Reduced masticatory function is related to lower satellite cell numbers in masseter muscle.

The physiology of masseter muscles is known to change in response to functional demands, but the effect on the satellite cell (SC) population is not known. In this study, the hypothesis is tested that a decreased functional demand of the masseter muscle causes a reduction of SCs. To this end, twelve 5-week-old male Sprague-Dawley rats were put on a soft diet (SD, n = 6) or a hard diet (HD, n = 6) and sacrificed after 14 days. Paraffin sections of the superficial masseter and the m. digastricus (control muscle) were stained with haematoxylin and eosin for tissue survey and with anti-myosin heavy chain (MHC) for slow and fast fibres. Frozen sections of both muscles were double-stained for collagen type IV and Pax7. Slow MHC fibres were equally distributed in the m. digastricus but only localized in a small area of the m. masseter. No differences between HD or SD for the m. digastricus were found. The m. masseter had more SCs per fibre in HD than in SD (0.093 ± 0.007 and 0.081 ± 0.008, respectively; P = 0.027). The m. masseter had more fibres per surface area than the m. digastricus in rats with an SD group (758.1 ± 101.6 and 568.4 ± 85.6, P = 0.047) and a HD group (737.7 ± 32.6 and 592.2 ± 82.2; P = 0.007). The m. digastricus had more SCs per fibre than the m. masseter in the SD group (0.094 ± 0.01 and 0.081 ± 0.008; P = 0.039). These results suggest that reduced masseter muscle function is related to a lower number of SCs. Reduced muscle function might decrease microdamage and hence the requirement of SCs in the muscle fibres.

Local variations in turnover of periodontal collagen fibers in rats.

BACKGROUND AND OBJECTIVE: The exact cause of orthodontic relapse is still unclear, although it is often suggested to be caused by periodontal collagen fibers. We hypothesize that long-lived collagen fibers in the periodontium cause relapse. The aim was to determine the half-life of periodontal collagen fibers around rat molars. MATERIAL AND METHODS: Thirty weanling rats were repeatedly injected with (3) H-proline, and autoradiography of histological sections was performed at 1, 4, 8, 15, 22, 29, 36, 57, 78 and 113 d after labeling. Grain densities determined in specific areas of the periodontium were used to calculate collagen half-life. RESULTS: The half-life (t(½) ) was found to decrease from the supra-alveolar region to the apical periodontal ligament region. It was longer in the supra-alveolar region (1.39 ± 0.14 wk) compared with the deeper regions (p < 0.05). The t(½) of the upper periodontal ligament region (0.78 ± 0.20 wk) was longer than that of the inter-radicular periodontal ligament region (0.42 ± 0.07 wk, p < 0.05). The t(½) of the apical periodontal ligament region was 0.61 ± 0.15 wk. CONCLUSION: The data indicate that long-lived collagen fibers do not exist in the soft tissues of the periodontium, and are probably not responsible for relapse. The differences in collagen half-life might be caused by local variations in compressive strain induced by normal function.

Validity of near-infrared light transillumination for the assessment of proximal caries in permanent teeth.

PURPOSE: The aim of this study was to evaluate the performance of DIAGNOcam (DC) in diagnosing proximal caries and to compare its effectiveness with the International Caries Detection and Assessment System (ICDAS) and bitewing radiography (BWR). METHODS: 118 premolars extracted for orthodontic reasons were included and examined using three detection methods and validated by histological sections as the gold standard. The sensitivity, specificity and areas under the ROC curve (Az value) at the outer half enamel (D1), inner half enamel (D2) and dentine (D3) thresholds were compared between different methods. RESULTS: At all categories, the specificity of DC was almost as high as ICDAS and BWR. DC showed a significantly higher sensitivity (0.68) than both visual (0.33) and radiographic examination (0.47) at the D1 threshold. DC presented the highest Az value (area under the ROC curve) at the D1 and D2 threshold (0.81, 0.86), while BWR showed the greatest Az values at D3 (0.94). Furthermore, DC had the highest association strength with the gold standard (Spearman's ρ = 0.80). CONCLUSIONS: It can be concluded that DC could detect proximal caries effectively and showed comparable or even better performance than ICDAS and BWR.

