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the liver is a vital metabolic organ and is the first of many locations for the development of cancer metastases due to dual feeding by the hepatic artery and portal vein .
hepatic resection is a safe method for some benign and malignant diseases , and the mortality rate of liver resection has decreased to < 5% due to advances in technology , surgical techniques , and a better understanding of hepatic physiology and anatomy . in particular , liver resection for colorectal cancer metastasis affects survival positively .
in fact , some patients have a potentially curative treatment . however , liver failure is one of the most common complications of liver resection surgery and the main cause of morbidity and mortality [ 35 ] .
diabetes mellitus , obesity , steatohepatitis , hepatitis b and c , malnutrition , renal failure , hyperbilirubinemia , thrombocytopenia , pulmonary disease , liver cirrhosis , and age > 65 years which are factors predictive of postoperative liver failure .
operative factors include a blood loss > 1200  ml during surgery , blood transfusion requirement , resection of the vena cava or other vessels , operative time > 240  min , resected liver volume > 50% , right lobe resection with major hepatectomy , a skeletonized hepatoduodenal ligament , and remnant liver < 25% of the liver mass .
portal vein embolization and two - stage hepatectomy methods are currently applied successfully to prevent postoperative liver failure
. however , estimated postresection liver volume is one of the most important factors limiting resection . a rapid regeneration method after hepatectomy could expand the indications for liver resection in selected patients .
the human amniotic membrane ( ham ) is currently used to treat corneal wounds in patients with burns injuries and intestinal anastomosis , as it has a positive healing effect [ 7 , 8 ] .
the ham also has hypoimmunogenic properties and has been used as a xenograft without tissue rejection [ 7 , 9 ] . because of this feature in many animal experiments study , human amniotic membrane was successfully used as xenograft and allograft [ 7 , 8 , 10 ] . in the present study , we investigated the effects of overlaying the ham on remnant liver and on its regeneration after liver resection .
this study was performed at the kocaeli university experimental animal research center with approval ( number = 7/2  2013 ) from the kocaeli university institutional ethics committee .
twenty female wistar rats ( weight range , 212275  g ; same age ) were used in this study .
the rats were maintained under a 12-h light : dark photoperiod and normal laboratory conditions .
all surgical procedures were performed in the afternoon to prevent an influence of the circadian rhythm .
the rats were divided randomly into control ( n = 10 ) and experimental groups ( n = 10 ) .
written consent was obtained from pregnant women who were negative for hepatitis b and c as well as human immunodeficiency virus .
the placenta was transported quickly after cesarean section to the laboratory in a sterile box with saline .
a 20  ml sterile syringe was immersed in the saline and used to separate the membrane with air .
the ham was dissected while immersed in dakin 's solution for 15  min to clean off the blood and other contaminants .
the ham was incubated with saline containing 50  mg / ml penicillin , 50  mg / ml streptomycin , 100  g / ml neomycin , and 2.5  mg / ml amphotericin b for 10  min .
the ham was sectioned into 1.5-  3-cm pieces and maintained between wet sterile cloths impregnated with saline .
anesthesia was induced by intraperitoneal administration of 50  mg / kg ketamine ( ketalar ; parke davis , istanbul , turkey ) and 5  mg / kg xylazine hydrochloride ( rompun ; bayer ag , leverkusen , germany ) .
the abdominal area was disinfected with 10% povidone - iodine solution , and a midline abdominal incision was made under sterile conditions .
the left lateral and medial lobes of the liver were cut , and a 70% hepatectomy was performed based on the method described by higgis [ 11 , 12 ] .
after the hepatectomy , the 1.5-  3-cm sections of ham were overlaid onto the remnant liver in the experiential group .
approximately 72  h later , the rats were anesthetized using the same method , and their weights ( g ) were measured .
blood ( 2  ml ) was drawn from the heart prior to excision of the remnant liver .
the remnant liver weight ( mg ) was measured , and a specimen was maintained in 10% formaldehyde for pathological examination .
the blood samples were centrifuged for 5  min at 3500  rpm , and the serum fraction was transferred to a separate tube for biochemical analyses .
