the sample collection was approved by the ethical board at the university of lund with reference number 513/2006 and by the regional ethical review board in stockholm with reference number 2014/1588  32/3 . written informed consent was obtained from all participants . for analysis of saliva samples , individuals with periodontitis ( n  =  37 ) and healthy controls ( n  =  39 ) from a population from southern sweden undergoing dental examination ( lundegren et  al . anamnestic data including diseases and smoking habits were collected . for the patients diagnosed with periodontitis , gingival inflammation , registered as bleeding on probing ( bop ) , was more than 30% and all patients had loss of supporting tissues exceeding 1/3 of the root length , determined through radiographical examinations . the majority of these patients ( 84% ) had pocket probing depth ( ppd )   6  mm and six patients ( 16% ) had ppd 45  mm . the healthy controls included individuals with no signs of bone loss on radiographs , ppd <  4  mm , and bop less than 30% . stimulated saliva samples were obtained by chewing paraffin tablets for 5  min while the saliva was collected into test tubes . the samples where then thawed and centrifuged at 500  g for 10  min at 4c . the supernatants were aliquoted into 1.5  ml eppendorf tubes and stored at 80c until analysis . the total protein concentrations were measured using the bradford assay ( biorad , hercules , ca , usa ) according to the manufacturer 's instructions , using bovine serum albumin as standard . the levels of muc4 and mmp7 were measured using commercially available elisa kits according to the respective manufacturer 's protocols ( muc4 : kamiya biomedical company , seattle , wa , usa , mmp7 : quantikine ; r&d systems , minneapolis , mn , usa ) . before analysis of muc4 and mmp7 , the saliva samples were diluted 1:2 in phosphate buffered saline ( pbs ) and in calibrator diluent buffer respectively . the sensitivities for the assays used were 0.134  ng / ml for muc4 , 0.084  ng / ml for mmp7 , and 0.08  ng / ml for mmp8 ( tuomainen et  al . 2007 ) . after optical density readings , all readings that fell below the assay sensitivity were set to the lowest point of the assay sensitivity . the levels of mmp8 were measured by a timeresolved immunofluorometric assay , using monoclonal mmp8antibody 8708 ( medix biochemica , kauniainen , finland ) as capture antibody and monoclonal mmp8antibody 8706 ( medix biochemica ) labelled with europiumchelate as tracer antibody . saliva samples were diluted 1:4 in assay buffer ( 20  mm trishcl [ ph 7.5 ] , 0.5  m nacl , 5  mm cacl2 , 50  m zncl2 , 0.5% bovine serum albumin , 0.05% sodium azide , and 20  mg / l diethylenetriaminepentaacetic acid ) and incubated with the capture antibody for 1  h , followed by incubation for 1  h with the tracer antibody . enhancement solution was then added and fluorescence was measured after 5  min using a 1234 delfia research fluorometer ( wallac , turku , finland ) ( hanemaaijer et  al . gingival crevicular fluid samples were collected from an additional cohort consisting of 20 periodontitis patients , enrolled at a periodontal clinic , and 20 healthy controls . for the patients diagnosed with periodontitis , gcf was collected from sites with ppd    6  mm . for the healthy controls , gcf was collected from sites with ppd    3  mm . before sample collection , supragingival plaque was removed from the tooth surface using a cotton pellet and the surface was gently dried with air . gcf samples were collected from the buccal ( mesial and distal ) sites from one tooth by inserting two separate paper strips ( periopaper , proflow inc . , amityville , ny , usa ) into the gingival crevice until slight resistance was felt , and leaving them for 30  s. gcf samples visibly contaminated with blood were discarded . immediately after gcf collection , thereafter , the two paper strips were placed into a vial containing 175  l pbs with 0.05% tween20 , vortexed for 30  s , and stored at 80c . the levels of muc4 ( diluted 1:2 in pbs buffer ) and mmp7 in the gcf solutions were determined using commercial elisa kits as described above for the saliva samples . if all cell values exceeded five , chisquare test was used , otherwise fisher 's exact test was used . for boxplots , univariate analyses of protein levels between patients with periodontitis and healthy controls were performed with mann multiple linear regression analyses were used with the concentrations of the proteins as dependent variables . in order to achieve normality , periodontitis , age , and smoking habits were included as independent variables . in order to evaluate the discriminative power of muc4 and mmp7 , the ratio of muc4 over mmp7 related to total protein concentrations , as well as age and smoking habits , was included as independent variables . this was followed by construction of a receiver operating characteristic ( roc ) curve and calculation of area under curve ( auc ) using the proc package ( robin et  al . all statistical analyses were performed using software r ( r core team 2015 ) and differences were considered significant at a pvalue <  0.05 . this study was performed in accordance with the declaration of helsinki and current swedish legislation . the sample collection was approved by the ethical board at the university of lund with reference number 513/2006 and by the regional ethical review board in stockholm with reference number 2014/1588  32/3 . for analysis of saliva samples , individuals with periodontitis ( n  =  37 ) and healthy controls ( n  =  39 ) from a population from southern sweden undergoing dental examination ( lundegren et  al . anamnestic data including diseases and smoking habits were collected . for the patients diagnosed with periodontitis , gingival inflammation , registered as bleeding on probing ( bop ) , was more than 30% and all patients had loss of supporting tissues exceeding 1/3 of the root length , determined through radiographical examinations . the majority of these patients ( 84% ) had pocket probing depth ( ppd )   6  mm and six patients ( 16% ) had ppd 45  mm . the healthy controls included individuals with no signs of bone loss on radiographs , ppd <  4  mm , and bop less than 30% . stimulated saliva samples were obtained by chewing paraffin tablets for 5  min while the saliva was collected into test tubes . the samples where then thawed and centrifuged at 500  g for 10  min at 4c . the supernatants were aliquoted into 1.5  ml eppendorf tubes and stored at 80c until analysis . the total protein concentrations were measured using the bradford assay ( biorad , hercules , ca , usa ) according to the manufacturer 's instructions , using bovine serum albumin as standard . the levels of muc4 and mmp7 were measured using commercially available elisa kits according to the respective manufacturer 's protocols ( muc4 : kamiya biomedical company , seattle , wa , usa , mmp7 : quantikine ; r&d systems , minneapolis , mn , usa ) . before analysis of muc4 and mmp7 , the saliva samples were diluted 1:2 in phosphate buffered saline ( pbs ) and in calibrator diluent buffer respectively . the sensitivities for the assays used were 0.134  ng / ml for muc4 , 0.084  ng / ml for mmp7 , and 0.08  ng / ml for mmp8 ( tuomainen et  al . 2007 ) . after optical density readings , all readings that fell below the assay sensitivity the levels of mmp8 were measured by a timeresolved immunofluorometric assay , using monoclonal mmp8antibody 8708 ( medix biochemica , kauniainen , finland ) as capture antibody and monoclonal mmp8antibody 8706 ( medix biochemica ) labelled with europiumchelate as tracer antibody . saliva samples were diluted 1:4 in assay buffer ( 20  mm trishcl [ ph 7.5 ] , 0.5  m nacl , 5  mm cacl2 , 50  m zncl2 , 0.5% bovine serum albumin , 0.05% sodium azide , and 20  mg / l diethylenetriaminepentaacetic acid ) and incubated with the capture antibody for 1  h , followed by incubation for 1  h with the tracer antibody . enhancement solution was then added and fluorescence was measured after 5  min using a 1234 delfia research fluorometer ( wallac , turku , finland ) ( hanemaaijer et  al . gingival crevicular fluid samples were collected from an additional cohort consisting of 20 periodontitis patients , enrolled at a periodontal clinic , and 20 healthy controls . for the patients diagnosed with periodontitis , gcf was collected from sites with ppd    6  mm . for the healthy controls , gcf was collected from sites with ppd    3  mm . before sample collection , supragingival plaque was removed from the tooth surface using a cotton pellet and the surface was gently dried with air . gcf samples were collected from the buccal ( mesial and distal ) sites from one tooth by inserting two separate paper strips ( periopaper , proflow inc . , amityville , ny , usa ) into the gingival crevice until slight resistance was felt , and leaving them for 30  s. gcf samples visibly contaminated with blood were discarded . immediately after gcf collection , the paper strips were frozen at 20c until further processing . thereafter , the two paper strips were placed into a vial containing 175  l pbs with 0.05% tween20 , vortexed for 30  s , and stored at 80c . the levels of muc4 ( diluted 1:2 in pbs buffer ) and