Mouse calvarial osteoblasts in monolayer culture were subjected to a cyclic out-of-plane deformation of 0.69% for 6 s, every 90 s for 2-48 h, and the levels of each cytokine plus their downstream targets RANKL and OPG measured in culture supernatants by ELISAs. Mouse osteoblasts constitutively synthesized IL-1ß, TNF-α, and IL-6, the production of which was significantly up-regulated in all three by cyclic mechanical strain. RANKL and OPG were also constitutively synthesized; mechanical deformation however, resulted in a down-regulation of RANKL and an up-regulation OPG synthesis. We next tested whether the immunoreactive RANKL and OPG were biologically active in an isolated osteoclast resorption pit assay - this showed that culture supernatants from mechanically deformed cells significantly inhibited osteoclast-mediated resorptive activity across the 48 h time-course. These findings are counterintuitive, because IL-1ß, TNF-α, and IL-6 have well-established reputations as bone resorptive agents. Nevertheless, they are pleiotropic molecules with multiple biological activities, underlining the complexity of the biological response of osteoblasts to mechanical deformation, and the need to understand cell-cell signaling in terms of cytokine networks. It is also important to recognize that osteoblasts cultured in vitro are deprived of the mechanical stimuli to which they are exposed in vivo - in other words, the cells are in a physiological default state that in the intact skeleton leads to decreased bone strains below the critical threshold required to maintain normal bone structure.

Short periods of cyclic mechanical strain enhance triple-supplement directed osteogenesis and bone nodule formation by human embryonic stem cells in vitro.

Human embryonic stem cells (hESCs) are uniquely endowed with a capacity for both self-renewal and multilineage differentiation. The aim of this investigation was to determine if short periods of cyclic mechanical strain enhanced dexamethasone, ascorbic acid, and ß-glycerophosphate (triple-supplement)-induced osteogenesis and bone nodule formation by hESCs. Colonies were cultured for 21 days and divided into control (no stretch) and three treatment groups; these were subjected to in-plane deformation of 2% for 5 s (0.2 Hertz) every 60 s for 1 h on alternate days in BioFlex plates linked to a Flexercell strain unit over the following periods (day 7-13), (day 15-21), and (day 7-21). Numerous bone nodules were formed, which stained positively for osteocalcin and type I collagen; in addition, MTS assays for cell number as well as total collagen assays showed a significant increase in the day 7-13 group compared to controls and other treatment groups. Alizarin Red staining further showed that cyclic mechanical stretching significantly increased the nodule size and mineral density between days 7-13 compared to control cultures and the other two experimental groups. We then performed a real-time polymerase chain reaction (PCR) microarray on the day 7-13 treatment group to identify mechanoresponsive osteogenic genes. Upregulated genes included the transcription factors RUNX2 and SOX9, bone morphogenetic proteins BMP1, BMP4, BMP5, and BMP6, transforming growth factor-ß family members TGFB1, TGFB2, and TGFB3, and three genes involved in mineralization-ALPL, BGLAP, and VDR. In conclusion, this investigation has demonstrated that four 1-h episodes of cyclic mechanical strain acted synergistically with triple supplement to enhance osteogenesis and bone nodule formation by cultured hESCs. This suggests the development of methods to engineer three-dimensional constructs of mineralized bone in vitro, could offer an alternative approach to osseous regeneration by producing a biomaterial capable of providing stable surfaces for osteoblasts to synthesize new bone, while at the same time able to be resorbed by an osteoclastic activity-in other words, one that can recapitulate the remodeling dynamics of a naturally occurring bone matrix.

Northcroft Memorial Lecture 2007. A century of progress: advances in orthodontics since the foundation of the British Society for the Study of Orthodontics.

The British Orthodontic Society invites outstanding contributors from the field of Orthodontics to give the guest lecture in memory of George Northcroft. In 2007 the guest lecturer was Professor Murray C. Meikle. The article that follows was presented as the Northcroft Memorial Lecture 2007 at the British Orthodontic Conference, Harrogate, UK, 24th September 2007.

The tissue, cellular, and molecular regulation of orthodontic tooth movement: 100 years after Carl Sandstedt.