Bone marrow-derived cells in palatal wound healing.

OBJECTIVE: Myofibroblasts are responsible for contraction and scarring after cleft palate repair. This leads to growth disturbances in the upper jaw. We hypothesized that cells from the bone marrow are recruited to palatal wounds and differentiate into myofibroblasts. METHODS: We transplanted bone marrow from green fluorescent protein (GFP)-transgenic rats into lethally irradiated wild-type rats. After recovery, experimental wounds were made in the palatal mucoperiosteum, and harvested 2 weeks later. GFP-expressing cells were identified using immunostaining. Myofibroblasts, activated fibroblasts, endothelial cells, and myeloid cells were quantified with specific markers. RESULTS: After transplantation, 89 ± 8.9% of mononuclear cells in the blood expressed the GFP and about 50% of adherent cells in the bone marrow. Tissue obtained during initial wounding contained only minor numbers of GFP-positive cells, like adjacent control tissue. Following wound healing, 8.1 ± 5.1% of all cells in the wound area were positive, and 5.0 ± 4.0% of the myofibroblasts, which was significantly higher than in adjacent tissue. Similar percentages were found for activated fibroblasts and endothelial cells, but for myeloid cells it was considerably higher (22 ± 9%). CONCLUSIONS: Bone marrow-derived cells contribute to palatal wound healing, but are not the main source of myofibroblasts. In small wounds, the local precursor cells are probably sufficient to replenish the defect.

Skin and oral mucosa equivalents: construction and performance.

The skin and the oral mucosa act as a barrier against the external environment. Loss of this barrier function causes dehydration and a high risk of infection. For the treatment of extensive skin wounds such as in severe burns, autologous skin for transplantation is often not available in sufficient amounts. Reconstructions in the oral cavity, as required after tumor resections or cleft palate repair, are often complicated by similar problems. In the last two decades, the field of tissue engineering has provided new solutions to these problems. Techniques have been developed for the culture of epithelial grafts, dermal substitutes, and the combination of these two to a 'functional' skin or mucosa equivalent. The present review focuses on developments in the field of tissue engineering of skin and oral mucosa. The performance of different types of engineered grafts in animal models and clinical studies is discussed. Recent developments such as the use of epithelial stem cells, and gene therapy with transduced skin grafts are also discussed.

Rate of orthodontic tooth movement after changing the force magnitude: an experimental study in beagle dogs.

OBJECTIVES: To study a possible dose-response relation between force magnitude and rate of orthodontic tooth movement by altering forces during bodily orthodontic tooth movement. SETTING AND SAMPLE POPULATION: Eight young adult beagle dogs were used. The experiments were carried out in the Central Animal Facility, and all analyses were conducted in the Department of Orthodontics and Oral Biology, Radboud University Nijmegen Medical Centre. MATERIALS AND METHODS: Orthodontic appliances were placed exerting a reciprocal force on the mandibular second premolars and first molars. A force of 10 or 300 cN was randomly assigned to each side of the dogs. After 22 weeks, all forces were changed to 600 cN. Based on intra-oral measurements, tooth movement rates were calculated. RESULTS: The premolars showed no difference in the rates of tooth movement with 10 or 300 cN. Replacing 10 for 600 cN increased the rate, but replacing 300 for 600 cN did not. Molars moved faster with 300 than with 10 cN, and changing both forces to 600 cN increased the rate of tooth movement. Data from all teeth were pooled considering their relative root surfaces, and a logarithmic relation was found between force and rate of tooth movement. CONCLUSIONS: Only in the very low force range, a positive dose-response relation exists, while in higher force ranges, no such relation could be established.

Orofacial Muscles: Embryonic Development and Regeneration after Injury.