mmp7 in the gcf solutions were determined using commercial elisa kits as described above for the saliva samples . if all cell values exceeded five , chisquare test was used , otherwise fisher 's exact test was used . for boxplots , univariate analyses of protein levels between patients with periodontitis and healthy controls were performed with mann multiple linear regression analyses were used with the concentrations of the proteins as dependent variables . in order to achieve normality , periodontitis , age , and smoking habits were included as independent variables . in order to evaluate the discriminative power of muc4 and mmp7 , the ratio of muc4 over mmp7 related to total protein concentrations , as well as age and smoking habits , was included as independent variables . this was followed by construction of a receiver operating characteristic ( roc ) curve and calculation of area under curve ( auc ) using the proc package ( robin et  al . all statistical analyses were performed using software r ( r core team 2015 ) and differences were considered significant at a pvalue <  0.05 . the characteristics of the study participants including patients with periodontitis ( n  =  37 ) and healthy controls ( n  =  39 ) from which saliva samples were obtained are presented in table  1 . the mean age of subjects was higher in the periodontitis group compared to the healthy controls ( 62.0 and 37.6  years , respectively , p  <  0.01 ) . in the periodontitis group ,  the number of sites with ppd 45  mm and 6  mm were significantly ( p  <  0.01 ) higher compared to the periodontally healthy individuals with no sites with ppd 4  mm or more . the majority ( 84% ) of the patients with periodontitis had ppd    6  mm and six patients ( 16% ) had ppd 45  mm . the number of sites with plaque was also significantly ( p  <  0.01 ) higher in the periodontitis group than in the control group ( mean plaque index 38% and 12% respectively ) . in addition , the number of sites with bop was significantly ( p  <  0.01 ) higher in the periodontitis group compared to the control group ( mean bop index 54% and 12% respectively ) . the number of individuals with high blood pressure or heart disease was significantly ( p  <  0.01 ) higher in the group of periodontitis patients than in the healthy control group , whereas the number of subjects with diabetes , bowel disease , or muscle and joint diseases were not significantly different between the groups ( table  1 ) . characteristics of study participants from whom saliva samples were obtained ppd , pocket probing depth ; bop , bleeding on probing . the levels of muc4 and mmp7 in saliva samples from patients with periodontitis and healthy controls are demonstrated in fig . 1 . salivary concentrations of muc4 were significantly ( p  <  0.01 ) lower in patients with periodontitis relative to healthy controls . mmp7 concentrations , on the other hand , were significantly ( p  <  0.05 ) higher in periodontitis patients . in addition , the total protein concentrations were significantly ( p  <  0.01 ) higher in patients with periodontitis ( table  s1 ) . when relating the levels of muc4 and mmp7 to the total protein concentrations , muc4 levels were also significantly ( p  <  0.01 ) lower in saliva samples from periodontitis patients . the ratio of mmp7 over total protein concentration was , however , not significantly different in saliva samples obtained from periodontitis patients relative to controls ( fig .  1 and table  s1 ) . salivary levels of mucin 4 ( muc4 ) and matrix metalloproteinase 7 ( mmp7 ) . boxplots demonstrating the concentrations of ( a ) muc4 ( ng / ml ) , ( b ) muc4/total protein concentrations ( ng / mg ) , ( c ) mmp7 ( ng / ml ) , and ( d ) mmp7/total protein concentrations ( ng / mg ) in saliva samples from periodontitis patients and healthy controls . * p  <  0.05 , * * p  <  0.01 . in order to validate our novel biomarkers of salivary proteins for periodontitis , the levels of mmp8 were also determined since it has repeatedly been reported to be upregulated in periodontitis compared to healthy controls . in agreement with mmp7 , the salivary levels of mmp8 were significantly ( p  <  0.01 ) higher in periodontitis patients relative to controls without periodontal disease ( fig .  2 and table  s1 ) however , the levels of mmp8 related to the total protein concentrations did not significantly differ between the two groups ( fig .  