Erosion -- diagnosis and risk factors Dental erosion is a multifactorial condition : The interplay of chemical , biological and behavioural factors is crucial and helps explain why some individuals exhibit more erosion than others . The erosive potential of erosive agents like acidic drinks or foodstuffs depends on chemical factors , e.g. pH , titratable acidity , mineral content , clearance on tooth surface and on its calcium-chelation properties . Biological factors such as saliva , acquired pellicle , tooth structure and positioning in relation to soft tissues and tongue are related to the pathogenesis of dental erosion . Furthermore , behavioural factors like eating and drinking habits , regular exercise with dehydration and decrease of salivary flow , excessive oral hygiene and , on the other side , an unhealthy lifestyle , e.g. chronic alcoholism , are predisposing factors for dental erosion . There is some evidence that dental erosion is growing steadily . To prevent further progression , it is important to detect this condition as early as possible . Dentists have to know the clinical appearance and possible signs of progression of erosive lesions and their causes such that adequate preventive and , if necessary , therapeutic measures can be initiated . The clinical examination has to be done systematically , and a comprehensive case history should be undertaken such that all risk factors will be revealed . Introduction Erosion has , for many years , been a condition of little interest to clinicians and researchers . This has changed during the last years , and there is some evidence that the presence of dental erosion is growing steadily . In the UK , the prevalence of erosion was shown to have increased from the time of the children 's dental health survey in year 1993 compared to 1996/1997 -LSB- 82 -RSB- . In another UK study , the progression of erosion was investigated : 1,308 children were examined at the age of 12 years and 2 years later . In this study , 4.9 % of the subjects at baseline and 13.1 % 2 years later had deep-enamel or dentine lesions . Approximately 12 % of erosion-free children at 12 years developed the condition over the subsequent 2 years . New or more advanced lesions were seen in 27 % of the children over the study period -LSB- 20 -RSB- . The progression of erosion seems to be greater in older adults -LRB- 52 -- 56 years -RRB- compared to younger -LRB- 32 -- 36 years -RRB- and has a skewed distribution -LSB- 64 -RSB- . In this study , the group with high-erosion progression was found to have four or more dietary acid intakes per day , a low buffering capacity of stimulated saliva and used a hard-bristle toothbrush . Intake frequency of the same magnitude was also associated with an increased risk for erosion in children . In this investigation , the erosion group ate fruits significantly more frequently and had different drinking habits , such as swishing , sucking or holding drinks in their mouths -LSB- 83 -RSB- . Dental erosion is a multifactorial condition . To prevent further progression , it is important to detect this condition as early as possible . It is fundamental to diagnose the possible risk factors such that preventive measures can be initiated . This overview is aimed to give some basic aspects about the diagnosis and the risk factors of erosion . A more detailed description can be found elsewhere -LSB- 67 -RSB- . Diagnosis Diagnosis of early forms of erosion is difficult , as it is accompanied by few signs and fewer if any symptoms . There is no device available in routine dental practice for the specific detection of dental erosion and its progression . Therefore , clinical appearance is the most important feature for dental professionals to diagnose this condition . This is of particular importance in the early stage of dental erosion -LSB- 68 -RSB- . At a more advanced stage , it can be very difficult to determine if dentine is exposed or not -LSB- 31 -RSB- . It is possible to use disclosing agents to render dentine involvement visible . The appearance of smooth silky-glazed , sometimes dull , enamel surface with the absence of perikymata and intact enamel along the gingival margin are some typical signs of enamel erosion on facial and oral sites . It has been hypothesized that the preserved enamel band along the oral and facial gingival margin could be due to some plaque remnants , which could act as a diffusion barrier for acids . This phenomenon could also be due to an acid-neutralizing effect of the sulcular fluid -LSB- 66 -RSB- . In the more advanced stages , further changes in the morphology can be found . These changes result in developing a concavity in enamel , the width of which clearly exceeds its depth . Facial erosion should be distinguished from wedge-shaped defects which are located at or apical to the enamel -- cementum junction . The coronal part of wedge-shaped defects ideally has a sharp margin and cuts at right angle into the enamel surface , whereas the apical part bottoms out to the root surface . Thereby , the depth of the defect exceeds its width . The initial features of erosion on occlusal and incisal surfaces are the same as described above . Further progression of occlusal erosion leads to a rounding of the cusps and restorations rising above the level of the adjacent tooth surfaces . In severe cases , the whole occlusal morphology disappears . Erosive lesions have to be distinguished from attrition and abrasion . The latter are often flat , have glossy areas with distinct margins and corresponding features at the antagonistic teeth . It is sometimes challenging to distinguish between the influences of erosion , attrition or abrasion during a clinical examination . Indeed , they may occur simultaneously with sometimes similar shape . The most commonly reported areas with this condition are occlusal surfaces -LSB- 6 -RSB- . Figures 1 , 2 , 3 , and 4 show typical pattern of dental erosion process . Fig. 1Facial erosion : The intact enamel border along the gingival margin of tooth 12 and some plaque remnants are clearly visible . Note the smooth silky-glazed appearance and the absence of perikymata on the enamel surfaceFig . 2Advanced facial erosion of teeth 43 , 44 and 45 with dentinal involvement . The width of the lesions exceeds its depthFig . 3Erosion with involvement of dentine on the oral surface of tooth 13 , 12 and 11 . Intact enamel borders along the gingival marginFig . 4a -- c Typical pattern of advanced occlusal erosion of teeth 45 and 46 of three different patients : The whole occlusal morphology disappears , and extensive exposed dentinal areas are visible The clinical examination should be done systematically using a simple but accurate index . This is a difficult task to achieve as an index with a too-fine grading shows a small inter - and intraexaminer reliability -LSB- 60 -RSB- . For a dental practitioner , the most important part is to recognize the condition and to describe its dimension and severity . It is important to search for a general pattern and not to over-interpret one single sign . For epidemiological purpose , an index with high detection capability and reliability is most important . Whenever possible , the clinical examination should be accomplished by a thorough history taking with respect to general health , diet and habits and by the assessment of saliva flow rates and buffer capacity . People who show signs and symptoms of erosion are often not aware of this condition . Only when a comprehensive case history is undertaken will all the risk factors be revealed . It is difficult to judge the activity and progression of dental erosion . One tool is the comparison of clinical photographs of tooth surfaces to estimate the possible substance loss over time . Thereby , the discoloration of the lesions and their sensitivity state may give some information about the activity of the tooth surface . Further , study casts as well as the examination of dental radiography , especially bitewings longitudinally taken , can provide information about the substance loss over time . For research purpose , computed controlled mapping -LSB- 13 -RSB- or profilometric measurements using acid-resistant markers -LSB- 6 , 92 -RSB- are tools to monitor progression . Risk factors When an acidic solution comes in contact with enamel , it has to diffuse first through the acquired pellicle , and only thereafter can it