preoperative liver weight was considered to be 3.4% of the preoperative body weight and calculated .
residual liver weight was calculated as the excised liver weight subtracted from the preoperative liver weight .
regenerative liver weights were calculated by subtracting the residual liver weight from the remnant liver weight during necropsy .
relative liver weight was calculated as the regenerative liver weight divided by the preoperative liver weight multiplied by 100 .
blood samples were collected from the heart , and the serum was separated by centrifugation ( 3000  rpm at 4c for 15  min ) and stored at 80c for biochemical analyses .
serum total protein ( t prot ) g / dl , total bilirubin ( t bil ) mg / dl , direct bilirubin ( d bil ) mg / dl , indirect bilirubin ( i bil ) mg / dl , and albumin ( alb ) g / dl levels were determined using an automated architect c-4000 analyzer ( abbott laboratories , abbott park , il , usa ) .
aspartate aminotransferase ( ast ) iu / l , alanine aminotransferase ( alt ) iu / l , and lactate dehydrogenase ( ldh ) iu / l levels were measured using an architect c-8000 autoanalyzer ( abbott laboratories ) at bozok university , school of medicine , central laboratory , yozgat , turkey .
all specimens were transported to the pathology laboratory in 10% neutral formalin . approximately 1-mm tissue pieces were sampled from each case .
all samples were processed in an automated system ( excelsior es ; thermo scientific , rockford , il , usa ) , and paraffin blocks were prepared using the histostar embedding station ( thermo scientific ) .
two tissue sections 36-m - thick were obtained from each case using a microtome ( shandon - finesse me+ ; thermo scientific ) .
one was stained with hematoxylin and eosin ( h&e ) using an automated slide staining machine ( varistain gemini ; thermo scientific ) , and the other one was stained using a ki-67 antibody ( 7  ml - rtu , mouse anti - human monoclonal antibody clone k2 ; leica biosystems , danvers , ma , usa ) in an automated immunohistochemical staining system ( leica - bond max , leica biosystems ) .
the h&e- and ki-67-stained slides were evaluated under a light microscope ( bx53f ; olympus , tokyo , japan ) by a pathologist .
the number of ki-67-stained cells was counted in three different areas containing most of the ki-67 positive cells using a 100 objective with immersion oil .
all h&e sections were evaluated for mitotic figures in 10 high power fields ( 40 objective ) and for histopathological changes .
the mann - whitney u test was used for normally distributed data of weight loss , mitotic count , and levels of ast , ldh , total and direct bilirubin , and c - reactive protein .
ki-67 expression , relative liver weight , alt , and total protein values were in the normal range .
two rats in the control group died during the surgical procedure due to vena cava injuries .
direct bilirubin value was > 7  mg / dl due to an iatrogenic bile duct obstruction in one rat in the experimental group , and the rat was excluded .
thus , nine rats were included in the experimental group and eight in the control group .
mean preoperative body weights in the experimental and control group were 246.33 and 256.63  g , and postoperative weights were 221.11 and 221.00  g , respectively .
weight loss was calculated to be 31.38  g. no difference was detected between the groups in terms of weight loss ( p = 0.277 ) .
but , in the study group , they were like fatty liver disease ( figure 1 ) .
the mean resected liver weight was 5.41  0.36  g in the experimental group and 5.82  0.43  g in the control group .
consequently , a mean of 64.83% of the liver tissue was excised in the experimental group and 66.76% in the control group ( p = 0.103 ) .
serum total protein and albumin levels were higher in the control group than in the experimental group ( p < 0.01 ) .
no significant differences were observed in the ast , alt , ldh , and total and direct bilirubin levels between the groups .
the mean ki-67 level in the postoperative remnant liver tissue was 54.67  8.99 in the experimental group and 51.75  9.00 in the control group ( p = 0.515 ) ( figure 2 ) .
relative liver weight tended to be higher in the experimental group ( 29.61  5.60  g ) than in the control group ( 27.34  3.77  g ) ( p = 0.348 ) .
