the matricellular proteins are a heterogeneous group of extracellular matrix ( ecm ) proteins that interacts with either other ecm proteins and with cell - surface receptors , growth factors and cytokines .
the periostin , also called osteoblast - specific factor 2 ( osf-2 ) , is a non - structural matricellular protein that directly interacts with type i collagen and fibronectin regulating the biomechanical properties of connective tissues .
it is an 811 amino acid , 93 kda secreted ecm protein and four main isoforms have been identified which are not uniformly but differentially expressed in various cell lines ; a human periodontal ligament specific isoform has been recently characterized .
periostin is present in collagen - rich connective tissues like bone , skeletal muscle , tendons , joint ligaments , periodontal ligament , heart valves , adipose tissue , or skin . at present
the functions of periostin in several biomedical areas like osteology , oncology , cardiovascular and respiratory systems , inflammatory diseases or dentistry , in both normal and pathological conditions , are rather well known . as a whole
its main functions seem to be during the development and tissue repair , because the predominant role in mesenchymal remode ling .
a series of recent studies have detected expression of periostin in developing and mature dental tissues , alveolar bone or periodontal ligament .
consistently , mice deficient in periostin display changes in ecm of dental tissues as well as defects in mineralized tissues .
by contrast little information is available about the occurrence and distribution of periostin in the gingiva .
it is known that gingival fibroblasts may be a source of periostin in response to some cytokines which are elevated in periodontitis , and decreased levels of periostin were found in gingival crevicular fluid proportionally with the progression and severity of periodontal disease . moreover ,
periostin is involved in signaling pathways of drug - induced gingival over - growth . since
no data are available about the distribution of periostin in adult human gingival in normal conditions , and because its potential role in gingival pathologies , we used western blot and immunohistochemistry to analyze the occurrence and localization of periostin in these tissues . to establish whether periostin is cellular or extracellular we labeled in parallel keratinocytes and dermal fibroblasts
samples of gingival tissues ( sized 2x2x2 mm , approximately ) were surgically excised from healthy patients undergoing orthodontic treatment from zones not exposed to orthodontic forces , and were obtained from the instituto asturiano de odontologia .
the age range was 16 to 32 years , and were males ( n=8 ) and females ( n=4 ) .
the tissue samples were washed with tap water followed with cold saline , then fixed in buffered 10% formalin for 24 h , and routinely processed for paraffin embedding .
the pieces were cut 10 m thick and the sections mounted on gelatine - coated microscope slides . moreover , fresh samples ( n=6 from different subject ) were quickly frozen , stored at -80c and used for western blot .
this study was approved by the ethics committee of instituto asturiano de odontologia ( oviedo , spain ) and informed consent was obtained from each subject .
lysates prepared from gingival homogenates were processed as follows : representative samples of the free gingival , attached gingival and non - bone attached periodontal ligament were pooled and homogenized ( 1:2 , w / v ) in tris - hcl buffered saline ( tbs , 0.1 m , ph 7.5 ) containing 1 m leupeptin , 10 m pepstatin and 2 mm phenylmethylsulfonyl fluoride .
the homogenates were centrifuged at 25,000 rpm for 15 min at 4c and the resulting pellet dissolved in tris hcl 10 mm ph 6.8 , 2% sds , 100 mm dithiothreitol , and 10% glycerol at 4c .
after electrophoresis , proteins were transferred to a nitrocellulose membrane and antibody non - specific binding was blocked by immersion for 3 h in pbs containing 5% dry milk , and 0.1% tween-20 .
the membranes were then incubated at 4c for 2h with a rabbit polyclonal antibody against a peptide from fasciclin domain 1 of mouse periostin ( ls - bl10443 , lifespan biosciences , inc . ,
after incubation , the membranes were washed with tbs ph 7.6 containing 20% tween-20 , and incubated again for 1 h with goat anti - rabbit igg ( diluted 1:100 ) at room temperature .
membranes were washed again and incubated with the pap complex diluted 1:100 for 1 h at room temperature . finally , the reaction was developed using a chemiluminescent reagent ( ecl , amersham pharmacia biotech , buckinghamshire , uk ) and exposed to hyperfilm .
relative values of periostin were estimated by imagej quantification in the three different segments of the gingiva .
deparaffinized and rehydrated sections were processed for immunohistochemical detection of periostin using the envision antibody complex kit ( dako , copenhagen , denmark ) following the manufacturer s recommendations .
