this study was approved by uludag university animal experiments local ethics committee and was carried out on 20 new zealand white rabbits , aged 3 months , weighing between 2.5 and 3.0 kg , obtained from the experimental animal breeding and research centre with the permission of the deanery of uludag university medical faculty .
the rabbits were randomly allocated into two groups of 10 as the immunonutrition group and the control group .
anaesthesia of 5 mg / kg xylazine and 35 mg / kg ketamine was administered intramuscularly to both groups .
after disinfection of the fibular area with 10% free alcohol containing 10 g povidone iodine , a 12 cm skin incision was made approximately 1 cm to the posterior of the fibula . by subcutaneous dissection , continuing to slide the skin over the fibula , the fibula shaft was reached .
an oblique fracture was created using tissue scissors on the fibula corpus ( figs . 1 and 2 ) .
povidone iodine was applied with a dressing , then transparent film dressing spray , leaving the wound uncovered . from the post - operative first day for 30 days , the immunonutrition group were given standard rabbit food and water with the addition of 2 ml / kg / day 20% l - alanine and l - glutamine solution via a gastric catheter .
the control group received 2 ml / kg / day isotonic via gastric catheter in addition to standard rabbit food and water .
all the rabbits were sacrificed at the end of 30 days . by disarticulating the lower extremity where the fracture had been made , from the hip joint ,
the fractures were examined clinically , radiologically , and histopathologically in respect to the degree of union .
these radiographs were evaluated by five specialist doctors from the orthopaedics and traumatology department and five separate specialist doctors from the radiology department using the lane and sandhu ( 9 ) radiological scoring system to give a numerical value . in this system ,
the radiological findings are scored 04 , where 0=no healing , 1=callus formation , 2=the start of bone union , 3=the fracture line is starting to disappear , and 4=full union .
the fibula union was subjectively evaluated by examination in two planes as described by dimar et al .
this scoring system grades the amount and quality of bone callus tissue through physical movement .
a score of 0 indicates movement in both planes and therefore no union , a score of 1 indicates movement in one plane with moderate union and with a score of 4 , there is no movement and full union .
after the clinical and radiological evaluations , tissue from the prepared samples from the fracture union site were fixed in 10% concentration formalin and stored for 3 days .
the electrolytic method was used for decalcification and accordingly the tissues were kept for 2 weeks in a decalcification solution , and changed every 2 days ( 11 ) .
the tissues were washed for 4 h in running water , then for 1 h in each of the following strengths of alcohol ; 70 , 80 , 90 , 96 , and 100% , kept overnight in xylol , then for 2 h in liquid paraffin before being fixed in paraffin blocks .
the lamina prepared from 5-micron thickness sections cut by rotary microtome were stained with haematoxylin and eosin and examined by light microscope .
whitney u - test was used for comparison of the two independent groups and the wilcoxon test for comparison of dependent groups .
spearman correlation analysis was used in the examination of the relationships between variables . a significance level of p<0.05
the rabbits were randomly allocated into two groups of 10 as the immunonutrition group and the control group .
anaesthesia of 5 mg / kg xylazine and 35 mg / kg ketamine was administered intramuscularly to both groups .
after disinfection of the fibular area with 10% free alcohol containing 10 g povidone iodine , a 12 cm skin incision was made approximately 1 cm to the posterior of the fibula . by subcutaneous dissection , continuing to slide the skin over the fibula , the fibula shaft was reached .
an oblique fracture was created using tissue scissors on the fibula corpus ( figs . 1 and 2 ) .
povidone iodine was applied with a dressing , then transparent film dressing spray , leaving the wound uncovered . from the post - operative first day for 30 days , the immunonutrition group were given standard rabbit food and water with the addition of 2 ml / kg / day 20% l - alanine and l - glutamine solution via a gastric catheter .
the control group received 2 ml / kg / day isotonic via gastric catheter in addition to standard rabbit food and water .
all the rabbits were sacrificed at the end of 30 days . by disarticulating the lower extremity where the fracture had been made , from the hip joint ,
the fractures were examined clinically , radiologically , and histopathologically in respect to the degree of union . anterior 
these radiographs were evaluated by five specialist doctors from the orthopaedics and traumatology department and five separate specialist doctors from the radiology department using the lane and sandhu ( 9 ) radiological scoring system to give a numerical value . in this system ,
the radiological findings are scored 04 , where 0=no healing , 1=callus formation , 2=the start of bone union , 3=the fracture line is starting to disappear , and 4=full union .