interact with enamel . The acquired pellicle is an organic film , free of bacteria , covering oral hard and soft tissues . It is composed of mucins , glycoproteins and proteins , including several enzymes -LSB- 41 -RSB- . On the surface of enamel , the hydrogen ion component of the acid will start to dissolve the enamel crystal . First , the prism sheath area and then the prism core are dissolved , leaving the well-known honey comb appearance -LSB- 74 -RSB- . Thereafter , fresh , unionized acid will eventually diffuse into the interprismatic areas of enamel and dissolve further mineral in the region underneath the surface -LSB- 23 , 27 , 65 -RSB- . This will lead to an outflow of ions -LRB- dissolution -RRB- and subsequently to a local pH rise in the tooth substance immediately below and in the liquid surface layer adjacent to the enamel surface -LSB- 65 -RSB- . The events in dentine are , in principle , the same but are even more complex . There are different predisposing factors and aetiologies of the erosive condition . The interplay of chemical , biological and behavioural factors is crucial and helps explain why some individuals exhibit more erosion than others , even if they are exposed to the same acid challenge in their diets . Comprehensive knowledge of the different risk factors is a prerequisite to initiate adequate preventive -LRB- non-interventive -RRB- and , if necessary , therapeutic -LRB- interventive -RRB- measures . When a restoration becomes inevitable , in all situations , the preparations have to follow the principles of minimally invasive treatment . Figure 5 is an attempt to reveal the multifactorial predisposing factors and aetiologies of the erosive condition . Fig. 5Interactions of the different factors for the development of dental erosion -LRB- adapted from -LSB- 68 -RSB- -RRB- Chemical factors Several in vitro and in situ studies show that the erosive potential of an acidic drink or foodstuff is not exclusively dependent on its pH value but is also strongly influenced by its mineral content , its titratable acidity -LRB- ` the buffering capacity ' -RRB- and by the calcium-chelation properties -LSB- 7 , 8 , 37 , 44 , 61 , 72 , 73 , 76 , 77 , 80 , 86 , 88 , 94 , 95 , 96 -RSB- . The pH value , calcium , phosphate and fluoride content of a drink or foodstuff determine the degree of saturation with respect to the tooth mineral , which is the driving force for dissolution . Solutions oversaturated with respect to dental hard tissue will not dissolve it . A low degree of undersaturation with respect to enamel or dentine leads to a very initial surface demineralization which is followed by a local rise in pH and increased mineral content in the liquid surface layer adjacent to the tooth surface . This layer will then become saturated with respect to enamel -LRB- or dentine -RRB- and will not demineralize further . Acids such as citric acid exist in water as a mixture of hydrogen ions , acid anions -LRB- e.g. citrate -RRB- and undissociated acid molecules , with the amounts of each determined by the acid-dissociation constant and the pH of the solution . The hydrogen ion directly attacks the crystal surface . Over and above the effect of the hydrogen ion , the citrate anion may complex with calcium , also removing it from the crystal surface . Each acid anion has a different strength of calcium complexation dependent on the structure of the molecule and how easily it can attract the calcium ion -LSB- 28 -RSB- . Consequently , acids such as citric acids have double actions and may be very damaging to the tooth surface . Up to 32 % of the calcium in saliva can be complexed by citrate at concentrations common in fruit juices , thus reducing the super-saturation of saliva and increasing the driving force for dissolution with respect to tooth minerals -LSB- 75 -RSB- . The dissolution with water of drinks containing organic acids with high buffering capacity will hardly reduce the pH but will reduce the titratable acidity . This is of some importance , as the greater the buffering capacity of the drink , the longer it will take for saliva to neutralize the acid . But dilution will also reduce concentrations of Ca and P -LRB- if present -RRB- , which have a protective effect -LSB- 10 , 61 , 62 -RSB- . The calcium and phosphate content of a foodstuff or beverage are important factors for the erosive potential as they influence the concentration gradient within the local environment of the tooth surface . The addition of calcium -LRB- and phosphate -RRB- salts to erosive drinks showed promising results . Addition of calcium to a low-pH blackcurrant juice drink has been shown to reduce the erosive effect of the drink -LSB- 48 -RSB- . In a follow-up study , a blackcurrant drink with added calcium was compared to a conventional orange drink in situ . Servings of 250 ml of each drink were consumed four times per day during 20 working days . Measurements of enamel loss were made by profilometry on enamel samples for up to 20 days . The experimental carbonated blackcurrant drink supplemented with calcium caused significantly less enamel loss than the conventional carbonated orange drink at all time points measured -LSB- 98 -RSB- . When Ca was added to a sports drink , a reduction of the erosive potential was found -LSB- 46 -RSB- . Today , several Ca-enriched orange juices and sports drinks are on the market which hardly soften the enamel surface . Yoghurt is another example of food with a low pH -LRB- ~ 4.0 -RRB- , yet it has hardly any erosive effect due to its high calcium and phosphate content , which makes it supersaturated with respect to apatite . A yoghurt or another milk-based food may have an erosive potential when it has a low content of Ca and/or P and a low pH. It has to be kept in mind that , with the added mineral , enamel dissolution could not always be completely prevented . But the progression can be retarded which has some implications for the patient and the clinician . Theoretically , fluoride has some protective effect in a drink with a pH higher than that indicated by the saturation curve of fluorapatite at given Ca and P concentrations . Lussi et al. -LSB- 61 , 62 -RSB- and Mahoney et al. -LSB- 71 -RSB- found an inverse correlation of the erosive potential of different beverages with their fluoride content . It is unlikely that fluoride at the concentration present in beverages alone has any great beneficial effect on erosion because the challenge is high . However , it is possible that , under conditions in which the other erosive factors are not excessive , fluoride in solution may exert some protective effect -LSB- 71 -RSB- . It appears that topical fluoride application can positively affect the tooth-wear process . The influence of immersion in fluoride solutions on brushing abrasion of eroded dentine slabs was investigated by Attin et al. -LSB- 3 -RSB- . Bovine dentine specimens were alternately stored in a demineralising and in a remineralising solution and brushed five times . Before each immersion in the remineralising solution , the specimens were not treated , treated with 250 ppm or treated with 2,000 ppm neutral sodium fluoride solution for 1 min . The least wear was found in the uneroded controls , whereas the greatest wear was found in the group without fluoride treatment . Significantly greater wear resistance was found as fluoride concentration increased . The same group evaluated the abrasion resistance of eroded enamel brushed with an acidified fluoride gel -LSB- 4 -RSB- . They found that treatment with a slightly acidic amine/sodium fluoride gel increased the abrasion resistance of bovine enamel compared to an unfluoridated gel . They speculated that the fluoride is incorporated into and deposited on the enamel during treatment with the acidic gel , which could be one reason for the higher resistance . The adhesiveness and displacement of the liquid are other factors to be considered in the erosive process . There appear to be differences in the ability of beverages to adhere to enamel based on their thermodynamic properties , e.g. the thermodynamic work of adhesion -LSB- 50 -RSB- . In summary , the two very often-cited parameters , the pH and the titratable acidity , do not readily explain the erosive potential of food and drink . The mineral content is also an important parameter , as is the ability of any of the components to complex calcium and to remove