general anaesthesia was achieved by an intraperitoneal injection of 0.1% xylazine ( 13  mg / kg ) and 0.1% ketamine ( 87  mg / kg ) .
a midcrestal incision was made on the maxillary alveolar ridge in the existing edentulous area between the 1st molar and the incisors .
a fullthickness flap was raised on each side of the incision and then repositioned and sutured with resorbable vycril 40 sutures ( ethicon inc . , johnson & johnson , nj , usa ) .
eight experimental groups were established : study groups : periopatch was placed on the incision line immediately following surgery and was replaced every 12  h for the following 3  days .
placebo patch control groups : protocol was similar as in the study groups only that a placebo patch ( similar in composition to the periopatch , but without the active herbal ingredients ) was applied . neither study nor placebo patches were present at the time of the new patch application .
the type of patch was coded ( a or b ) and was not available to the researchers until the results were available , therefore , the study was designed as blind . to minimize the number of animals without compromising results , each hemimaxilla served as a sample in a group .
a single type of patch was applied only on one side of each animal while in half of them , the contralateral side served as negative control and in the remaining as intact group .
half of the animals were killed after 5  days and the remaining ones after 12  days .
initially , each one of the eight groups consisted of 12 sites , therefore , altogether , a total of 96 samples were available .
animals were killed under anaesthesia using co2 and the maxillae were excised and fixed in 4% paraformaldehyde for 7  days at room temperature .
after decalcification in a 10% edta solution for 30  days , the specimens were embedded in paraffin and cut into serial sections of 4  m thickness in a coronal plane , perpendicular to the line of incision .
conventional histology ( haematoxylin & eosin ) for measurement of epithelial gap distance : slides were stained with haematoxylin & eosin with the standard methods .
epithelial gap was measured under a 40 magnification using an olympus bh2 ( olympus america inc , new york , usa ) light microscope .
a gap zone was defined by lack of wound tissue and its width was measured at three epithelial heights within the incision line : a. deepest ( close to the connective tissue ) , b. midway between the epithelial surfaces and c. most superficial portion of the epithelial wound .
a mean of all measurements in two sections was computed . the mean  standard deviation ( sd ) for each of the eight groups was then calculated .
quantitation of collagen fibre content with picrosirius red ( psr ) stain under polarized light microscopy : adjacent sections were stained with picrosirius red solution ( scytek kit srs250x ( scytek laboratories inc . , logan , usa ) . using polarized light microscopy and a 200 objective ,
the wound site was photographed and the area of the wound occupied by collagen fibres was determined as percent of the total area of the connective tissue .
total connective tissue pixels adjacent to incision site were calculated as:[total photomicrograph pixels][epithelial pixels+alveolar crest pixels ] 
  the percentage of collagen in connective tissue was calculated as : connective tissue collagen fibres pixels ( red , yellow , green)*100/total connective tissue pixels . 

macrophages stained with cd68 were counted and expressed as number of cells / mm of gingival connective tissue adjacent to the incision site .
the number of ki67 positive cells was determined in the connective tissue below the epithelial area of interest for all eight groups .
cells stained for ki67 were counted and expressed as number of cells / mm of gingival connective tissue adjacent to the incision site .
the number of blood vessels was expressed per mm of gingival connective tissue adjacent to the incision site .
statistical analysis was performed using ibm spss statistics ( armonk , new york , u.s . ) .
a twoway anova analysis was performed to examine changes between groups for each dependent parameter .
animals were killed under anaesthesia using co2 and the maxillae were excised and fixed in 4% paraformaldehyde for 7  days at room temperature .
after decalcification in a 10% edta solution for 30  days , the specimens were embedded in paraffin and cut into serial sections of 4  m thickness in a coronal plane , perpendicular to the line of incision .
conventional histology ( haematoxylin & eosin ) for measurement of epithelial gap distance : slides were stained with haematoxylin & eosin with the standard methods .
epithelial gap was measured under a 40 magnification using an olympus bh2 ( olympus america inc , new york , usa ) light microscope .
a gap zone was defined by lack of wound tissue and its width was measured at three epithelial heights within the incision line : a. deepest ( close to the connective tissue ) , b. midway between the epithelial surfaces and c. most superficial portion of the epithelial wound . a mean of all measurements in two sections was computed . the mean  standard deviation ( sd ) for each of the eight groups
quantitation of collagen fibre content with picrosirius red ( psr ) stain under polarized light microscopy : adjacent sections were stained with picrosirius red solution ( scytek kit srs250x ( scytek laboratories inc . , logan , usa ) . using polarized light microscopy and a 200 objective ,
the wound site was photographed and the area of the wound occupied by collagen fibres was determined as percent of the total area of the connective tissue .
total connective tissue pixels adjacent to incision site were calculated as:[total photomicrograph pixels][epithelial pixels+alveolar crest pixels ] 
  the percentage of collagen in connective tissue was calculated as : connective tissue collagen fibres pixels ( red , yellow , green)*100/total connective tissue pixels . 