moreover , anti - vimentin ( clone 334 , boehringer - mannheim , mannheim , germany ) , anti - pancytokeratin ( clone pck-26 , sigma - aldrich quimica , sl , madrid , spain ) mouse monoclonal antibodies , and anti - collagen type i rabbit monoclonal antibody ( erp778 , abcam , cambridge , uk ) were used to label dermal fibroblasts , epithelial cells and type i collagen fibrils , respectively .
the sections were processed for simultaneous detection of periostin and vimentin , and periostin and pan - cytokeratin , as follows : the non - specific binding was reduced by incubation for 30 min with a solution of 1% bovine serum albumin in tbs .
the sections were then incubated overnight , at 4c in a humid chamber with a 1:1 mixture of anti - periostin and anti - vimentin antibodies ( both diluted 1:100 in the blocking solution ) ; or anti - periostin and anti - pancytokeratin antibodies ( diluted 1:200 and 1:100 , respectively , in the blocking solution ) ; or anti - vimentin and anti - type i collagen ( both diluted 1:100 in the blocking solution ) . after rinsing with tbs
, the sections were incubated for 1 hour with alexa fluor 488-conjugated goat anti - rabbit igg ( serotec , oxford , uk ) , diluted 1:1000 in tbs containing 5% mouse serum ( serotec ) , then rinsed again , and incubated for another hour with cy3-conjugated donkey anti - mouse antibody ( jackson - immunoresearch , baltimore , md , usa ) diluted 1:50 in tbs .
both steps were performed at room temperature in a dark humid chamber . finally , to ascertain structural details sections were counterstained and mounted with dapi diluted in glycerol medium ( 10 ng / ml ) .
triple fluorescence was detected using a leica dmr - xa automatic fluorescence microscope ( photonic microscopy service , university of oviedo ) coupled with a leica confocal software , ver .
2.5 ( leica microsystems gmbh , heidelberg , germany ) and the images captured were processed using the software image j version 1.43 g master biophotonics facility , mac master university ontario ( www.macbiophotonics.ca ) . for control purposes
representative sections were processed in the same way as described above using non - immune rabbit or mouse sera instead of the primary antibodies , or omitting the primary antibodies in the incubation . under these conditions
samples of gingival tissues ( sized 2x2x2 mm , approximately ) were surgically excised from healthy patients undergoing orthodontic treatment from zones not exposed to orthodontic forces , and were obtained from the instituto asturiano de odontologia .
the age range was 16 to 32 years , and were males ( n=8 ) and females ( n=4 ) .
the tissue samples were washed with tap water followed with cold saline , then fixed in buffered 10% formalin for 24 h , and routinely processed for paraffin embedding .
the pieces were cut 10 m thick and the sections mounted on gelatine - coated microscope slides . moreover , fresh samples ( n=6 from different subject ) were quickly frozen , stored at -80c and used for western blot .
this study was approved by the ethics committee of instituto asturiano de odontologia ( oviedo , spain ) and informed consent was obtained from each subject .
lysates prepared from gingival homogenates were processed as follows : representative samples of the free gingival , attached gingival and non - bone attached periodontal ligament were pooled and homogenized ( 1:2 , w / v ) in tris - hcl buffered saline ( tbs , 0.1 m , ph 7.5 ) containing 1 m leupeptin , 10 m pepstatin and 2 mm phenylmethylsulfonyl fluoride .
the homogenates were centrifuged at 25,000 rpm for 15 min at 4c and the resulting pellet dissolved in tris hcl 10 mm ph 6.8 , 2% sds , 100 mm dithiothreitol , and 10% glycerol at 4c .
after electrophoresis , proteins were transferred to a nitrocellulose membrane and antibody non - specific binding was blocked by immersion for 3 h in pbs containing 5% dry milk , and 0.1% tween-20 .
the membranes were then incubated at 4c for 2h with a rabbit polyclonal antibody against a peptide from fasciclin domain 1 of mouse periostin ( ls - bl10443 , lifespan biosciences , inc . , seattle , wa , usa ) , used diluted 1:200 .
after incubation , the membranes were washed with tbs ph 7.6 containing 20% tween-20 , and incubated again for 1 h with goat anti - rabbit igg ( diluted 1:100 ) at room temperature .
membranes were washed again and incubated with the pap complex diluted 1:100 for 1 h at room temperature . finally , the reaction was developed using a chemiluminescent reagent ( ecl , amersham pharmacia biotech , buckinghamshire , uk ) and exposed to hyperfilm .