the fibula union was subjectively evaluated by examination in two planes as described by dimar et al .
this scoring system grades the amount and quality of bone callus tissue through physical movement .
a score of 0 indicates movement in both planes and therefore no union , a score of 1 indicates movement in one plane with moderate union and with a score of 4 , there is no movement and full union .
after the clinical and radiological evaluations , tissue from the prepared samples from the fracture union site were fixed in 10% concentration formalin and stored for 3 days .
the electrolytic method was used for decalcification and accordingly the tissues were kept for 2 weeks in a decalcification solution , and changed every 2 days ( 11 ) .
the tissues were washed for 4 h in running water , then for 1 h in each of the following strengths of alcohol ; 70 , 80 , 90 , 96 , and 100% , kept overnight in xylol , then for 2 h in liquid paraffin before being fixed in paraffin blocks .
the lamina prepared from 5-micron thickness sections cut by rotary microtome were stained with haematoxylin and eosin and examined by light microscope .
whitney u - test was used for comparison of the two independent groups and the wilcoxon test for comparison of dependent groups .
spearman correlation analysis was used in the examination of the relationships between variables . a significance level of p<0.05
in the first week of the study , two rabbits in the immunonutrition group aspirated the dipeptiven solution given by gastric catheter .
one of the rabbits died within minutes of aspiration and the other on the following day . in the control group , in the first week ,
one rabbit trapped its right lower extremity in the cage while it was being cleaned and sustained a femoral fracture .
a pelvipedal plaster was applied but the rabbit died 3 days later . in the second week , one rabbit in the control group was found dead in its cage of unknown causes .
the study continued with eight rabbits in the immunonutrition group and eight rabbits in the control group . throughout the study no surgical site infection developed in any of the rabbits .
apart from the rabbit that sustained a femoral fracture , no functional problems were observed in any rabbit other than the created fibula fracture .
the radiological evaluation results determined the control group points from the orthopaedic and traumatology specialists to be mean 2.5 ( minimum 0.4maximum 3.6 ) , and from the radiology specialists , mean 2.65 ( minimum 0.6maximum 3.8 ) ( figs . 3 and 4 ) . control group anterior 
the immunonutrition group points from the orthopaedic and traumatology specialists were determined to be mean 3.42 ( minimum 3.0maximum 3.8 ) , and from the radiology specialists , mean 3.35 ( minimum 2.4maximum 4.0 ) ( p=0.13 orthopaedists , p=0.279 radiologists , fig . 5 ) .
an orthopaedic doctor who was not involved in the experimental study made a subjective clinical examination of the relevant disarticulated extremities after removal from the soft tissue . in the clinical evaluation , the control group mean points were 1.875 ( minimum 1.0maximum 2.0 ) , and the immunonutrition group mean points were 2.0 ( minimum 2.0maximum 2.0 ) ( p=0.721 , fig . 6 ) .
the control group mean points were 8.5 ( minimum 8.0maximum 9.0 ) , and the immunonutrition group mean points were 9.0 ( minimum 9.0maximum 9.0 ) ( figs . 
 711 
 ) .
control group stage 8 recovery : image shows immature bone ( ib ) and little cartilage ( c ) tissue ( haemotoxylin and eosin staining 100 magnification ) .
control group stage 8 recovery : image shows immature bone ( ib ) and little cartilage ( c ) tissue ( haemotoxylin and eosin staining 200 magnification ) .
immunonutrition group stage 9 recovery : section from fracture line ( fl ) showing complete immature bone tissue ( ib ) ( haemotoxylin and eosin staining40 magnification ) .
immunonutrition group stage 9 recovery : complete immature bone tissue ( ib ) ( haemotoxylin and eosin staining 100 magnification ) .
the radiological evaluation results determined the control group points from the orthopaedic and traumatology specialists to be mean 2.5 ( minimum 0.4maximum 3.6 ) , and from the radiology specialists , mean 2.65 ( minimum 0.6maximum 3.8 ) ( figs . 3 and 4 ) . control group anterior 
the immunonutrition group points from the orthopaedic and traumatology specialists were determined to be mean 3.42 ( minimum 3.0maximum 3.8 ) , and from the radiology specialists , mean 3.35 ( minimum 2.4maximum 4.0 ) ( p=0.13 orthopaedists , p=0.279 radiologists , fig . 5 ) .