2 and table  s1 ) . boxplots demonstrating the concentrations of ( a ) mmp8 ( ng / ml ) , and ( b ) mmp8/total protein concentrations ( ng / mg ) in saliva samples from patients with periodontitis and healthy controls . * multiple linear regression analyses were performed to investigate the impact of periodontitis on the salivary levels of muc4 and mmp7 , adjusted for age and smoking habits ( table  2 ) . the analyses were performed using the total concentrations of muc4 and mmp7 as well as their concentrations related to the total protein levels . the results revealed that periodontitis contributed significantly ( p  <  0.05 ) to the levels of muc4 in saliva samples , when adjusting for age and smoking . muc4 remained significant ( p  <  0.01 ) also when related to the total protein concentrations in the saliva samples ( table  2 ) . in addition , the analysis revealed that smoking contributed significantly to the levels of mmp7 as well as to mmp7 related to total protein concentrations ( table  2 ) . multiple linear regression analyses of the association between salivary protein levels and periodontitis , adjusted for age and smoking statistically significant pvalues ( <  0.05 ) are indicated with boldface . furthermore , a logistic regression analysis was performed with periodontitis as dependent variable and the ratio of muc4 over mmp7 related to total protein concentrations , as well as age and smoking habits as independent variables ( table  s2 ) . the results from this analysis revealed that the ratio of muc4 over mmp7 related to the total protein concentration , as well as age , was significantly associated with periodontitis ( p  <  0.05 and p  <  0.01 , respectively , table  s2 ) . the logistic regression analysis was thereafter used to investigate the diagnostic potential of the combination of the novel biomarkers , through construction of a roc curve . the roc curve analysis showed that the area under curve was 0.931 ( fig . diagnostic power of mucin 4 ( muc4 ) and matrix metalloproteinase 7 ( mmp7 ) by receiver operating characteristic curve ( roc curve ) . the roc curve is based on a binary logistic regression model with periodontitis as dependent variable and muc4/mmp7 levels related to the total protein concentrations , age , and smoking habits as independent variables . in addition to saliva samples , the levels of muc4 and mmp7 were also investigated in gcf samples from a cohort of 40 subjects with periodontitis and healthy controls . the mean age of subjects was significantly ( p  <  0.01 ) higher in the periodontitis group compared to the healthy controls ( 67.1    9.5 and 38.5    10.9  years respectively ) . the periodontitis group , comprising 10 females and 10 males , had significantly ( p  <  0.01 ) higher ppd ( mean 6.9    2.0  mm ) and bop ( 45% ) , than the healthy control group , comprising 14 females and six males ( ppd    3  mm , bop 5% ) ( table  not shown ) . in agreement with our findings in saliva , the total concentrations of muc4 as well as muc4 related to total protein concentration were significantly ( p  <  0.05 , and p  <  0.01 respectively ) lower in the gcf from periodontitis patients relative to controls ( fig .  4 and table  s3 ) . moreover , the levels of mmp7 , as well as the total protein concentrations , were significantly ( p  <  0.01 ) higher in gcf from periodontitis patients ( fig .  4 and table  s3 ) . mucin 4 ( muc4 ) and matrix metalloproteinase 7 ( mmp7 ) levels in gingival crevicular fluid ( gcf ) samples . boxplots demonstrating the concentrations of ( a ) muc4 ( ng / ml ) , ( b ) muc4/total protein concentrations ( ng / mg ) , ( c ) mmp7 ( ng / ml ) , and ( d ) mmp7/total protein concentrations ( ng / mg ) in gcf samples from periodontitis patients and healthy controls . * p  <  0.05 , * * p  <  0.01 . multiple linear regression analyses were also performed with the total concentrations of muc4 and mmp7 as well as in relation to protein concentrations as dependent variables , adjusted for age and smoking habits ( table  3 ) . the analyses revealed that periodontitis was significantly ( p  <  0.01 ) associated with the gcf levels of muc4 , and muc4 related to total protein concentrations , as well as with the levels of mmp7 ( table  3 ) . multiple linear regression analyses of the association between protein levels of gingival crevicular fluid ( gcf ) and periodontitis , adjusted for age and smoking statistically significant pvalues ( <  0.05 ) are indicated with boldface . the characteristics of the study participants including patients with periodontitis ( n  =  37 ) and healthy controls ( n  =  39 ) from which saliva samples were obtained are presented in table  1 . the mean age of subjects was higher in the periodontitis group compared to the healthy controls ( 62.0 and 37.6  years , respectively , p  <  0.01 ) . in the periodontitis group ,  the number of sites with ppd 45  mm and 6  mm were significantly ( p  <  0.01 ) higher compared to the periodontally healthy individuals with no sites with ppd 4  mm or more . the majority ( 84% ) of the patients with periodontitis had ppd    6  mm and six patients ( 16% ) had ppd 45  mm . the number of sites with plaque was also significantly ( p  <  0.01 ) higher in the periodontitis group than in the control group ( mean plaque index 38% and 12% respectively ) . in addition , the number of sites with bop was significantly ( p  <  0.01 ) higher in the periodontitis group compared to the control group ( mean bop index 54% and 12% respectively ) . the number of individuals with high blood pressure or heart disease was significantly ( p  <  0.01 ) higher in the group of periodontitis patients than in the healthy control group , whereas the number of subjects with diabetes , bowel disease , or muscle and joint diseases were not significantly different between the groups ( table  1 ) . characteristics of study participants from whom saliva samples were obtained ppd , pocket probing depth ; bop , bleeding on probing . the levels of muc4 and mmp7 in saliva samples from patients with periodontitis and healthy controls are demonstrated in fig . 1 . salivary concentrations of muc4 were significantly ( p  <  0.01 ) lower in patients with periodontitis relative to healthy controls . mmp7 concentrations , on the other hand , were significantly ( p  <  0.05 ) higher in periodontitis patients . in addition , the total protein concentrations were significantly ( p  <  0.01 ) higher in patients with periodontitis ( table  s1 ) . when relating the levels of muc4 and mmp7 to the total protein concentrations , muc4 levels were also significantly ( p  <  0.01 ) lower in saliva samples from periodontitis patients . the ratio of mmp7 over total protein concentration was , however , not significantly different in saliva samples obtained from periodontitis patients relative to controls ( fig .  1 and table  s1 ) . salivary levels of mucin 4 ( muc4 ) and matrix metalloproteinase 7 ( mmp7 ) . boxplots demonstrating the concentrations of ( a ) muc4 ( ng / ml ) , ( b ) muc4/total protein concentrations ( ng / mg ) , ( c ) mmp7 ( ng / ml ) , and ( d ) mmp7/total protein concentrations ( ng / mg ) in saliva samples from periodontitis patients and healthy controls . * p  <  0.05 , * * p  <  0.01 . in order to validate our novel biomarkers of salivary proteins for periodontitis , the levels of mmp8 were also determined since it has repeatedly been reported to be upregulated in periodontitis compared to healthy controls . in agreement with mmp7 , the salivary levels of mmp8 were significantly ( p  <  0.01 ) higher in periodontitis patients relative to controls without periodontal disease ( fig .  2 and table  s1 ) however , the levels of mmp8 related to the total protein concentrations did not significantly differ between the two groups ( fig .  2 and table  s1 ) . boxplots demonstrating the concentrations of ( a ) mmp8 ( ng / ml ) , and ( b ) mmp8/total protein concentrations ( ng / mg ) in saliva samples from patients with periodontitis and healthy controls . * multiple linear regression analyses were performed to investigate the impact of periodontitis on the salivary levels of muc4 and mmp7 , adjusted for age and smoking habits ( table  2 ) . the analyses were performed using the total concentrations of muc4 and mmp7 as well as their concentrations related to the total protein levels . the results revealed that periodontitis contributed significantly ( p  <  0.05 ) to the levels of muc4 in saliva samples , when adjusting for age and smoking . muc4 remained significant ( p  <  0.01 ) also when related to the total protein concentrations in the saliva samples ( table  2 ) . in addition , the analysis revealed that smoking contributed significantly to the levels of mmp7 as well as to mmp7 related to total protein concentrations ( table  2 ) . multiple linear regression analyses of the association between salivary protein levels and periodontitis , adjusted for age and smoking statistically significant pvalues ( <  0.05 ) are indicated with boldface . furthermore , a logistic regression analysis was performed with periodontitis as dependent variable and the ratio of muc4 over mmp7 related to total protein concentrations , as well as age and smoking habits as independent variables ( table  s2 ) . the results from this analysis revealed that the ratio of muc4 over mmp7 related to the total protein concentration , as well as age , was significantly associated with periodontitis ( p  <  0.05 and p  <  0.01 , respectively , table  s2 ) . the logistic regression analysis was thereafter used to investigate the diagnostic potential of the combination of the novel biomarkers , through construction of a roc curve . the roc curve analysis showed that the area under curve was 0.931 ( fig . diagnostic power of mucin 4 ( muc4 ) and matrix metalloproteinase 7 ( mmp7 ) by receiver operating characteristic curve ( roc curve ) . the roc curve is based on a binary logistic regression model with periodontitis as dependent variable and muc4/mmp7 levels related to the total protein concentrations , age , and smoking habits as independent variables . in addition to saliva samples , the levels of muc4 and mmp7 were also investigated in gcf samples from a cohort of 40 subjects with periodontitis and healthy controls . the mean age of subjects was significantly ( p  <  0.01 ) higher in the periodontitis group compared to the healthy controls ( 67.1    9.5 and 38.5    10.9  years respectively ) . the periodontitis group , comprising 10 females and 10 males , had significantly ( p  <  0.01 ) higher ppd ( mean 6.9    2.0  mm ) and bop ( 45% ) , than the healthy control group , comprising 14 females and six males ( ppd    3  mm , bop 5% ) ( table  not shown ) . in agreement with our findings in saliva , the total concentrations of muc4 as well as muc4 related to total protein concentration were significantly ( p  <  0.05 , and p  <  0.01 respectively ) lower in the gcf from periodontitis patients relative to controls ( fig .  4 and table  s3 ) . moreover , the levels of mmp7 , as well as the total protein concentrations , were significantly ( p  <  0.01 ) higher in gcf from periodontitis patients ( fig .  4 and table  s3 ) . mucin 4 ( muc4 ) and matrix metalloproteinase 7 ( mmp7 ) levels in gingival crevicular fluid ( gcf ) samples . boxplots demonstrating the concentrations of ( a ) muc4 ( ng / ml ) , ( b ) muc4/total protein concentrations ( ng / mg ) , ( c ) mmp7 ( ng / ml ) , and ( d ) mmp7/total protein concentrations ( ng / mg ) in gcf samples from periodontitis patients and healthy controls . multiple linear regression analyses were also performed with the total concentrations of muc4 and mmp7 as well as in relation to protein concentrations as dependent variables , adjusted for age and smoking habits ( table  3 ) . the analyses revealed that periodontitis was significantly ( p  <  0.01 ) associated with the gcf levels of muc4 , and muc4 related to total protein concentrations , as well as with the levels of mmp7 ( table  3 ) . multiple linear regression analyses of the association between protein levels of gingival crevicular fluid ( gcf ) and periodontitis , adjusted for age and smoking statistically significant pvalues ( <  0.05 ) are indicated with boldface . we have recently reported that muc4 and mmp7 are highly associated with periodontitis , as identified by rna sequencing analysis of gingival tissue biopsies from periodontitis patients and healthy controls ( lundmark et  al . , we report , for the first time , significantly different levels of these two proteins in saliva and gcf samples from patients with periodontitis relative to healthy controls . furthermore , in this study group , we also show that the combination of the salivary levels of muc4 and mmp7 has the potential of discriminating between individuals with and without periodontitis . in this study , protein levels of muc4 , determined by elisa using specific antibodies , were significantly lower in saliva and gcf samples of patients with periodontitis as compared to healthy controls . muc4 has previously been implicated in cancer , including pancreatic , breast , and lung ( reviewed in carraway et  al . regarding periodontitis , previous studies have reported higher levels of mucins in general in saliva samples from periodontitis patients than in healthy subjects ( sanchez et  al . these studies used , however , the alcian blue method , which stains glycoproteins in general and can therefore not distinguish different mucin family members , up to date 20 members ( frenkel & ribbeck 2015 ) . at the mrna level , our previous sequencing study , investigating the whole transcriptome in gingival tissue biopsies from periodontitis patients and healthy controls , identified higher expression of muc4 in gingival tissue biopsies from patients with periodontitis ( lundmark et  al . the contrasting findings in tissue of gingiva versus oral fluids may be due to the fact that muc4 exists in both secreted and membranebound form ( hilkens & buijs 1988 , sheng et  al . 2001 , linden et  al . 