macrophages stained with cd68 were counted and expressed as number of cells / mm of gingival connective tissue adjacent to the incision site .
the number of ki67 positive cells was determined in the connective tissue below the epithelial area of interest for all eight groups .
cells stained for ki67 were counted and expressed as number of cells / mm of gingival connective tissue adjacent to the incision site .
the number of blood vessels was expressed per mm of gingival connective tissue adjacent to the incision site .
statistical analysis was performed using ibm spss statistics ( armonk , new york , u.s . ) .
a twoway anova analysis was performed to examine changes between groups for each dependent parameter .
clinical healing was not evaluated , however , in most animals ; partial soft tissue healing could be appreciated by 5  days , whereas it was complete and uneventful by the end of the study at day 12 .
five days after the surgical procedure , the smallest epithelial gap was found in the study group . however , the difference between the three groups where surgery was performed , was not statistically significant ( p  =  0.701 ) .
in contrast , on day 12 , the epithelial gap was significantly smaller in the study group compared to the placebo group ( p  =  0.073 ) and to the negative control group ( p  =  0.056 ) ( figs  1 , 2 , 3 ) .
s = study group ( pp ) ; p , placebo patch group ; nc , negative control ( no patch ) group . # ,
p  =  0.07 ( versus the respective study group ) . * , p  =  0.05 ( versus the respective study group ) .
( a ) alveolar crest , ( b ) gingival connective tissue,(c ) epithelium and incision site ( arrow ) .
( a ) alveolar crest , ( b ) gingival connective tissue , ( c ) epithelium and incision site ( arrow ) .
a statistically significant higher collagen content was found in the study group both at 5 and 12  days after the surgical procedure ( p  <  0.001 ) ( fig . 
connective tissue collagen content ( % , mean and sd ) in the various experimental groups .
s , study group ( pp ) ; p , placebo patch group ; nc , negative control ( no patch ) group .
i , intact ( no surgery ) group . * * , p  <  0.001 ( versus the respective study group ) .
although macrophage counts were highest in the study group both at 5 and 12  days , differences between groups were not statistically significant ( fig . 
number of connective tissue macrophages ( cd68 positive cells ) ( /mm , mean and sd ) in the various experimental groups .
s , study group ( pp ) ; p , placebo patch group ; nc , negative control ( no patch ) group .
. a larger number of proliferating cells was found in the study group . at 5  days , the effects of the type of patch approached statistical significance ( p  =  0.054 ) ( fig . 
6  +  figures s4a , s4b ) . on day 12 , although the mean number of proliferating cells was highest in the study group , differences between groups were not statistically significant .
number of proliferating ( ki67 positive ) cells ( /mm , mean and sd ) in the various experimental groups .
s , study group ( pp ) ; p , placebo patch group ; nc , negative control ( no patch ) group .
the mean number of blood vessels was highest in the study group , however , at 5  days , differences were not statistically significant .
( p  =  0.354 ) , reaching significance only at 12  days ( p  =  0.014 ) ( fig . 
number of blood vessels ( tgii positive ) ( /mm , mean and sd ) in the various experimental groups .
s , study group ( pp ) ; p , placebo patch group ; nc , negative control ( no patch ) group .
i , intact ( no surgery ) group . * * , p  =  0.01 ( versus the respective study group ) .
our results showed that application of the topical herbal patch improved gingival healing compared to control and the placebo patch , thus , suggesting that the active herbal ingredients ( centella asiatica ,  echinacea purpurea and  sambucus nigra ) play an important role in this effect .
asiaticoside and madecassoside , found in centella asiatica , promote fibroblast proliferation and extracellular matrix synthesis in wound healing ( lu et  al . 2004 ) , alleviate infiltration of inflammatory cells , enhance epithelialization resulting from fibroblasts proliferation and promote angiogenesis ( liu et  al .
sambucus nigra extract was found to potently inhibit  proinflammatory activities ( harokopakis et  al .
certain components in echinacea species display remarkable wound healing and antiinflammatory activities ( tumen et  al .
degree of epithelialization , number of blood vessels , proliferating cells and collagen deposition are the most generally evaluated parameters to demonstrate positive effects on wound healing .