Orofacial congenital defects such as cleft lip and/or palate are associated with impaired muscle regeneration and fibrosis after surgery. Also, other orofacial reconstructions or trauma may end up in defective muscle regeneration and fibrosis. The aim of this review is to discuss current knowledge on the development and regeneration of orofacial muscles in comparison to trunk and limb muscles. The orofacial muscles include the tongue muscles and the branchiomeric muscles in the lower face. Their main functions are chewing, swallowing, and speech. All orofacial muscles originate from the mesoderm of the pharyngeal arches under the control of cranial neural crest cells. Research in vertebrate models indicates that the molecular regulation of orofacial muscle development is different from that of trunk and limb muscles. In addition, the regenerative ability of orofacial muscles is lower, and they develop more fibrosis than other skeletal muscles. Therefore, specific approaches need to be developed to stimulate orofacial muscle regeneration. Regeneration may be stimulated by growth factors such fibroblast growth factors and hepatocyte growth factor, while fibrosis may be reduced by targeting the transforming growth factor ß1 (TGFß1)/myofibroblast axis. New approaches that combine these 2 aspects will improve the surgical treatment of orofacial muscle defects.

The anti-epileptic drug valproic acid causes malformations in the developing craniofacial skeleton of zebrafish larvae.

Valproic acid (VPA) is an anti-epileptic drug known to cause congenital craniofacial abnormalities, including orofacial clefts (OFC). The exact mechanisms by which VPA leads to craniofacial skeletal malformations are poorly understood. In this study, we investigated the effects of VPA on cartilage and bone formation in the zebrafish larval head during 1-13 hpf (early) and 25-37 hpf (late) development in which cranial neural crest cells (CNCCs) arise and then proliferate and differentiate, respectively. Double-staining for cartilage and bone at 5 dpf revealed that VPA reduced cartilage and bone formation in a dose-dependent manner after both early or late exposure. Several different CNCC-derived cartilage and bone elements were affected in both groups. In the early group (100 µM VPA), the posterior head length and the ethmoid plate were reduced in length (both p < 0.01), while mineralization of 4 out of 9 bone elements was often lacking (all p < 0.01). In the late group (100 µM VPA), also the posterior head length was reduced as well as the length of the ceratohyals (both p < 0.01). Similar to early exposure, mineralization of 3 out of 9 bone elements was often lacking (all p < 0.01). These results indicate that both CNCC formation (early) and differentiation (late) are hampered by VPA treatment, of which the consequences for bone and cartilage formation are persistent at 5 dpf. Indeed, we also found that the expression of several genes related to cartilage and bone was upregulated at 5 dpf. These data indicate a compensatory reaction to the lack of cartilage and bone. Altogether, VPA seems to induce craniofacial malformations via disturbed CNCC function leading to defects in cartilage and bone formation.

Matrix metalloproteinase inhibitors reduce collagen gel contraction and alpha-smooth muscle actin expression by periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Orthodontic tooth movement requires remodeling of the periodontal tissues. The matrix metalloproteinases (MMPs) degrade the extracellular matrix components of the periodontal ligament, while the tissue inhibitors of metalloproteinases (TIMPs) control their activity. Synthetic MMP inhibitors have been developed to inhibit MMP activity. In this study, periodontal ligament cells in contracting collagen gels served as a model for enhanced periodontal remodeling. The effect of MMP inhibitors on gel contraction and on MMP and TIMP expression was analyzed. MATERIAL AND METHODS: Human periodontal ligament cells were cultured in three-dimensional collagen gels and incubated with the MMP inhibitors BB94, CMT-3, doxycycline and Ilomastat. Gel contraction was determined using consecutive photographs. The relative amounts of MMPs and TIMPs were analyzed using substrate zymography and mRNA expression using quantitative polyermase chain reaction. RESULTS: All MMP inhibitors reduced MMP activity to about 20% of the control activity. They all reduced contraction, but CMT-3 and doxycycline had the strongest effect. These inhibitors also reduced MMP-2, MMP-3 and alpha-smooth muscle actin mRNA expression. The expression of MMP-1 mRNA seemed to be increased by CMT-3. No effects were found on the amounts of MMPs and TIMPs. CONCLUSION: Synthetic MMP inhibitors strongly reduced gel contraction by periodontal ligament cells. This was primarily caused by an inhibitory effect on MMP activity, which reduces matrix remodeling. In addition, alpha-smooth muscle actin expression was reduced by CMT-3 and doxycycline, which limits the contractile activity of the fibroblasts.

Increased expression of integrin alpha2 and abnormal response to TGF-beta1 in hereditary gingival fibromatosis.