an orthopaedic doctor who was not involved in the experimental study made a subjective clinical examination of the relevant disarticulated extremities after removal from the soft tissue . in the clinical evaluation ,
the control group mean points were 1.875 ( minimum 1.0maximum 2.0 ) , and the immunonutrition group mean points were 2.0 ( minimum 2.0maximum 2.0 ) ( p=0.721 , fig . 6 ) .
following the clinical and radiological examinations , the preparations were histopathologically evaluated according to the criteria described by huo et al .
the control group mean points were 8.5 ( minimum 8.0maximum 9.0 ) , and the immunonutrition group mean points were 9.0 ( minimum 9.0maximum 9.0 ) ( figs . 
 711 
 ) .
control group stage 8 recovery : image shows immature bone ( ib ) and little cartilage ( c ) tissue ( haemotoxylin and eosin staining 100 magnification ) .
control group stage 8 recovery : image shows immature bone ( ib ) and little cartilage ( c ) tissue ( haemotoxylin and eosin staining 200 magnification ) . immunonutrition group stage 9 recovery : section from fracture line ( fl ) showing complete immature bone tissue ( ib ) ( haemotoxylin and eosin staining40 magnification ) .
immunonutrition group stage 9 recovery : complete immature bone tissue ( ib ) ( haemotoxylin and eosin staining 100 magnification ) .
in recent years several scientific studies have focussed on glutamine as a non - essential amino acid as it has been determined to have significant physiological roles .
glutamine facilitates the process of rapid cell reproduction as it is a leading source of both energy and biosynthesis ( 13 , 14 ) . particularly in critical cases such as multiple injuries , sepsis , or burns ,
glutamine is used as an essential amino acid as it undertakes the precursor task for nucleic acids , nucleotides , amino sugars , and proteins ( 3 ) . under catabolic conditions , glutamine takes on the aspects of an amino acid ( 3 , 15 ) . when muscle tissue is exposed to excessive stress , the most important of the amino acids which provide a repository of some amino acids , is glutamine ( 16 ) .
a fall in the level of glutamine is associated with poor clinical results , as starting with a decreased amount in the process of muscle destruction , morbidity and mortality rates may increase .
parenteral or enteral administration of glutamine to critical patients leads to decreased complications associated with sepsis and better functional results ( 17 , 18 ) .
glutamine has the essential properties of an amino acid in respect to affecting lymphocyte proliferation in the most appropriate way by neutrophil and macrophage functions 1922 ) . by eliminating a lack of glutamine
, it is possible to restore nitrogen balance and improve immunosuppression ( 17 ) . to the best of our knowledge
there are no studies on the effect of orally administered glutamine on fracture healing . in this study , taking various beneficial effects into consideration , it was thought that glutamine could make a positive contribution to fracture healing , and in this context , the similarity of wound healing to fracture healing was the guide .
the dipeptide structure formed when glutamine is combined with alanine may remain stable in aqueous solutions .
l - amino acids are more easily absorbed than d - amino acids ( 16 ) .
therefore , in this study an l - alanine l - glutamine solution was used to achieve absorption with active transport of the glutamine and to prevent rapid denaturing . in an experimental study of 50 rabbits with fibula fracture and femoral condyle defects , and orally nourished with essential amino acids containing lactose , fini et al .
although the current study investigated the effect on rabbit fibula fracture of glutamine as a different essential amino acid , both studies showed a similarity in respect to the chosen animal model and the choice of amino acid given . in the fracture model created in the rabbits , cartilage islands started to form on the sixth day of fracture healing , on the 12th day the centre was filled with cartilage tissue , and in the third and fourth week bone tissue replaced the cartilage showing complete union .
thus , in the current study , the experiment was terminated in the fourth week as a sufficient time for fracture healing in rabbits .
although various techniques are used for the radiological evaluation of fracture healing , most of these have been shown to be inadequate as they are not objective and vary from person to person . in some studies ,
the bridging status between the fractured ends has been evaluated from direct radiographs ( 9 , 24 ) . in the current study ,
the widely used lane and sandhu ( 9 ) scoring system was used in the radiological evaluation by more than one orthopaedic , traumatology , and radiology specialist ( 2528 ) .
we are of the opinion that this provides a more objective evaluation . in clinical application , bone union
similarly , in the current study , the presence of bone union was investigated by forced movements to the fractured fibula in two planes , and radiology provided an additional evaluation .