in contrast , the mitotic index in the remnant liver tissue was 4.22  2.91 in the experimental group and 0.50  1.07 in the control group ( p < 0.01 ) ( table 2 , figure 3 ) .
histopathologically , there was no specific change , tumor cell , and significant difference in inflammatory cells in the liver of study and control groups .
however , macrovesicular steatosis with large and small droplets was detected in all postoperative remnant liver tissues .
thus , severe hepatic macrovesicular steatosis was observed in residual liver tissue from the experimental group compared with the control group ( figure 4 ) .
the liver is capable of regenerating and can reach its previous size and function quickly .
thus , hepatic resections are often completed easily without complications , although posthepatectomy liver failure may arise in cases of massive hepatic resection .
the liver regeneration process in rats and mice is completed within 57 days after surgery .
the incidence rate of liver failure is reported to be 10% in patients undergoing major liver resection .
posthepatectomized liver failure is a major cause of mortality and morbidity in patients undergoing liver resection . according to shoup et al .
, posthepatectomy liver failure results in a threefold increased risk of death in patients with a postresection volume <
25% of the preoperative liver volume . methods such as portal vein embolization and two - stage hepatectomy have been used successfully to increase residual volume .
techniques that enable rapid regeneration of residual liver volume can be effective for preventing posthepatectomy failure and expand resection indications .
intercellular communication , growth factors , and cytokines , particularly hepatocyte growth factor , transforming growth factor- , epidermal growth factor , tumor necrosis factor , and interleukin-6 , play important roles in liver regeneration .
some studies have shown that these factors accelerate regeneration and growth of the liver mass [ 19 , 20 ] .
ham has the potential for cells to differentiate into hepatocytes , cardiomyocytes , chondrocytes , pancreatic b - cells , and neuronal cells .
laminin is a major component of the ham that plays an important role in cell differentiation and shaping . .
resected 70% of the rat liver and showed a positive effect on liver regeneration by placing ham on the abdominal wall muscles .
we found no difference in ast , alt , ldh , or total and direct bilirubin levels between the groups in our study .
total protein and albumin levels were significantly lower in the experimental group than in the control group .
no differences in relative liver weights or ki-67 levels were detected between the groups on postoperative day 3 .
based on these findings , we concluded that applying the ham was ineffective for enhancing liver regeneration and could lead to liver cirrhosis .
previous studies used the ham as a xenograft without tissue rejection reactions in organs such as the intestines , skin , and cornea [ 7 , 8 , 21 ] .
it has low levels of hla class ia and high levels of class ib and hla - g [ 9 , 22 ] .
jia et al . showed that placing ham in the abdominal muscles increased the rate of liver regeneration .
we believe that our negative results were caused by immune response mechanisms , cytokines , and cell interactions . however , the evaluation parameters were inadequate in our study .
the number of mitoses was higher in the experimental group than in the control group , which was a sign of increased liver regeneration induced by the ham .
however , the increase in mitosis was not reflected by an increase in relative liver weight .
spreading the ham on rat liver negatively affected liver regeneration . nevertheless , stem cell research , such as studies using the ham , continues to provide hope for postoperative liver regeneration .
introduction . operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy / physiology and developments in operation technology . 
 surgery can cure some patients with liver metastasis of some tumors . nevertheless , postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass . 
 the human amniotic membrane has regenerative effects . 
 thus , we investigated the effects of the human amniotic membrane on regeneration of the resected liver . methods . 
 twenty female wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy . 
 the human amniotic membrane was placed over the residual liver in the experimental group . 
 relative liver weight , histopathological features , and biochemical parameters were assessed on postoperative day 3 . 
 results . 
 total protein and albumin levels were significantly lower in the experimental group than in the control group . 
 no difference in relative liver weight was observed between the groups . 
 hepatocyte mitotic count was significantly higher in the experimental group than in the control group . 
 hepatic steatosis was detected in the experimental group . 
 conclusion . applying the amniotic membrane to residual 
 liver adversely affected liver regeneration . 
 however , mesenchymal stem cell research has the potential to accelerate liver regeneration investigations .