OBJECTIVE: To investigate the possible correlation between integrin alpha1, alpha2, and beta1 expression and excessive collagen synthesis in fibroblasts from 3 unrelated Chinese families with hereditary gingival fibromatosis (HGF). DESIGN: Gingival fibroblasts from three Chinese HGF patients and three healthy subjects were included. The expression of alpha1, alpha2, and beta1 integrin subunits was examined by immunohistochemistry, quantitative PCR, and flow cytometry. We also investigated the effects of transforming growth factor-beta1 (TGF-beta1) on the expression of these integrin subunits. RESULTS: Our results demonstrate that the expression of alpha2 was significantly higher in HGF fibroblasts compared with control fibroblasts (P < 0.01). No significant differences in the expression of alpha1 and beta1 were detected. Furthermore, TGF-beta1 promoted the expression of alpha1 and alpha2 in fibroblasts from both HGF patients and controls. However, it had a larger effect on the expression of alpha2 in HGF fibroblasts than in control cells. In contrast, alpha1 expression was stimulated more in control fibroblasts. CONCLUSION: The increased expression of integrin alpha2 and the increased response to TGF-beta1 of HGF fibroblasts may be related to the excessive collagen deposition in HGF patients.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement.

Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate differences in MMP and TIMP levels in the gingival crevicular fluid (GCF) at the resorption and apposition sides of orthodontically moved teeth, and to compare these with control teeth. GCF samples were collected from eight orthodontic patients wearing fixed appliances with superelastic nickel-titanium coil springs. The samples were analysed by gelatin zymography, which allows detection of both active and latent MMPs, and reverse zymography for analysis of TIMPs. Western blotting was performed to confirm the identity of MMPs. The data were analysed using either the one-way analysis of variance or the Kruskal-Wallis test. In general, higher levels of MMPs and TIMPs were found at both the resorption and apposition sides compared with the control teeth. Remarkably, partially active MMP-1 was found in GCF from both the resorption and the apposition side but was barely present at the control teeth. TIMP-1 was strongly increased at the apposition side. Gelatinases were mainly present at the resorption side, while gelatinolytic fragments were exclusively detected at the apposition side. MMP-9, which is known to be involved in bone degradation, and a 48 kDa gelatinase were increased at the resorption side. The small increase in TIMP-1 at the resorption side might stimulate bone resorption, whereas the large increase at the apposition side reduces bone resorption. The analysis of MMPs and TIMPs may contribute to the improvement of orthodontic treatment regimens.

Fgf8a mutation affects craniofacial development and skeletal gene expression in zebrafish larvae.

Craniofacial development is tightly regulated and therefore highly vulnerable to disturbance by genetic and environmental factors. Fibroblast growth factors (FGFs) direct migration, proliferation and survival of cranial neural crest cells (CNCCs) forming the human face. In this study, we analyzed bone and cartilage formation in the head of five dpf fgf8ati282 zebrafish larvae and assessed gene expression levels for 11 genes involved in these processes. In addition, in situ hybridization was performed on 8 and 24 hours post fertilization (hpf) larvae (fgf8a, dlx2a, runx2a, col2a1a). A significant size reduction of eight out of nine craniofacial cartilage structures was found in homozygous mutant (6-36%, P<0.01) and heterozygous (7-24%, P<0.01) larvae. Also, nine mineralized structures were not observed in all or part of the homozygous (0-71%, P<0.0001) and heterozygous (33-100%, P<0.0001) larvae. In homozygote mutants, runx2a and sp7 expression was upregulated compared to wild type, presumably to compensate for the reduced bone formation. Decreased col9a1b expression may compromise cartilage formation. Upregulated dlx2a in homozygotes indicates impaired CNCC function. Dlx2a expression was reduced in the first and second stream of CNCCs in homozygous mutants at 24 hpf, as shown by in situ hybridization. This indicates an impairment of CNCC migration and survival by fgf8 mutation.

Cleft palate cells can regenerate a palatal mucosa in vitro.