it from the mineral surface . Biological factors Biological factors such as saliva , acquired pellicle , tooth structure and positioning in relation to soft tissues and tongue are related to dental erosion development . A very important biological parameter is saliva . Several salivary protective mechanisms come into play during an erosive challenge : dilution and clearance of an erosive agent from the mouth , neutralisation and buffering of acids , and slowing down the rate of enamel dissolution through the common ion effect by salivary calcium and phosphate -LSB- 104 -RSB- . Practical experience demonstrates the importance of saliva in patients suffering from salivary-flow impairment . Studies have shown that erosion may be associated with low salivary flow or/and low buffering capacity -LSB- 53 , 64 , 89 -RSB- . The dry mouth condition is usually related to aging -LSB- 18 , 81 , 84 -RSB- , even though some other studies have not found this correlation -LSB- 9 , 43 -RSB- . It is well established that patients taking medication can also present decreased saliva output -LSB- 101 -RSB- , as well as those who have received radiation therapy for neck and head cancer -LSB- 19 -RSB- . Tests of the stimulated and unstimulated flow rate as well as of the buffer capacity of saliva may provide some information about the susceptibility of an individual to dental erosion . However , it has to be kept in mind that these parameters are two of a multifactorial condition . Sialometric evaluations should be carried at a fixed time point or in a limited time interval in the morning , avoiding intra-individual variations due to the circadian cycle -LSB- 29 -RSB- . Studies have shown that sour foodstuff has a strong influence on the anticipatory salivary flow -LSB- 15 , 55 -RSB- , which can be significantly increased when compared to the normal unstimulated flow rate -LSB- 25 -RSB- . Hypersalivation also occurs in advance of vomiting as a response from the ` vomiting center ' of the brain -LSB- 56 -RSB- , as frequently seen in individuals suffering from anorexia and bulimia nervosa , rumination or chronic alcoholism . It is suggested that this could minimize the erosion caused by acids of gastric origin . On the other hand , patients with symptoms of gastro-esophageal reflux disease -LRB- GERD -RRB- should not expect the salivary output to increase before the gastric juice regurgitation because this is an involuntary response not co-coordinated by the autonomic nervous system -LSB- 42 , 90 -RSB- . Therefore , there may be insufficient time for saliva to act before erosion occurs . The influence of saliva on the remineralisation/rehardening of erosive damaged dental hard tissue is discussed controversially . There is evidence that acid-softened enamel can reharden after exposure to saliva or remineralisation solution and that dietary products and fluoride can enhance the rehardening process -LSB- 2 , 26 , 33 -RSB- . Other investigations could not find a significant rehardening effect of saliva in situ -LSB- 16 , 32 , 34 , 58 -RSB- . Some limited increase of the abrasion resistance of softened enamel was found after intraoral exposure to saliva -LSB- 5 , 51 -RSB- . It seems that in vitro , some rehardening could be expected when supersaturated solution or saliva with no protein added are used -LSB- 23 , 24 -RSB- , whereas in situ , this is only the case to a very small amount . The salivary acquired pellicle is a protein-based layer which is rapidly formed on dental surfaces after its removal by tooth brushing with dentifrice , other prophylaxis , measures or chemical dissolution . This organic layer becomes detectable on dental surfaces after few minutes of exposure to the oral environment -LSB- 39 , 93 -RSB- . Enzymatic activity is also detectable at early stages of pellicle formation -LSB- 40 , 41 -RSB- . It is suggested that it grows until reaching an equilibrium between protein adsorption and de-sorption within 2 h -LSB- 57 -RSB- . The acquired pellicle may protect against erosion by acting as a diffusion barrier or a perm-selective membrane preventing the direct contact between the acids and the tooth surface -LSB- 39 , 102 , 103 -RSB- , reducing the dissolution rate of hydroxyapatite -LSB- 57 -RSB- . Millward et al. -LSB- 78 -RSB- monitored the pH at the surface of teeth of healthy volunteers after drinking 1 % citric acid . They observed that the pH recovered to above pH 5.5 within 2 min from a site adjacent to the palatal surface of the upper central incisor and within 4 -- 5 min from another palatal surface of the upper first molar . Other observations have revealed a longer clearance time on upper incisors for patients with active erosions and normal saliva values compared to patients with no erosion -LRB- Lussi , unpublished data -RRB- . These differences could be due to the anatomy of the teeth and soft tissues that may influence the retention/clearance pattern of erosive agents . Furthermore , soft tissue movements by the tongue and buccal mucosa and swallowing pattern can influence clearance rate . The importance of the tongue in modifying the tooth-wear process has long been the subject of speculation . Holst and Lange -LSB- 45 -RSB- considered mechanical abrasion caused by tongue to be a contributing factor in erosion caused by vomiting . Observations from animal studies also provide support in that beverages produced erosion mainly on the lingual surfaces of rat molar teeth in areas where the tongue contacts the teeth -LSB- 96 -RSB- . Dugmore and Rock -LSB- 21 -RSB- found that orthodontic anomaly appeared to confer a protective effect . Behavioural factors During and after an erosive challenge , behavioural factors play a role in modifying the extent of tooth wear . The manner that dietary acids are introduced into the mouth will affect which teeth are contacted by the erosive challenge and possibly the clearance pattern . As lifestyles have changed through the decades , the total amount and frequency of consumption of acidic foods and drinks have also changed . Soft drink consumption in the USA increased by 300 % in 20 years -LSB- 11 -RSB- , and serving sizes increased from 185 g -LRB- 6.6 oz -RRB- in the 1950s to 340 g -LRB- 12 oz -RRB- in the 1960s and to 570 g -LRB- 20 oz -RRB- in the late 1990s . Around the year 1995 , between 56 and 85 % of children at school in the USA consumed at least one soft drink daily , with the highest amounts ingested by adolescent males . Of this group , 20 % consumed four or more servings daily -LSB- 36 -RSB- . Studies in children and adults have shown that this number of servings per day is associated with the presence and the progression of erosion when other risk factors exist -LSB- 64 , 83 -RSB- . High erosion was associated with a method of drinking whereby the drink was kept in the mouth for a longer period -LSB- 54 -RSB- . One study investigated a randomly selected group of Swiss adults and diagnosed the cause of tooth wear . It showed that the consumption of erosive drinks and foodstuffs was strongly associated with erosion on facial and occlusal surfaces . Severe palatal erosions were scarce and highly associated with chronic vomiting -LSB- 60 -RSB- . The presence of calculus -LRB- odds ratio 0.48 -RRB- or eating fruit other than apples or citrus fruit -LRB- 0.48 -RRB- reduced the chances of erosion . High consumption of carbonated drinks increased the odds of erosion being present at 12 years by 252 % and was a strong predictor of the amount of erosion found at age 14 -LSB- 21 -RSB- . In a sample of 987 children -LRB- 2 to 5 year-olds -RRB- , consumption of vitamin C supplements , carbonated drinks and fruit syrup from a feeding bottle at bed - or naptime were related to erosion -LSB- 1 -RSB- . Considerable risk of erosion was found when citrus fruits were eaten more than twice a day and soft drinks were drunk daily -LSB- 53 -RSB- . Excessive consumption of acidic candies combined with a low salivary buffering capacity may aggravate erosive lesions -LSB- 17 , 63 -RSB- . The high intake of herbal teas , widely perceived as a healthy drink , may have an erosive potential exceeding that of orange juice -LSB- 85 -RSB- . An increase in agitation -LRB- e.g. when a patient is swishing his/her drink in the mouth -RRB- will enhance the dissolution process because the solution on the surface layer adjacent to tooth mineral will be readily renewed . Further , the amount of