relative values of periostin were estimated by imagej quantification in the three different segments of the gingiva .
deparaffinized and rehydrated sections were processed for immunohistochemical detection of periostin using the envision antibody complex kit ( dako , copenhagen , denmark ) following the manufacturer s recommendations .
the anti - periostin antibody was the same described above . moreover , anti - vimentin ( clone 334 , boehringer - mannheim , mannheim , germany ) , anti - pancytokeratin ( clone pck-26 , sigma - aldrich quimica , sl , madrid , spain ) mouse monoclonal antibodies , and anti - collagen type i rabbit monoclonal antibody ( erp778 , abcam , cambridge , uk ) were used to label dermal fibroblasts , epithelial cells and type i collagen fibrils , respectively .
the sections were processed for simultaneous detection of periostin and vimentin , and periostin and pan - cytokeratin , as follows : the non - specific binding was reduced by incubation for 30 min with a solution of 1% bovine serum albumin in tbs .
the sections were then incubated overnight , at 4c in a humid chamber with a 1:1 mixture of anti - periostin and anti - vimentin antibodies ( both diluted 1:100 in the blocking solution ) ; or anti - periostin and anti - pancytokeratin antibodies ( diluted 1:200 and 1:100 , respectively , in the blocking solution ) ; or anti - vimentin and anti - type i collagen ( both diluted 1:100 in the blocking solution ) . after rinsing with tbs
, the sections were incubated for 1 hour with alexa fluor 488-conjugated goat anti - rabbit igg ( serotec , oxford , uk ) , diluted 1:1000 in tbs containing 5% mouse serum ( serotec ) , then rinsed again , and incubated for another hour with cy3-conjugated donkey anti - mouse antibody ( jackson - immunoresearch , baltimore , md , usa ) diluted 1:50 in tbs .
finally , to ascertain structural details sections were counterstained and mounted with dapi diluted in glycerol medium ( 10 ng / ml ) .
triple fluorescence was detected using a leica dmr - xa automatic fluorescence microscope ( photonic microscopy service , university of oviedo ) coupled with a leica confocal software , ver .
2.5 ( leica microsystems gmbh , heidelberg , germany ) and the images captured were processed using the software image j version 1.43 g master biophotonics facility , mac master university ontario ( www.macbiophotonics.ca ) . for control purposes
representative sections were processed in the same way as described above using non - immune rabbit or mouse sera instead of the primary antibodies , or omitting the primary antibodies in the incubation . under these conditions
the expression of periostin at the protein level in the human gingiva was analyzed using western blot and immunohistochemistry . in gingival homogenates western
blot associated to the anti - periostin antibody used throughout this study detected a single protein band with an estimated molecular weight of 94 kda ( figure 1a ) which is consistent with than expected for the human periostin .
imagej densitometric analysis demonstrated that the higher relative levels of periostin with respect to -actin corresponded to the non - bone attached periodontal ligament segment , followed by free gingiva and attached gingiva ( figure 1b ) . to map the histological distribution of periostin in the human gingiva we used double immunofluorescence coupled with laser - confocal microscopy in three different sectors : the free gingiva , the attached gingiva , and the segment of connective tissue related to the junctional epithelium ( which is especially rich in fibers of the periodontal ligament ) .
the results were compared with the pattern of localization of cytokeratin and vimentin . as a rule periostin
was never localized within the cells but always in the extracellular space . in the sections processed for the simultaneous demonstration of periostin plus cytokeratin ( figure 1 c , d , g , h )
it was observed that periostin - immunoreactivity is concentrated at the epithelium - connective tissue junction , presumably associated to ecm proteins of the basal membrane and was never found within the cytoplasm of the basal epithelial cells . on the other hand ,
the distribution of the immunoreactivity for periostin and type i collagen were similar at the epithelium - connective tissue junction ( figure 1 e , f ) although a faint widespread immunostaining for type i collagen was also detected in the connective tissue .
double immunofluorescence was also carried out for periostin and vimentin in order to determine the relation of periostin with the connective tissue fibroblasts .
independently of the gingival segment analyzed , free ( figure 2 a - c ) or attached gingival ( figure 2 d - f ) , periostin immunoreactivity was dissociated from the vimentin positive fibroblast and was restricted to the epithelial - connective tissue junction .
regarding to the gingival segment containing the non - bone attached periodontal ligament , disposed under the junctional epithelium , the distribution of the periostin immunoreactivity was irregular , and never co - localized with the vimentin - positive fibroblasts ( figure 2 g - i ) .