Cleft palate repair leaves full-thickness mucosal defects on the palate. Healing might be improved by implantation of a mucosal substitute. However, the genetic and phenotypic deviations of cleft palate cells may hamper tissue engineering. The aim of this study was to construct mucosal substitutes from cleft palate cells, and to compare these with substitutes from normal palatal cells, and with native palatal mucosa. Biopsies from the palatal mucosa of eight children with cleft palate and eight age-matched control individuals were taken. Three biopsies of both groups were processed for (immuno)histochemistry; 5 were used to culture mucosal substitutes. Histology showed that the substitutes from cleft-palate and non-cleft-palate cells were comparable, but the number of cell layers was less than in native palatal mucosa. All epithelial layers in native palatal mucosa and mucosal substitutes expressed the cytokeratins 5, 10, and 16, and the proliferation marker Ki67. Heparan sulphate and decorin were present in the basal membrane and the underlying connective tissue, respectively. We conclude that mucosal cells from children with cleft palate can regenerate an oral mucosa in vitro.

Interplay of mechanical loading and growth factors in the mandibular condyle.

The mandibular condyle is an important growth site in the developing mandible. The growth of the condyle is known to be highly adaptable to functional factors. This property is exploited in orthodontics for the treatment of class II malocclusions and mandibular asymmetries. However, there is an ongoing debate on the efficacy of functional appliances. The comparison of experimental studies is complicated by the lack of detailed analyses of the load distribution within the condyle. In spite of this, there is a large body of evidence showing that mechanical manipulation of the condyle induces metabolic changes, and changes in the expression of growth factors and other signalling molecules. This review aims to give an overview of the role of growth factors in the condyle with special emphasis on their responsiveness to mechanical perturbation.

Relaxin stimulates MMP-2 and alpha-smooth muscle actin expression by human periodontal ligament cells.

The main cells in the periodontal ligament (PDL) are the fibroblasts, which play an important role in periodontal remodelling. Matrix metalloproteinases (MMPs) are largely responsible for the degradation of extracellular matrix proteins in the PDL. Previous studies have indicated that MMP production can be stimulated by the hormone relaxin. This hormone facilitates delivery by softening the connective tissues of the reproductive tract, and it prepares the mammary gland for lactation. Periodontal remodelling takes place during orthodontic tooth movement, which might be enhanced by relaxin. Therefore, we investigated the effects of relaxin on gelatinase expression of human PDL cells. Cultures of human PDL cells were incubated with relaxin. Gelatinase (MMP-2 and -9) expression, alpha-smooth muscle actin expression (alpha-SMA), total MMP activity and DNA content were measured. Both proMMP-2 and active MMP-2 was identified in the cultures. There was a clear trend showing a dose-dependent increase of MMP-2 production, which was significant at 250 ng/ml. Total MMP activity was not affected. A stimulation of alpha-SMA expression was found at 50 ng/ml. The results indicate that relaxin activates human PDL cells by the stimulation of MMP-2 and alpha-smooth muscle actin.

Mechanobiology of tooth movement.

This review describes the mechanical and biological signalling pathways during orthodontic tooth movement and provides an update of the current literature. A theoretical model is introduced to elucidate the complex cascade of events after the application of an orthodontic force to a tooth. In this model, the events are divided into four stages: matrix strain and fluid flow, cell strain, cell activation and differentiation, and remodelling. Each stage is explained in detail and discussed using recent literature.

CMT-3 inhibits orthodontic tooth displacement in the rat.

OBJECTIVE: Orthodontic tooth movement requires extensive remodeling of the periodontal ligament (PDL) and the alveolar bone. Osteoclasts resorb bone, allowing teeth to migrate in the direction of the force. Matrix metalloproteinases (MMPs) are able to degrade the extracellular matrix of the periodontal tissues. Chemically modified tetracyclines (CMTs) can inhibit MMPs, but lack antimicrobial activity. We hypothesize that CMT-3 will decrease the rate of orthodontic tooth movement in the rat. DESIGN: Eighteen Wistar rats received a standardized orthodontic appliance at one side of the maxilla. During 14 days, three groups of six rats received a daily dose of 0, 6 or 30mg/kg CMT-3, and tooth displacement was measured. Thereafter, osteoclasts were counted on histological sections using an ED-1 staining. Multi- and mononuclear ED-1-positive cells in the PDL were also counted. In addition, sections were stained for MMP-9. RESULTS: CMT-3 significantly inhibited tooth movement (p=0.03) and also decreased the number of osteoclasts at the compression sides in the 30mg/kg group (p<0.05). Significantly more mono- than multinuclear ED-1-positive cells were present in the PDL, but no significant differences were found between the dosage groups. Osteoclasts in the 30mg/kg group seemed to contain less MMP-9 than in the control. CONCLUSIONS: CMT-3 inhibits tooth movement in the rat, probably by reducing the number of osteoclasts at the compression side. This might be due to induction of apoptosis in activated osteoclasts or reduced osteoclast migration. Reduced MMP activity by CMT-3 might also directly inhibit degradation of the organic bone matrix.