2008 ) and that the balance between these two forms may be altered due to reprogramming of signalling pathways of the muc4 gene ( andrianifahanana et  al . 2005 ) in response to inflammation . as a result of the biofilm formed adjacent to the gingival tissue , the membranebound form of muc4 may increase in order to prevent the bacteria to access the cell surface and to protect the gingival tissue . this suggestion is in line with our previous results demonstrating increased muc4 expression in oral epithelial cells stimulated with lps ( lundmark et  al . another explanation for the decreased levels of muc4 in saliva samples might be due to proteolytic degradation , either by enzymes upregulated by the host or expressed by microbes ( linden et  al . the decreased levels of muc4 in saliva might lead to a lower capability to agglutinate and cleanse oral pathogens , allowing biofilm formation on the teeth , leading to sustained inflammatory response in periodontitis patients . our findings demonstrating higher levels of mmp7 in saliva and gcf samples from patients with periodontitis relative to healthy controls are in agreement with our rna sequencing results , which revealed overexpression of mmp7 in gingival tissue biopsies of patients with periodontitis ( lundmark et  al . levels of mmp7 have , to our knowledge , not previously been reported in saliva of periodontitis patients . with regard to gcf , mmp7 has been reported to be elevated in samples from patients with adult periodontitis relative to gcf samples collected from patients with localized juvenile periodontitis and controls ( tervahartiala et  al . another study showed no significant differences in levels of total mmp7 in gcf samples from 20 patients with different periodontal diseases and healthy controls . after correcting for the volume of gcf obtained from each site , however , the levels of mmp7 were identified as significantly lower in periodontitis patients than in healthy controls ( emingil et  al . nonetheless , one limitation with this study as well as our current study is the small number of gcf samples and additional studies should be performed in order to validate these findings . another limitation with our study is the relatively high age of subjects in the periodontitis group relative to the healthy control group . nevertheless , our multiple regression analyses were adjusted for age and therefore the results would be applicable to younger patients as well . in addition to demonstrating that the levels of muc4 and mmp7 differ significantly between oral fluids of patients with periodontitis and healthy controls , in this study population , we also confirm elevated levels of mmp8 in saliva samples from patients with periodontitis . mmp8 levels have repeatedly been demonstrated to be upregulated in periodontitis and the elevated levels of mmp8 in our cohort of saliva samples indicate a concordance of our results with those of previous studies ( miller et  al . regarding proinflammatory periodontal tissue destruction cascades , it should be noted that mmp7 can activate prommp8 , thereby potentially promoting periodontal disease progression ( balbin et  al . are involved in tissue homoeostasis by promoting cell proliferation and repressing apoptosis ( ii et  al . , we also show that the combination of the salivary levels of muc4 and mmp7 might have a diagnostic application . a logistic regression analysis identified the ratio of muc4 over mmp7 as significantly associated with periodontitis , and a subsequent roc curve analysis had a high auc , indicating that the combination of muc4 and mmp7 is able to discriminate patients with periodontitis from healthy controls , in this study population . a combination of biomarkers will likely be more specific than a single marker and future studies should focus on panels of biomarkers , including muc4 and mmp7 , for predicting periodontitis . in conclusion , this study is , to the best of our knowledge , the first to identify significantly different levels of muc4 and mmp7 in saliva and gcf of patients with periodontitis in comparison to healthy controls , suggesting that muc4 and mmp7 , alone or in combination , might have the potential to act as novel diagnostic markers for periodontitis . table  s1 . levels of muc4 , mmp7 , mmp8 , and total protein in saliva samples from patients with periodontitis and healthy controls . logistic regression of the saliva samples with periodontitis as dependent variable and muc4/mmp7 related to total protein concentrations as independent variable , adjusted for age and smoking . levels of muc4 , mmp7 , and total protein in gingival crevicular fluid samples from patients with periodontitis and healthy controls .