although some images occasionally might suggest co - localization of periositin witn vimentin or cytokeratin 2d cytofluorograms from the two detection channels of periostin ( green ) and cytokeratin or vimentin ( red ) demonstrated absence of co - localization ( figure 2 j - l ) . as a summary , in human normal gingiva periostin is extracellular and is restricted to the epithelial - connective tissue junction , and among the fibroblasts forming the non - bone attached segments of the periodontal ligament .
this study was designed to investigate the occurrence and distribution of the segregated matricellular protein periostin in the human gingiva .
the localization of periostin in the dental tissues and the periodontal ligament in now rather well known , but no information is available about its distribution in the gingiva in spite of its potential role in the pathophysiology of some gingival disorders . the protein we have identified by western blot has a molecular weight of 94 kda .
this is in good agreement with the expected molecular mass of this protein ( 93.3 kda ) , and was similar to that found in other human epithelial tissues like the cornea .
the small differences between our results and others could be related to occurrence of tissue specific periostin isoforms , or post - translational processes of the protein .
based on image densitometry the relative level of periostin varies among different segments of the gingiva .
the reason for these differences might be related to the density of cells producing periostin , and the functional significance of these findings if any remain to be elucidated .
we have regularly observed that periostin in normal gingiva is restricted to the epithelium - connective tissue junction , and is also present around the fibers of the non - bone attached portion of the periodontal ligament .
as far as we know this is the first study reporting the distribution of periostin in the human gingiva . in agreement with the identification of periostin as a matricellular protein we observed that it is localized extracellularly
nevertheless , other studies on epithelial tissues reported the presence of periostin immunoreactivity in the basal keratinocytes , basal lamina , and dermal fibroblasts in healthy human skin , or in the basal layer cells of the human corneal epithelium . according to those authors
periostin only becomes extracellular during tissue remodeling of wound repair . since in normal gingiva periostin
is exclusively extracellular , its function may be distinct from that of skin , and the presence of intracellular periostin probably reflects non - secreted protein .
the role of periostin in the interactions of the connective tissue and the epithelial cells has been extensively studied , however it remains still unknown in the gingiva . in the skin periostin plays pivotal functions in collagen fibrillogenesis , collagen cross - linking , and the formation of ecm meshwork via interactions with other ecm components .
whether or not it is the same for the gingiva remains to be investigated . in the skin periostin expression
this is indicative of the physiologically protective functions of periostin , which promotes wound repair producing myofibroblast differentiation , keratinocytes proliferation and fibroblast proliferation and migration .
the localization of periostin in the gingiva and the since it is an ecm secreted protein suggest it could signal in both epithelial cells .
matricellular proteins appear to be of importance in collagen assembly , and the expression of periostin in the epithelial - connective tissue junction suggest it may influence the biology of the basal membrane , including collagen fibrillogenesis ( especially type i collagen which co - localizes ) but probably its presence may be also related to cell migration , proliferation and adhesion of fibroblasts . on the other hand ,
the free and sub - junctional epithelium segments of the gingival contain fibers of the periodontal ligament subject to mechanical stress which in turn may activate latent tgf- 1 and to increase periostin mrna in periodontal ligament fibroblasts .
therefore , periostin may participate in the maintenance of the structure and reparative processes in adult human gingival as it occurs in the skin , although in these tissues have different localization , extracellular vs intracellular . as a summary
, our study has demonstrated the localization of periostin in the ecm of the periodontal ligament and the cell - free zone of the dental pulp in adult human teeth .
 the periostin is a matricellular protein expressed in collagen - rich tissues including some dental and periodontal tissues where it is regulated by mechanical forces , growth factors and cytokines . 
 interestingly the expression of this protein has been found modified in different gingival pathologies although the expression of periostin in normal human gingiva was never investigated . 
 here we used western blot and double immunofluorescence coupled to laser - confocal microscopy to investigated the occurrence and distribution of periostin in different segments of the human gingival in healthy subjects . by western blot a protein band with an estimated molecular mass of 94 kda was observed . 
 periostin was localized at the epithelial - connective tissue junction , or among the fibers of the periodontal ligament , and never co - localized with cytokeratin or vimentin thus suggesting it is an extracellular protein . 
 these results demonstrate the occurrence of periostin in adult human gingiva ; its localization suggests a role in the bidirectional interactions between the connective tissue and the epithelial cells , and therefore in the physiopathological conditions in which these interactions are altered . 