( 10 ) emphasised clinical examination in two planes for stability evaluation and the approach in the current study supports this .
some histopathological evaluations related to fracture healing have given qualitative results rather than numerical values ( 29 , 30 ) .
( 12 ) recommended a more comprehensive method , which was used in the current study . by examination of 10 stages in this system ,
physiological and pathological events in living organisms occur as a result of substances which may have harmful effects .
the most important of these are free oxygen radicals which are balanced by the body 's antioxidant system ( 36 ) .
the effects of antioxidant molecules on fracture healing have been examined with particular focus on some vitamins and essential amino acids ( 4 , 31 , 33 , 3739 ) . as part of the antioxidant system ,
glutamine being a precursor of the glutathione - peroxidase enzyme , also serves as an antioxidant .
( 31 ) emphasised that the effect of antioxidants in reducing free oxygen radicals found in fracture haematoma may have a positive effect on fracture healing .
therefore , in the current study it was thought that as well as other effects , glutamine as an antioxidant could positively influence fracture healing by reducing free oxygen radicals found in the fracture area , but indicators related to this were not examined . in a study on rabbits by sinha and goal ( 41 ) , where the amino acids lysine and arginine
were administered orally , increased callus vascularisation and mineralisation was reported and the healing period was accelerated to 2 weeks . in an experimental study by kdolsky et al .
( 42 ) a femoral diaphysis defect was created in guinea pigs and intramedullary fixation was applied to the femur with a k - wire .
the findings were evaluated histologically , mechanically , and radiologically and it was concluded that the animals which had received l - arginine had better fracture healing and mechanical stability . comparing the current study to that one , the amino acid used was glutamine , the fibula was used rather than the femur , and there was no mechanical evaluation . therefore , although the idea of the effect of amino acids on fracture healing is similar , from a technical aspect there is no similarity to the current study . in the histological evaluation of the current study ,
all the rabbits that had received glutamine were seen to be at stage 9 full recovery , whereas half of the control group were at stage 8 recovery with cartilage and immature bone together and the others were at stage 9 recovery .
similarly , in the radiological evaluation , the immunonutrition group was determined as mean 3.42 points from a total of 5 by the orthopaedic and traumatology specialists and mean 3.35 points by the radiology specialists whereas the control group points were low at mean 2.5 and 2.65 respectively . despite the difference between them
this is thought to be because of the low number of animals used in this study .
it is also thought that histological evaluation at an earlier stage of healing may yield significant results . in conclusion , despite the beneficial effects of glutamine on the speed of wound healing and reducing morbidity and mortality rates in sepsis , and the positive effect observed on bone healing in the immunonutrition group clinically , radiologically , and histopathologically , no statistically significant difference was determined compared to the control group in this study .
additional light will be shed on this subject in literature by subsequent research considering different parameters .
 backgroundthere have been various studies related to fracture healing . 
 glutamine is an amino acid with an important role in many cell and organ functions . 
 this study aimed to make a clinical , radiological , and histopathological evaluation of the effects of glutamine on fracture healing.methodstwenty rabbits were randomly allocated into two groups of control and immunonutrition . 
 a fracture of the fibula was made to the right hind leg . 
 all rabbits received standard food and water . from post - operative first day for 30 days , the study group received an additional 2 ml / kg / day 20% l - alanine l - glutamine solution via a gastric catheter , and the control group received 2 ml / kg / day isotonic via gastric catheter . at the end of 30 days 
 , the rabbits were sacrificed and the fractures were examined clinically , radiologically , and histopathologically in respect to the degree of union.resultsradiological evaluation of the control group determined a mean score of 2.5 according to the orthopaedists and 2.65 according to the radiologists . in the clinical evaluation , the mean score was 1.875 for the control group and 2.0 for the study group . 
 histopathological evaluation determined a mean score of 8.5 for the control group and 9.0 for the study group.conclusionone month after orally administered glutamine  
 alanine , positive effects were observed on fracture healing radiologically , clinically , and histopathologically , although no statistically significant difference was determined . 
Materials and methods

Study plan  surgical technique
Results
Radiological findings
Clinical examination results
Histopathological results
Discussion and conclusion
Conflict of interest and funding