An update on the diagnosis and treatment of dens invaginatus.

Dens invaginatus is a malformation with varying anatomical features, posing challenges to treatment. Early and accurate diagnosis plays a significant role in selecting the appropriate treatment. The diagnosis of teeth with a complex root canal system including dens invaginatus has made progress with the application of three-dimensional imaging techniques in endodontics. Advanced treatment options provide hope for teeth that could not be saved before. This review discusses diagnostic methods and treatment options for teeth with dens invaginatus, and provides guidelines for the management of dens invaginatus cases in clinic. Current as well as traditional diagnostic techniques are summarized. Treatment options including state-of-the-art alternatives are presented for coronal dens invaginatus and radicular dens invaginatus.

Neonatal Satellite Cells Form Small Myotubes In Vitro.

Although palatal muscle reconstruction in patients with cleft palate takes place during early childhood, normal speech development is often not achieved. We hypothesized that the intrinsic properties of head satellite cells (SCs) and the young age of these patients contribute to the poor muscle regeneration after surgery. First, we studied the fiber type distribution and the expression of SC markers in ex vivo muscle tissue from head (branchiomeric) and limb (somite-derived) muscles from neonatal (2-wk-old) and young (9-wk-old) rats. Next, we cultured SCs isolated from these muscles for 5, 7, and 9 d, and investigated the in vitro expression of SC markers, as well as changes in proliferation, early differentiation, and fusion index (myotube formation) in these cells. In our ex vivo samples, we found that virtually all myofibers in both the masseter (Mass) and the levator veli palatini (LVP) muscles contained fast myosin heavy chain (MyHC), and a small percentage of digastric (Dig) and extensor digitorum longus myofibers also contained slow MyHC. This was independent of age. More SCs were found in muscles from neonatal rats as compared with young rats [17.6 (3.8%) v. 2.3 (1.6%); P < 0.0001]. In vitro, young branchiomeric head muscle (BrHM) SCs proliferated longer and differentiated later than limb muscle SCs. No differences were found between SC cultures from the different BrHMs. SC cultures from neonatal muscles showed a much higher proliferation index than those from young animals at 5 d (0.8 v. 0.2; P < 0.001). In contrast, the fusion index in neonate SCs was about twice as low as that in SCs from young muscles at 9 d [27.6 (1.4) v. 62.8 (10.2), P < 0.0001]. In conclusion, SCs from BrHM differ from limb muscles especially in their delayed differentiation. SCs from neonatal muscles form myotubes less efficiently than those from young muscles. These age-dependent differences in stem cell properties urge careful consideration for future clinical applications in patients with cleft palate.

Tissue inhibitors of metalloproteinases (TIMPs): their biological functions and involvement in oral disease.

Several families of enzymes are responsible for the degradation of extracellular matrix (ECM) proteins during the remodeling of tissues. An important family of such enzymes is that of the matrix metalloproteinases (MMPs). To control MMP-mediated ECM breakdown, tissue inhibitors of metalloproteinases (TIMPs) are able to inhibit MMP activity. A disturbed balance of MMPs and TIMPs is found in various pathologic conditions, such as cancer, rheumatoid arthritis, and periodontitis. The role of MMPs in pathology has been extensively described in the literature. The main focus of this review lies in the biological functions of TIMPs and their occurrence in disease, especially in the head and neck area. Their biological functions and their role in diseases like oral cancers and periodontitis, and in the development of cleft palate, will be discussed. Finally, the diagnostic and therapeutical opportunities of TIMPs will be evaluated.