an animal study ( ganjali et  al . 2013 ) investigated if the methanolic extract of the otostegia persica can accelerate the healing process of burn wound because of its antiinflammatory and antioxidant effects .
it was concluded that methanolic extract of otostegia persica exhibited significant healing activity when topically applied on rats . in another example , glucan exerted various positive effects in burn wound healing , including immunomodulatory effects , antioxidant effects ( freeradical scavenging activity ) and effects associated with the reduction in the inflammatory response ( firat et  al .
glucan application led to higher fibroblast proliferation , angiogenesis and reepithelialization on the 7th day .
in particular , reepithelialization on the 21st day was significantly better where glucan was administered .
in yet another study , topical treatment with curcumin ( cur ) on burn wound healing in rats showed positive effects ( kulac et  al .
other studies have also reported that curtreated wounds were found to heal much faster as indicated by improved rates of inflammatory cells , collagen deposition , angiogenesis , granulation tissue formation and epithelialization which were also confirmed by histopathological and biochemical examinations ( akbik et  al .
previous controlled clinical studies have proven that periopatch application is useful to reduce gingival inflammation following periodontal nonsurgical treatment ( grbic et  al .
2011 , samuels et  al . 2012 ) and that its application , following scaling and root planing and oral hygiene instructions , together with a mouth rinse containing the herbal active ingredients may decrease gingival recession and gingival index scores with increased gingival thickness ( levine et  al .
this study evaluated , for the first time , the effects of this device on the various processes essential for soft tissue surgical wound healing : reepithelialization , inflammation , angiogenesis cellular proliferation and matrix deposition .
our results showed that the topical herbal patch application enhanced the epithelial bridging and improved healing .
although , the effect was noted already at day 5 , it only approached statistical significance on day 12 .
however , the placebo patch , which provided only mechanical protection , did not exert a positive healing effect .
one may speculate that the study model was based on a surgical wound which is clean , therefore , resulting mainly in a reparative process with minimal inflammation .
periopatch application resulted in an improved healing as evaluated by an increased number of proliferating cells .
this effect only approached statistical significance by day 5 ; however , by day 12 results were statistically similar in the different groups , coinciding with the pattern of increased cellular proliferation during the healing process . increased collagen fibre quantity was appreciated in the study groups which was statistically significant both at 5 and 12  days
blood vessels were significantly more numerous in the study group compared to placebo patch group at day 12 .
effective wound healing requires a highly organized series of events that comprise inflammation , reepithelialization , keratinocyte and fibroblast proliferation , matrix deposition , angiogenesis and wound contraction .
together , these processes result in the restoration of tissue integrity and functional healing . the clinical investigation in search for therapeutic tools to improve the wound healing process has led to the development of novel treatment strategies ( hackam & ford 2002 ) among which this herbal topical patch may be suitable for some clinical applications .
in this preclinical , animal study , application of the periopatch and not the placebo patch improved wound healing in the described model .
the positive effects on soft tissue wound healing were also appreciated by increased number of proliferating cells , collagen production and number of blood vessels .
midcrestal incision on the maxillary alveolar ridge in the existing edentulous area between the 1st molar and the incisors .
following fullthickness flaps raised and repositioned on each side of the incision , resorbable sutures were performed to replace the soft tissues .
periopatch or placebo patch was placed on the incision line immediately following surgery and replaced every 12  h for the following 3  days .
 abstractaimthis study evaluated the effects of a topical herbal patch ( periopatch ) for gingival wound healing in a rat model.materials and methodsa midcrestal incision was performed on each side of the edentulous anterior maxilla in 48 , 6monthold , wistar rats . 
 fullthickness flaps were raised , repositioned and sutured . 
 four experimental groups were established : herbal patch , placebo patch , no patch and no patch and no surgery . 
 patches were placed immediately after surgery and replaced every 12  h for the following 3  days . 
 half of the animals were killed after 5 and the remaining ones after 12  days . 
 tissue blocks were retrieved and processed for histological and immunohistochemical evaluation . 
 epithelial gap , collagen contents , amount of macrophages , cellular proliferation and vascular contents were evaluated in the central incision area . 
 statistical analysis consisted of twoway anova.resultsthe herbal patch group presented the smallest epithelial gap at 12  days , the highest collagen content both at 5 and 12  days , a larger number of proliferating cells at day 5 and more numerous blood vessels at day 12 . 
 macrophage number was similar in all groups.conclusionherbal patch improved wound healing in this animal model . 
Materials and methods
Tissue harvesting
Histological evaluation
Statistical analysis
Results
Discussion
Conclusions
Supporting information