the drink in the mouth in relation to the amount of saliva present will modify the dissolution process -LSB- 69 -RSB- . Several authors have suggested that using a straw is beneficial , since the straw directs drinks past the anterior teeth and towards the pharynx -LSB- 22 , 38 , 49 -RSB- . However , the placing of a straw labial to the anterior teeth can be destructive -LSB- 70 -RSB- . Nighttime exposure to erosive agents may be particularly destructive because of the absence of salivary flow . Two examples of this are bedtime baby bottle-feeding with acidic beverages and gastroesophageal reflux with regurgitation during sleeping . Healthier lifestyle paradoxically can lead to dental health problems in the form of dental erosion , as it often involves regular exercise and what is considered healthy diets with more fruits and vegetables . A lactovegetarian diet , which includes the consumption of acidic foods , has been associated with a higher prevalence of dental erosion -LSB- 30 -RSB- . The benefits of exercise are well-proven ; however , exercise increases the loss of body fluids and may lead to dehydration and decreased salivary flow . A few case reports and studies have reported an association between sports activities and dental erosion . The cause could be direct acid exposure or strenuous exercise which may increase gastroesophageal reflux . Risk groups are swimmers exercising in water with low pH and athletes consuming frequently erosive sport drinks . In a study with 25 swimmers and 20 cyclists , the latter showed significantly more tooth wear into dentine . However , no association between erosion and sports drink consumption was found -LSB- 79 -RSB- . Professional swimmers train several hours in the water , which should have proper pH regulation . The main disinfection techniques used are gas chlorination and sodium hypochlorite . In the Netherlands , where the sodium hypochlorite method is used , only 0.14 % of the tested pools were found in the year 2001 to have low pH values -LSB- 59 -RSB- . Another case report confirmed these findings -LSB- 91 -RSB- . In a review by Geurtsen -LSB- 35 -RSB- , an increased prevalence of dental erosion among intensive swimmers due to low-pH gas-chlorinated pool water was described . The recommended pH for swimming pools is between pH 7.2 and 8.0 . Swimming activities in pH-adjusted pools do not harm the teeth -LSB- 100 -RSB- . However , erosion among competitive swimmers was found in 39 % of swim-team members who trained in a pool with a pH of 2.7 which is a H concentration 100,000 times higher than that recommended for swimming pools -LSB- 12 -RSB- . Sports drinks are often erosive -LSB- 46 , 47 , 95 , 97 -RSB- , and when consumed during strenuous activity when the person is in a state of some dehydration , the possible destructive effects may be enhanced further . Health-conscious individuals also tend to have better than average oral hygiene . While good oral hygiene is of proven value in the prevention of periodontal disease and dental caries , frequent tooth brushing with abrasive oral hygiene products may enhance dental erosion . At the other end of the spectrum , an unhealthy lifestyle may also be associated with dental erosion -LSB- 105 -RSB- . Alcoholics may be at particular risk for dental erosion and tooth wear . Robb and Smith -LSB- 87 -RSB- reported significantly more tooth wear in 37 alcoholic patients than in age - and sex-matched controls . Tooth wear was most pronounced in men and those with frequent alcohol consumption . Wine has properties such as low pH and low content of P and Ca , which renders it to have an erosive potential . Professional wine tasting is very common all over the world . In some countries -LRB- e.g. Sweden , Finland -RRB- , wine tasters are employed by the state to support their state-owned wine shops . Full-time Swedish wine tasters test on average 20 -- 50 different wines nearly 5 days a week . Wiktorsson et al. -LSB- 99 -RSB- investigated the prevalence and severity of tooth erosion in 19 qualified wine tasters in relation to number of years of wine tasting , salivary flow rate and buffer capacity . Salivary flow rate and buffer capacity of unstimulated and stimulated saliva were measured . Data on occupational background and dental and medical histories were collected . Fourteen subjects had tooth erosion mainly on the labio-cervical surfaces of maxillary incisors and canines . The severity of the erosion tended to increase with years of occupational exposure . Caries activity in all subjects was low . It was concluded that full-time wine tasting is an occupation associated with an increased risk for tooth erosion . In a cross-sectional comparative study , the prevalence and severity of tooth-surface loss between winemakers -LRB- exposed -RRB- and their spouses -LRB- non-exposed -RRB- was examined . There was a difference in the prevalence and severity of tooth surface loss between the two groups -LSB- 14 -RSB- . On the other hand , many other studies were not able to find an association between dental erosion and behavioural factors -LSB- 30 , 52 -RSB- , or they found only a weak association -LSB- 82 -RSB- . One can only speculate about the reasons . A possible explanation is the mode of questioning the persons -LRB- orally vs written questionnaire -RRB- , the statistics employed -LRB- multivariate vs univariate -RRB- and the population group under study -LRB- selected vs randomly -RRB- . Although no detrimental effects were described on a population level , one has to keep in mind that factors like sports-drink consumption and occupation can be , for some patients , a cofactor in the development of or in the increase in dental erosion when other factors are present . It is unlikely that one or two isolated factors -LRB- e.g. sports drink , dehydration -RRB- will be responsible for a multifactorial condition like erosion . Conclusion This overview shows the importance of early diagnosis of dental erosion and of accurate detection of possible risk factors and their interplay . These facts are prerequisites to initiate adequate preventive -LRB- and therapeutic -RRB- measures .

B O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O


Effect of thrombin peptide 508 -LRB- TP508 -RRB- on bone healing during distraction osteogenesis in rabbit tibia Thrombin-related peptide 508 -LRB- TP508 -RRB- accelerates bone regeneration during distraction osteogenesis -LRB- DO -RRB- . We have examined the effect of TP508 on bone regeneration during DO by immunolocalization of Runx2 protein , a marker of osteoblast differentiation , and of osteopontin -LRB- OPN -RRB- and bone sialoprotein -LRB- BSP -RRB- , two late markers of the osteoblast lineage . Distraction was performed in tibiae of rabbits over a period of 6 days . TP508 -LRB- 30 or 300 μg -RRB- or vehicle was injected into the distraction gap at the beginning and end of the distraction period . Two weeks after active distraction , tissue samples were harvested and processed for immunohistochemical analysis . We also tested the in vitro effect of TP508 on Runx2 mRNA expression in osteoblast-like -LRB- MC3T3-E1 -RRB- cells by polymerase chain reaction analysis . Runx2 and OPN protein were observed in preosteoblasts , osteoblasts , osteocytes of newly formed bone , blood vessel cells and many fibroblast-like cells of the soft connective tissue . Immunostaining for BSP was more restricted to osteoblasts and osteocytes . Significantly more Runx2 - and OPN-expressing cells were seen in the group treated with 300 μg TP508 than in the control group injected with saline or with 30 μg TP508 . However , TP508 failed to increase Runx2 mRNA levels significantly in MC3T3-E1 cells after 2 -- 3 days of exposure . Our data suggest that TP508 enhances bone regeneration during DO by increasing the proportion of cells of the osteoblastic lineage . Clinically , TP508 may shorten the healing time during DO ; this might be of benefit when bone regeneration is slow . Introduction Distraction osteogenesis -LRB- DO -RRB- is a well-established technique , originally developed in orthopedic surgery for bone lengthening -LRB- Kojimoto et al. 1988 -RRB- and later also used to treat hereditary malformations in the craniomaxillofacial region -LRB- Amir et al. 2006 -RRB- . DO is characterized by the formation of new bone between two osteotomized bone segments , which are separated by gradual traction . One of the clinical phases of the distraction technique is the bone consolidation period , defined as the time between the end of active distraction and the removal of the distraction device . It represents the time needed for a complete bridging of the distraction gap by bone and a further maturation of this bone . Typically , the bone consolidation phase takes approximately 6 -- 12 weeks in the craniomaxillofacial region and 3 -- 6 months in long bones -LRB- Fischgrund et al. 1994 ; Smith et al. 1999 ; Felemovicius et al. 2000 ; Amir et al. 2006 -RRB- . Although DO is believed to be superior to other bone augmentation techniques , many attempts have been made to improve the technique to enhance the bone regeneration process in the distraction gap -LRB- Hagiwara and Bell 2000 ; Li et al. 2002 ; Mofid et al. 2002 ; Takamine et al. 2002 ; Schortinghuis et al. 2005 -RRB- . Such improvements would provide the opportunity to shorten the bone consolidation period and hence minimize complications such as the development of non-union , infection or fracture . Various methods have been tested to promote bone formation in the distraction gap , e.g. electrical and mechanical stimulation -LRB- Hagiwara and Bell 2000 ; Mofid et al. 2002 -RRB- , transplantation of osteoblast-like cells -LRB- Takamine et al. 2002 -RRB- or administration of growth factors such as bone morphogenetic proteins -LRB- Li et al. 2002 -RRB- or fibroblast growth factor 2 -LRB- FGF-2 ; Okazaki et al. 1999 -RRB- . Another procedure to accelerate bone regeneration involves the application of thrombin-related peptide 508 -LRB- TP508 ; Ryaby et al. 2000 ; Sheller et al. 2004 ; Li et al. 2005a , b ; Wang et al. 2005 -RRB- . TP508 is a synthetic peptide consisting of 23 amino acids , which represent the natural amino acid sequence of the receptor-binding domain of human thrombin -LRB- pro-thrombin amino acids 508 -- 530 ; Sower et al. 1999 ; Stiernberg et al. 2000 -RRB- . Thrombin is an important factor in blood homeostasis , inflammation and wound healing -LRB- Coughlin et al. 1992 -RRB- . It plays a role in the formation of fibrin clots and platelet activation . Thrombin also stimulates the cell proliferation , migration and/or differentiation of various cell types , such as fibroblasts , endothelial cells and lymphocytes . All these cell types contain one or more thrombin receptors -LRB- Chen and Buchanan 1975 ; Belloni et al. 1992 -RRB- . TP508 appears to mimic and accelerate many of the effects of native thrombin in initiating wound healing through a non-proteolytic pathway -LRB- Sower et al. 1999 -RRB- . Histological studies have indicated that TP508 is chemotactic for neutrophils , lymphocytes and monocytic cells ; it enhances the formation of granulation tissue , neovascularization and other wound-healing events -LRB- Sower et al. 1999 ; Ryaby et al. 2000 ; Naldini et al. 2004 -RRB- . TP508 has also been tested for its potential to enhance bone formation -LRB- Ryaby et al. 2000 ; Sheller et al. 2004 ; Li et al. 2005a , b ; Wang et al. 2005 -RRB- . A single injection of 1 μg TP508 accelerates fracture healing in rat long bone fracture . The breaking strength of bones of young and aged rats injected with TP508 has been enhanced over that of controls -LRB- Ryaby et al. 2000 -RRB- . One recent study of TP508 treatment during DO has revealed a significantly greater bone mineral density in TP508-treated groups than in saline-treated controls -LRB- Li et al. 2005a , b -RRB- . Histological analysis has also shown advanced bone consolidation and remodeling after TP508 treatment . In the present study , we report the effect of TP508 in the regulation of bone regeneration during DO in more detail . We have examined Runx2/Cbfa1 , osteopontin -LRB- OPN -RRB- and bone sialoprotein -LRB- BSP -RRB- protein expression in tissue sections of rabbit long bones undergoing DO . Runx2 is an essential transcription factor for osteoblast differentiation and for extracellular matrix gene expression -LRB- Komori et al. 1997 ; Karsenty et al. 1999 -RRB- . In Runx2 knockout mice , both intramembranous and endochondral ossification are blocked because of the maturational arrest of osteoblasts -LRB- Komori et al. 1997 -RRB- . Runx2 can directly stimulate the transcription of the collagen type I and osteoblast-related genes , such as those for OPN and BSP , both major non-collagenous proteins found in the extracellular matrix of bone -LRB- Karsenty et al. 1999 -RRB- . We have hypothesized that TP508 treatment during DO enhances the expression of Runx2 , OPN and BSP . Materials and methods Animal model of DO The paraffin blocks containing distracted rabbit long bones were those prepared previously -LRB- Li et al. 2005a , b -RRB- from 30 adult male New Zealand White rabbits -LRB- age 24 weeks , body weight 2.6 -- 3.5 kg -RRB- . External fixators were applied to stabilize the osteotomized mid-tibia for 7 days of the latency period and were activated for 6 days , once a day at a rate of 1.4 mm . This distraction rate was intentionally chosen in order to copy the poor bone healing condition as previously described -LRB- Li et al. 1997 -RRB- . During the experiment , seven animals were excluded in the study because of soft tissue complications -LRB- n = 2 -RRB- , pinhole fracture -LRB- n = 3 -RRB- and anaesthetic death -LRB- n = 2 -RRB- . The remaining 23 rabbits were randomly divided into three experimental groups , each group consisting of minimally seven rabbits . TP508 -LRB- OrthoLogic , Tempe , USA -RRB- at two different concentrations -LRB- 30 and 300 μg TP508 -RRB- or saline were injected percutanously at the beginning and at the end of the lengthening phase . The first group -LRB- n = 8 -RRB- received injections of 300 μl saline containing 300 μg TP508 , the second group -LRB- n = 8 -RRB- received injections of 300 μl saline containing 30 μg TP508 and the third group -LRB- n = 7 -RRB- received injections of 300 μl saline alone and served as a control group . The injections were given in three different areas -LRB- 100 μl in each area -RRB- : the proximal , central and distal area of the distraction gap . Two weeks after the lengthening phase , all animals were killed . Tissue samples consisting of the distraction gap and 5 mm proximal and distal to the pre-existing bone were then harvested and immediately fixed in 95 % ethanol for further examination . Tissues were decalcified with EDTA and embedded in paraffin . Serial sections -LRB- 5 μm thick -RRB- were cut and mounted on poly-lysine-coated glass slides . The peripheral quantitative computed tomography and histological examination of these bones has been published previously -LRB- Li et al. 2005a , b -RRB- . All animal experimental procedures were approved and performed following the guidelines for animal scientific procedures -LRB- Animal Scientific Procedures Act 1986 , British Home Office -RRB- . Antibodies The mouse recombinant monoclonal antibody Pebp2alphaA was kindly donated by Dr. K. Sasaguri , Kanawaga Dental School , Japan . This antibody has been used and characterized previously and reacts with all isotypes of Runx2 -LRB- Bronckers et al. 2001 , 2005 -RRB- . The mouse anti-rat OPN monoclonal antibody -LRB- MPIIIB101 -RRB- was obtained from the Developmental Studies Hybridoma Bank -LRB- developed under the auspices of the NICHD and maintained by the University of Iowa , Department of Biological Sciences Iowa City , IA 52242 , USA -RRB- . The mouse anti-human BSP -LRB- monoclonal antibody A4232 .1 -RRB- was purchased from Immunodiagnostik , Bensheim , Germany . Both anti-OPN and anti-BSP antibodies have been used and specified previously -LRB- Ibrahim et al. 2000 ; Bronckers et al. 2005 ; Zerbo et al. 2005 -RRB- . Immunohistochemical procedures Tissue sections were de-paraffinized in xylene and rehydrated through a graded series of aqueous ethanol solutions . Endogenous peroxidase was quenched by incubation in 3 % H2O2 in 100 % ethanol -LRB- 5 min -RRB- . A pre-incubation in hot citrate buffer -LRB- 0.01 M , pH 6.0 , 95 °C , 20 min -RRB- was performed for antigen retrieval prior to incubation with 30 % normal horse serum -LRB- 30 min -RRB- . Sections were then incubated overnight with primary monoclonal anti-Runx2 antibody -LRB- 1:200 -RRB- , anti-BSP antibody -LRB- 1:400 -RRB- or anti-OPN antibody -LRB- 1:600 -RRB- at 4 °C in a humidified chamber . After washes with phosphate-buffered saline -LRB- PBS -RRB- , biotinylated horse anti-mouse IgG -LRB- ABC-peroxidase Elite kit ; Vector Laboratories , Burlingame , Calif. , USA -RRB- was added at a dilution of 1:200 and the sections were incubated at room temperature for a minimum of 60 min . The sections were washed again with PBS , incubated with ABC peroxidase complex for another 60 min followed by a maximum of 20 min in 3,3 ′ diaminobenzidine substrate solution -LRB- Vectastain kit -RRB- to visualize the immunoreaction , counterstained with methyl green for 2 -- 3 min , mounted in 50 % glycerol and covered with a glass coverslip . As negative controls , sections were incubated with non-immune mouse IgG in place of the primary antibody . Scoring Sections were examined by using a Leica DM RA microscope equipped with a Leica DC 200 digital camera . The central areas of the gap were selected as the region of interest -LRB- ROI -RRB- . First , the boundaries of the distraction gap -LRB- indicated by the osteotomy lines -RRB- were determined and subsequently a grid was positioned over the ROI halfway between the two osteotomy lines . The grid was 609 μm in width and 479 μm in length . The ROI consisted of some newly formed bone -LRB- mostly in the group treated with 300 μg TP508 -RRB- and fibrous tissue -LRB- Fig. 1a -RRB- . Counting was performed by using the grid at a magnification of 200 × , with a minimum of three sections , at least 30 μm apart , per animal . Some tissue blocks were of poor quality or no longer contained a complete intact gap area with an ROI ; this reduced the number of animals per group to 5 -- 6 animals . The total number of cells was counted -LRB- immunopositive and immunonegative cells -RRB- . The sections were coded and randomized . Quantification was performed by one investigator -LRB- L.A. -RRB- who was unaware of the origin of the sections . Numbers of immunopositive cells were noted in absolute terms and were expressed as the percentage of all -LRB- immunopositive and negative -RRB- cells present in the ROI . Fig. 1Immunolocalization of Runx2 and osteopontin proteins in TP508-treated groups -LRB- a-c , e 300 μg TP508 group , d 30 μg TP508 group -RRB- . a Overview of immunoreactions in a biopsy taken from rabbit long bone after distraction osteogenesis Runx2 staining -LRB- FT fibrous tissue , NB new bone , P periosteum , PC periosteal callus -RRB- . A white grid was used to count the number of immunopositive and negative cells in the region of interest . × 50 . b Runx2 staining in the nuclei and cytoplasm of the cells . Runx2 was strongly expressed in osteoblasts -LRB- Ob -RRB- . Note that young osteocytes -LRB- Ocy -RRB- , which had recently embedded in the bone matrix , expressed Runx2 protein . Cells adjacent to the osteoblast layer , presumably pre-osteoblasts -LRB- Pre-Ob -RRB- , also expressed Runx2 , but with lower intensity -LRB- B bone -RRB- . × 400 . c Immunostaining for osteopontin . Positive staining was found in the cytoplasm of the cells -LRB- white arrows -RRB- . Note that expression was stronger towards the newly formed bone -LRB- NB -RRB- . × 200 . d Runx2 immunostaining in tissue of a rabbit injected with 30 μg TP508 . Expression was much lower than that after injection with 300 μg TP508 and was similar to saline-injected control tissue -LRB- not shown -RRB- . Fewer fibrocartilage-like cells -LRB- FCC -RRB- were stained than after injection with 300 μg TP508 -LRB- HC hypertrophic cartilage-like cells -RRB- . × 200 . e Negative control stained with non-immune IgG instead of primary antibodies to Runx2 . Note the lack of positive staining . Counter-staining with methyl green . × 200 . Bars 20 μm -LRB- b , d -RRB- , 50 μm -LRB- c , e -RRB- , 300 μm -LRB- a -RRB- Cell cultures The mouse osteoblast-like MC3T3-E1 cell line , derived from newborn mouse calvaria , was routinely maintained in α-MEM -LRB- Gibco , Paisley , UK -RRB- supplemented with 10 % fetal bovine serum -LRB- HyClone -RRB- , 120 μg/ml penicillin -LRB- Sigma , St Louis , Mo. , USA -RRB- , 100 μg/ml streptomycin sulphate -LRB- Sigma -RRB- , 1.25 μg/ml fungizone -LRB- Gibco -RRB- , 50 μg/ml sodium ascorbate -LRB- Merck , Darmstadt , Germany -RRB- , 10 mM β-glycerophosphate -LRB- Sigma -RRB- and 300 μg/ml glutamine -LRB- Sigma -RRB- at 37 °C in a humidified atmosphere of 5 % CO2 in air . This cell line is capable of expressing Runx2 mRNA -LRB- Tsuji et al. 1998 -RRB- . To stimulate cells to express osteoblastic markers , they were first exposed to 10 nM dexamethasone in 25 cm2 flasks -LRB- Greiner BioOne , Solingen , Germany -RRB- for 1 week . Upon confluency -LRB- typically after 5 days -RRB- , cells were harvested by using 0.25 % trypsin and 0.1 % EDTA in PBS , centrifuged at 600g for 10 min , washed and plated in 24-well culture dishes -LRB- Greiner BioOne -RRB- at three different cell densities -LRB- 3 × 104 , 1.5 × 104 and 7.5 × 103 cells/well -RRB- in order to obtain a comparable number of cells at the end of each experiment . After 1 day of culture , the medium was replaced by media containing various concentrations of TP508 -LRB- 0 , 10 or 100 μg/ml -RRB- . Throughout the experiment , all media were supplemented with 10 nM dexamethasone . Cells were exposed to TP508 for 24 , 48 and 72 h , after which times , the cells were collected . The experiment was carried out in quadruplicate and repeated once . RNA analysis and quantitative real-time polymerase chain reaction Total RNA from cultured cells was isolated by using TRIZOL reagent -LRB- Gibco -RRB- according to the manufacturer 's instructions . The RNA content was determined by measuring the absorbance in water at 260 nm by means of an Ultrospec III spectrophotometer -LRB- Amersham , Buckinghamshire , England -RRB- . cDNA synthesis was performed by using 750 ng total RNA in a final reaction volume of 20 μl containing 5 U transcriptor -LRB- Roche Applied Science -RRB- , 5 × polymerase chain reaction -LRB- PCR -RRB- buffer , 4 U random primers -LRB- Roche -RRB- , 20 U protector RNase inhibitor -LRB- Roche -RRB- , 1 mmol each dNTP and 10 μl template . The reverse transcription step was performed on a Gene Amp 9700 Thermocycler -LRB- Applied Biosystems , Foster City , Calif. , USA -RRB- at 55 °C for 30 min followed by 85 °C for 5 min . Real-time PCR was performed on the ABI PRISM 7700 sequence detection system -LRB- Applied Biosystems -RRB- . The phosphobilinogen deaminase gene -LRB- PBGD -RRB- served as the endogenous reference -LRB- de Vries et al. 1999 -RRB- to normalize Runx2 expression . The primers for the amplification of Runx2 mRNA were 5 ′ - ATGCTTCATTCGCCTCAC-3 ′ and 5 ′ - ACTGCTTGCAGCCTTAAAT-3 ′ -LRB- GenBank database , accession no . NM 001024630 -RRB- . The PCR primers for PBGD , amplified as the internal reference , were 5 ′ - AGTGATGAAAGATGGGCAACT-3 ′ and 5 ′ - TCTGGACCATCTTCTTGCTGA-3 ′ -LRB- accession no . BC 003861 -RRB- . For the amplification of the Runx2 and PBGD products , 37.5 ng cDNA was added to the PCR mixture containing SYBR Green PCR Master Mix consisting of SYBR Green I Dye , AmpliTaq Gold DNA polymerase , dNTPs with dUTP , a passive reference and buffer -LRB- Applied Biosystems -RRB- and 300 nM of each primer , in a final volume of 25 μl . The enzyme was activated by preheating the samples for 10 min at 95 °C , followed by a two-step PCR procedure consisting of a denaturation step at 95 °C for 15 s and an annealing and extension step at 60 °C for 1 min for 45 cycles . Relative expression was calculated by using the comparative Ct method . Samples were normalized for the expression of PBGD by calculating ΔCt -LRB- CtRunx2 − CtPBGD -RRB- ; subsequently , the ΔΔCt values were calculated as ΔCtsample − ΔCtcalibrator , where the calibrator was the control sample -LRB- without TP508 incubation -RRB- . Relative expression of the Runx2 gene was expressed as 2 - -LRB- ΔΔ Ct -RRB- -LRB- Livak and Schmittgen 2001 -RRB- . Statistical analysis Values obtained from the scoring of tissue sections and normalized mRNA levels determined by PCR analysis -LRB- presented as means and standard deviations -RRB- were analysed by using GraphPad Prism 4 for Windows . For the analysis of the immunostaining , an average value was calculated per animal for each protein and treatment ; these average values were used to determine the mean and standard deviation for each treatment group -LRB- 5 -- 6 animals/group -RRB- . Analysis of variance -LRB- ANOVA -RRB- was used to test for statistical significance , which was accepted when P < 0.05 -LRB- two-tailed -RRB- . Tukey 's post test was performed to compare individual pairs of groups if P < 0.05 . Linear regression analysis was used to examine the correlation between Runx2 mRNA expression and culture time . Results Immunohistochemistry demonstrated different localization patterns for Runx2 , OPN and BSP proteins . Cells expressing these proteins were detected throughout the distraction gap and in the periosteum , the periosteal callus and the old -LRB- predistraction -RRB- bone -LRB- Fig. 1a -RRB- . Runx2 staining was located in nuclei and cytoplasm , whereas strong staining for OPN and BSP was localized in the cytoplasm -LRB- Figs. 1b , c ,2 i ,3 c -RRB- ; weak staining for BSP and OPN was also seen in the extracellular bone matrix . Figures 1 , 2 , 3 present the different staining patterns and Table 1 summarizes the average intensity of immunostaining in the ROI . Fig. 2Runx2 -LRB- a , d , g -RRB- , OPN -LRB- b , e , h -RRB- and BSP -LRB- c , f , i -RRB- expression in the distraction gap -LRB- B bone , FT fibrous tissue , FC fibrous cartilage-like tissue , asterisks central area of distraction gap , hatch layers of cells adjacent to the newly formed bone , arrows osteoblasts , arrowheads osteocytes -RRB- of the various groups -LRB- a-c 0 μg TP508 group , d-f 30 μg TP508 group , g-i 300 μg TP508 group -RRB- . Inset -LRB- i -RRB- : Newly formed bone in the gap . Original magnifications : a 50 × ; d , g 100 × ; b , e , h 100 × ; c , f , i , inset 200 × . Bars 20 μm -LRB- c , f , i -RRB- , 50 μm -LRB- b , d , e , g , h -RRB- , 100 μm -LRB- a -RRB- Fig. 3Immunolocalization of Runx2 , OPN and BSP in the 300 μg TP508 group . a Runx2 staining of a vessel wall -LRB- EC endothelial cells , SMC smooth muscle cells -RRB- . Note that some cells near the blood vessel weakly express Runx2 -LRB- white arrows -RRB- . × 400 . b Osteopontin staining of a vessel wall . Note that the cells surrounding the blood vessel are immunonegative and only stain weakly for methyl green -LRB- white arrows -RRB- . × 400 . c BSP staining is restricted to the layer of osteoblasts -LRB- Ob -RRB- and hardly occurs in fibrous tissue × 400 . d OPN staining is expressed in osteoblasts -LRB- Ob -RRB- and osteocytes of new bone -LRB- NB -RRB- and some fibrous tissue cells close to osteoblasts , presumably pre-osteoblasts -LRB- Pre-Ob -RRB- . × 200 . Bars 20 μm -LRB- b -RRB- , 50 μm -LRB- c , d -RRB- , 100 μm -LRB- a -RRB- Table 1Intensity scores of immunostaining in region of interest -LRB- ± weak , + moderate , + + strong , + + + very strong -RRB- . The intensity of immunostaining was scored in the central area of the gap -LRB- region of interest -RRB- at 40 × final magnification . Variation exists between sections in each animal and between animals within groupsMarkerGroup with 0 μg TP508Group with 30 μg TP508Group with 300 μg TP508RUNX2 + to + + + to + + + + to + + + OPN ± to + + ± to + + + + BSP ± to + + + ± + to + + + Expression of Runx2 , OPN and BSP in the group injected with saline -LRB- control group -RRB- was similar to that in the group treated with 30 μg TP508 -LRB- Figs. 1d , 2a -- f -RRB- . The intensity of the staining in both groups was however much lower than that in the 300 μg TP508 group -LRB- Figs. 1c ,2 g , h ,3 c , d -RRB- . Runx2 and OPN were detected in active osteoblasts , in fibroblasts next to the osteoblastic layer -LRB- presumably pre-osteoblasts -RRB- , in osteocytes in the old -LRB- predistraction -RRB- and newly formed bone , in fibroblasts of the periosteum , in the central area of the gap , in endothelial and smooth muscle cells of the vessels and in chondrocyte-like cells next to new bone . We found two types of cartilage-like tissue in the group treated with 30 μg TP508 and in control group , i.e. fibrocartilage-like -LRB- collagen fibres between lacunae -RRB- and hypertrophic-like -LRB- evenly dispersed chondrocytes , without clear fibres ; Fig. 1d -RRB- . Higher numbers of cells associated with these two tissues were found in the group treated with 30 μg TP508 and in the control group than in the group treated with 300 μg TP508 -LRB- Fig. 2a , d , g -RRB- . More fibrocartilage-like cells than hypertrophic cartilage-like cells were positive for Runx2 -LRB- Fig. 1d -RRB- . Sections in which primary antibodies had been replaced with non-immune antibodies were negative -LRB- Fig. 1e -RRB- . The Runx2 - and OPN-positive blood vessel cells -LRB- both the endothelial and smooth muscle cells -RRB- were more evident in the group with 300 μg TP508 -LRB- Fig. 3a , b -RRB- . They appeared to occur at locations with high bone formation activity . Connective tissue cells in close vicinity to Runx2-positive blood vessels also stained faintly for Runx2 but not for OPN . Immunostaining for BSP was more restricted than that for Runx2 and OPN and was seen in osteoblasts and young osteocytes -LRB- Fig. 3c , d -RRB- . Weak expression was found in some of the cells considered to be pre-osteoblasts . Fibroblast-like cells in the central fibrous area of the distraction gap expressed Runx2 -LRB- Fig. 2a , d , g -RRB- and OPN -LRB- Fig. 2b , e , h -RRB- but were negative for BSP . The number and the intensity of Runx2 and OPN in the gap increased from fibrous tissue towards the newly formed bone -LRB- Fig. 1c -RRB- . Weak BSP staining was seen in the area within the gap in which bone trabecules started to be formed -LRB- Fig. 2c , f , i -RRB- . In the group injected with 300 μg TP508 , immunostaining for Runx2 , OPN and BSP was distributed throughout the regenerating areas . The immunostaining for Runx2 , OPN and BSP in the connective tissue in the gap -LRB- endosteal area -RRB- was relatively high and as intense as in the periosteal callus tissue . In the 30 μg TP508 group and in the control group , staining was less intense in the central gap area than in the periosteal callus area . Quantitative immunohistochemistry was performed to investigate the effect of TP508 injection on the total number of cells and on the number of differentiated bone cells in the central area of the gap . The total number of cells -LRB- immunostained and unstained -RRB- in the ROI was not significantly different between the three groups . Mean values and the standard deviation of the total number of cells in the tissues treated with 0 , 30 μg TP508 and 300 μg TP508 were 958 ± 217 , 1140 ± 137 and 1161 ± 282 , respectively -LRB- ANOVA , P = 0.5 -RRB- . This indicated that TP508 injection had no overall effect on total number of cells in the distraction gap . Next , we counted the number of cells , in the ROI , expressing each of the three antibodies tested . Absolute values of immunopositive cells in the 0 , 30 μg TP508 and 300 μg TP508 groups were as follows : for Runx2 , 263 ± 61 , 288 ± 171 , 560 ± 115 , respectively ; for OPN , 299 ± 201 , 244 ± 40 , 677 ± 216 , respectively ; for BSP , 255 ± 111 , 252 ± 73 , 480 ± 31 , respectively . We also expressed the number of immunopositive cells as a percentage of the total number of cells -LRB- Fig. 4 -RRB- . The number of Runx2 - and OPN-expressing cells was significantly higher in the group treated with 300 μg TP508 than in the control group that received only saline -LRB- P < 0.05 ; Fig. 4 -RRB- . The number of BSP-expressing cells was also higher in the 300 μg TP508 group than in the 30 μg TP508 group -LRB- P < 0.05 -RRB- . No significant differences were found between the 30 μg TP508 group and the control group receiving saline -LRB- P > 0.05 -RRB- . Fig. 4Effect of TP508 injection on the number of immunopositive cells in the central portion of the distraction gap -LRB- black bars Runx2-positive cells , grey bars OPN-positive cells , white bars BSP-positive cells -RRB- . Data are presented as percentage cells immunopositive for each of the three proteins examined -LRB- means ± SD -RRB- . * The percentages of Runx2 - , OPN - and BSP-positive cells in the 300 μg TP508 group were significantly higher than for cells with the corresponding proteins in the saline-treated control group ; the percentage of BSP-positive cells in the 300 μg group was also significantly higher than that in the 30 μg group -LRB- ANOVA , P < 0.05 -RRB- The previous data suggested that TP508 increased the differentiation of osteogenic cells into osteoblasts in situ . To determine whether TP508 had any direct effect on osteogenic cells that were in the process of turning into osteoblasts , we performed in vitro experiments with the -LRB- mouse -RRB- osteoblast-cell line MC3T3-E1 after pre-culture in the presence of dexamethasone . After a 24-h stimulation with TP508 , we found a significant -LRB- but slight -RRB- downregulation of Runx2 mRNA at 10 μg/ml TP508 -LRB- P = 0.008 -RRB- but no change at 100 μg/ml TP508 -LRB- Fig. 5 -RRB- . Fig. 5Quantitative reverse transcription/polymerase chain reaction analysis of Runx2 mRNA expression in mouse osteoblast-like -LRB- MC3T3-E1 -RRB- cells cultured for 24 , 48 and 72 h in the presence of TP508 -LRB- black bars control group with 0 μg/ml TP508 , hatched bars 10 μg/ml TP508 , white bars 100 μg/ml TP508 -RRB- . Data are presented as means ± SD -LRB- n = 8 -RRB- and are representative of two experiments . * The 10 μg/ml TP508 group at 24 h of incubation was significantly lower than the control group -LRB- one-way ANOVA ; Tukey 's multiple comparison test , P < 0.01 -RRB- . Correlation analysis between mRNA and culture time : r2 = 0.94 , P = 0.06 for 10 μg/ml TP508 ; r2 = 0.99 , P = 0.06 for 100 μg/ml TP508 Discussion This study demonstrates a positive effect of TP508 on the synthetic activity of bone-forming cells in the distraction gap in rabbit long bones . After injection of a high dose of TP508 , significantly more cells expressing Runx2 , OPN and BSP are located in the central part of the distraction gap , predominantly consisting of soft connective tissue . We have also found that these cells stain more intensely for Runx2 , OPN and BSP suggesting that TP508 not only proportionally increases the number of osteogenic cells in the distraction gap but also their expression level . The finding that TP508 enhances bone-forming activity in the soft tissue gap area is in agreement with previous morphometric data indicating that TP508 stimulates bone regeneration in the distraction gap -LRB- Li et al. 2005a , b -RRB- . The underlying molecular mechanism of the stimulation of Runx2 expression in the distraction gap after TP508 treatment is not clear . Our in vitro experiment with osteoblast-like MC3T3-cells suggests that TP508 does not have a significant positive effect on Runx2 mRNA . However , we can not rule out that the MCT3C3 cells are too far advanced along the osteoblast differentiation pathway to respond to TP508 for they had been pre-cultured in dexamethasone-containing medium before they were exposed to TP508 . No definitive conclusions can therefore be drawn from these in vitro data . In vivo , TP508 may have stimulated the differentiation of osteogenic cells though the activity of other non-osteogenic cell types . Injection of TP508 into a bone fracture model upregulates FGF-2 mRNA in -LRB- not further specified -RRB- tissues at the fracture site -LRB- Wang et al. 2005 -RRB- . Since FGF-2 is known to be an upstream regulator of Runx2 -LRB- Franceschi and Xiao 2003 -RRB- , one explanation for the increased Runx2 expression in the gap is via an enhanced cytokine release by inflammatory cells , which in turn stimulates osteoblast differentiation -LRB- Okazaki et al. 1999 ; Stiernberg et al. 2000 ; Franceschi and Xiao 2003 ; Naldini et al. 2004 -RRB- . Alternatively , TP508 stimulates blood vessel growth in vivo -LRB- Norfleet et al. 2000 ; Li et al. 2005a , b ; Vartanian et al. 2005 ; Wang et al. 2005 -RRB- and this subsequently enhances bone formation . TP508 increases recruitment of human aortic and microvascular endothelial cells through chemotaxis -LRB- Norfleet et al. 2000 ; Li et al. 2002 -RRB- , upregulates angiogenesis-related genes -LRB- Wang et al. 2005 -RRB- and acts directly on microvascular cells to accelerate sprouting -LRB- Vartanian et al. 2005 -RRB- . Vascularization provides the oxygen that is required for the metabolic activity of the osteoblasts -LRB- Tuncay et al. 1994 ; Salim et al. 2004 -RRB- ; it provides homes for circulating stem cells that , under proper stimulation , differentiate into osteoblastic cells -LRB- Kuznetsov et al. 2001 -RRB- . In addition , vessel cells secrete paracrine factors that can regulate bone cell metabolism -LRB- Villars et al. 2000 ; Street et al. 2002 -RRB- . Thus , the angiogenic properties of TP508 might help to enhance bone growth . In the present study , we have also found Runx2 protein expression in endothelial cells , vascular smooth muscle cells and loose connective cells surrounding developing blood vessels at sites of active bone and tissue formation . Recent studies have presented evidence for transient Runx2 mRNA and protein expression during angiogenesis in embryonic human orofacial tissues -LRB- Bronckers et al. 2005 -RRB- , in human primary bone marrow endothelial cells and in endothelial cell lines -LRB- Namba et al. 2000 ; Sun et al. 2001 -RRB- . Thus , our data suggest that , with respect to Runx2 expression , the formation of blood vessels in regenerating adult tissues during DO resembles vessel formation in embryonic tissues . Of note , under certain conditions such as inflammation , cells associated with vessels -LRB- e.g. pericytes , endothelial cells and smooth muscle cells -RRB- or their precursors are capable of differentiating into osteoblast-like cells -LRB- Schor et al. 1995 ; Reilly et al. 1998 ; Kinner and Spector 2002 -RRB- suggesting that the forming microvasculature is a potential source for osteogenic cells . The distribution and staining intensity of Runx2 , OPN and BSP varies between the different groups . In the control group and in the group treated with a low dose of TP508 , staining is more pronounced in the periosteal callus than in the tissue in the distraction gap . However , in the group treated with a high dose of TP508 , we have found the same high staining in both periosteal callus and gap tissue . Periosteal callus formation is a common finding during fracture healing -LRB- Gerstenfeld et al. 2003 -RRB- . New woven bone is formed rapidly around the fractured segments , because of stimulation of the periosteum that contains many osteoprogenitor cells . In the same section , a high staining intensity in the periosteal callus can therefore be considered as an internal reference . Hence , as the staining for Runx2 , OPN and BSP in the distraction gap is as intense as that in the periosteal callus at the high dose of TP508 , this is a clear indication that the high dose of TP508 stimulates bone formation in the distraction gap . This observation is in accordance to a previous histological study that has demonstrated advanced bone consolidation in a group treated with a high dose of TP508 -LRB- Li et al. 2005b -RRB- . TP508 might stimulate the migration of osteoblast precursors to the distraction gap -LRB- Li et al. 2005a -RRB- . Li and colleagues have shown that human periosteal-derived osteoblasts and human microvascular endothelial cells are chemotactic to TP508 -LRB- Li et al. 2005a -RRB- . Therefore , the presence of TP508 in the gap might attract osteoprogenitor cells in the periosteum to migrate into the distraction gap . Runx2 is a critical gene for osteoblast differentiation and for the function of the fully differentiated osteoblast -LRB- Komori et al. 1997 ; Karsenty et al. 1999 -RRB- . The Runx2 protein binds to the osteoblast-specific element 2 that is present in the promoter regions of all major extracellular matrix genes expressed in osteoblasts , including the genes for osteocalcin -LRB- OC -RRB- , OPN and BSP and it regulates the expression of these proteins -LRB- Karsenty et al. 1999 -RRB- . OPN and BSP belong to the Small Integrin-Binding LIgand , N-linked Glycoprotein -LRB- or SIBLING -RRB- family -LRB- Fisher et al. 2001 -RRB- . Among other functions , members of this family are known to have a role in mineralization , as they bind strongly to hydroxyapatite -LRB- Fisher et al. 2001 -RRB- . In conclusion , we have demonstrated that the increase of bone regeneration by thrombin-related peptide TP508 is associated with an increase in the immunostaining for Runx2 , an essential transcription factor of the osteoblastic lineage , and for the bone matrix proteins BSP and OPN . TP508 may thus be a candidate for enhancing bone regeneration when bone regeneration is slow , as occurs in elderly patients .

O O B I O O O O O O O O B I O O O O O O O O O O B I O B I O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O

bone regeneration
distraction osteogenesis
thrombin peptide
immunohistochemistry
runx2
osteopontin
bone sialoprotein