The first experimental investigation of orthodontic tooth movement was published by Sandstedt in 1904-1905. After 100 years, there is a good understanding of the sequence of events at both tissue and cellular levels and now the current focus of research is at the molecular level. The techniques of reverse transcription-polymerase chain reaction and in situ hybridization to detect mRNAs of interest have revolutionized tooth movement studies and an expanding list of antibodies and enzyme-linked immunosorbent assays directed against human and animal proteins will facilitate their identification in tissue sections and/or culture supernatants. Nevertheless, although this technology has greatly simplified research for the clinical and laboratory investigator, message is not always translated into protein, and the presence of a protein does not necessarily mean it is biologically active. In vivo and in vitro methods have been widely used in tooth movement studies. However, data from in vitro models, in which the mechanical stimulus can be carefully controlled (tension versus compression; intermittent versus continuous), should be correlated with in vivo data from animal models. The current evidence suggests that downstream from the initial mechanotransduction event at focal adhesions which link the extracellular matrix to the cytoskeleton, mechanically induced remodelling is mediated by a complex feedback mechanism involving the synthesis of cytokines such as interleukin-1 (IL-1), IL-6, and receptor activator of nuclear factor k B ligand by cells of the osteoblast and/or fibroblast lineages. These in turn act in an autocrine/paracrine fashion to regulate the expression of transcription factors, cytokines, growth factors, enzymes, and structural molecules involved in the differentiation, proliferation, and function of mesenchymal and other cell types. Contrary to the impression gained from the literature, tooth movement is not confined to events within the periodontal ligament. Orthodontic tooth movement involves two interrelated processes: (1) deflection or bending of the alveolar bone and (2) remodelling of the periodontal tissues.

Guest editorial: what do prospective randomized clinical trials tell us about the treatment of class II malocclusions? A personal viewpoint.

The prospective randomized clinical trial (RCT) is seen by many to be the 'gold standard' for analysing treatment outcome and the only valid source of clinical data. In orthodontics, most RCTs have been designed to resolve the controversy surrounding the ability of functional appliances to significantly modify dentofacial growth. Given the variability in the timing, magnitude and duration of pubertal dentofacial growth, differing levels of motivation and patient compliance, the inherent inaccuracy of cephalometry and the questionable validity of the measurements themselves used to quantitate change, it is not surprising that the conclusions have not been as clear-cut as anticipated. Unlike a laboratory experiment, in which it is possible to limit the differences between experimental and control groups to the single factor being investigated, in a clinical trial an orthodontic appliance is just one of several variables affecting the outcome. Furthermore, RCTs are expensive and time-consuming; by the time the end-point of the study has been reached, the appliance may no longer be in widespread use, the fate of more than one recent well-publicized RCT. What RCTs have shown is that functional appliances such as the Herbst and twin block, based on the principle of 'jumping the bite', are more effective at modifying dentofacial growth and reducing overjets than headgear and more passive appliances such as the Andresen activator and its variants. However, if one asks whether RCTs have achieved their objective, or provided knowledge not previously available from retrospective studies or animal experimentation, then the answer would have to be no; it is also hard to justify the cost. What is particularly interesting is that knowledge based on years of clinical experience has been disregarded and then announced as if it was something completely new.

Cytokines, matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 in gingival crevicular fluid from patients with Papillon-Lefèvre syndrome.

The aim of the present study was to compare concentrations of cytokines, matrix metalloproteinases (MMPs) and a metalloproteinase inhibitor (TIMP-1) in gingival crevicular fluids (GCF) from sites with gingival inflammation in 28 young patients with Papillon-Lefèvre syndrome (PLS), and in age- and gender-matched controls. Each group consisted of 17 females and 11 males with a mean age of 11.0 years (range 4-22 years). In both groups, anterior upper sites with a clinical diagnosis of gingival inflammation and with pockets < or = 3 mm were selected for sampling of GCF, which was carried out with filter disks inserted into the gingival crevice until saturated. The concentrations of cytokines (IL-1alpha, IL-1beta, TNF-alpha, and IL-8), matrix metalloproteinases (MMP-1, MMP-3, MMP-8, and MMP-9), and their tissue inhibitor (TIMP-1) were analysed using commercial ELISA kits. Significantly higher levels of IL-1beta (P < 0.001) and MMP-8 (P < 0.05) were disclosed among the PLS patients compared with their controls, while the opposite was found for IL-8 (P < 0.05) and MMP-1 (P < 0.001). The individual variations were considerable in both groups. When comparing the expression of cytokines, MMPs, and TIMP-1 in PLS patients with clinically active and non-active periodontitis, the non-active PLS patients showed significantly higher values of IL-1beta than the patients with active periodontal disease (ANOVA, P < 0.01). In conclusion, this study was unable to demonstrate a clear-cut pathognomonic expression of cytokines or MMPs in patients with PLS, but further studies on cytokine and MMP output are warranted.