each of the options has limitations , such as the need for second site of surgery , limited supply , inadequate size and shape , and the morbidity associated with donor site ; thus , there remains a need for the new option .
synthetic hydroxyapatite is widely used as a bone substitute material because it can bond directly to the living bone and has excellent biocompatibility and bioactivity .
however , hydroxyapatite does not have enough mechanical properties to act as substitute in loading - bearing parts of the human skeleton .
it has been desired to develop a material that has mechanical properties analogous to those of natural bone .
it is known that human bone has a three - dimensional woven apatite - polymer structure made of inorganic apatite crystals and organic collagen fibers .
bone consists of an organic - inorganic hybrid with a characteristic structure that leads to specific mechanical properties such as high fracture toughness and flexibility .
it is a good strategy to mimic bone structure in the design of bone - repairing materials . from this point of view
, the fabrication of hybrid materials consisting of apatite and natural organic polymer can be expected to be a good strategy to obtain bone repairing materials that have both a bone bonding ability and mechanical properties similar to those of natural bone .
previous study   reported that apatite - coated silk scaffolds can combine the osteoconductive properties of bioceramics with the mechanical resilience of polymer .
silk scaffolds combined with apatite promoted cellular attachment and bone formation in vitro , providing an appropriate osteogenic environment for tissue engineering . in vivo research on mongrel dogs by zhao
.   showed that apatite - silk scaffolds could be successfully used to repair mandibular critical size border defects .
the premineralization of the porous silk protein scaffold provided an increase osteoconductive environment for the cells to regenerate sufficient new bone tissue .
silks are fibrous proteins with remarkable mechanical properties produced in fiber formed by silkworms and spiders .
sericin comprised granular and high - molecular , water soluble glycoproteins and it acted as a protein glue to fix fibroin fibers together in the cocoon . in silk textile processing , sericin is usually removed , resulting in fine silk fibers .
the protein which resists oxidation is antibacterial , uv - resistant , and absorbs and releases moisture easily .
sericin can be cross - linked , copolymerized , and blended with other macromolecular materials , especially artificial polymers , to produce materials with the improved properties .
the protein is also used as an improving reagent or a coating material for natural and artificial fibers , fabrics , and articles .
sericin composites are useful as degradable biomaterials , biomedical materials , polymers for forming articles , functional membranes , fibers , fabrics , and articles [ 8 , 9 ] .
sericin has a potential to facilitate apatite deposition and can be useful as polymer material in the fabrication of hybrid materials analogous to bone through biomimetic processes .
indonesia is well known for its silk textile derived from wild silkworm cocoon of cricula triphenestrata .
cricula triphenestrata is one of the world wild species of silkworms which only habitats in java island .
the use of discarded sericin of cricula triphenestrata cocoon extract from the water waste of silk textile industry as biomaterials will be beneficial for the local silk textile industry and also the development of natural biomaterials as bone substitute .
biomaterials may have low , medium , or high potential risk to human safety depending on the type and the extent of patient contact .
one of the international standards   recommends the appropriate steps for the biological assessment of medical devices in vitro assessment of cytotoxicity of new biomaterials . in this primary screening
, we aimed to evaluate the cytotoxicity of cricula triphenestrata cocoon extract on human gingival fibroblasts .
the cocoon shells of the silkworm cricula triphenestrata ( figure 1 ) were obtained from pt yarsilk gora mahottama textile industri at yogyakarta , indonesia .
the cocoon shells were taken from karang tengah forest at kabupaten bantul , yogyakarta , indonesia .
the medium of rpmi 1640 , dulbecco 's modified eagle 's minimum essential medium ( dmem ) , penicillin , streptomycin , amphotericin , and trypsin were obtained from gibco ( carlsbad , ca , usa ) .
all other chemicals were analytical or pharmaceutical grade and obtained from sigma - aldrich chemicals ( bornem , belgium ) .
the cocoon shells of the silkworm cricula triphenestrata , were mixed with an aqueous solution of 0.3% na2co3 ( w / v ) at 98c for 1 hour .
the solution was then dialyzed in cellulose membranes ( mw = 3500  g / mol ) against deionized water for 3 days by changing the water daily to remove the ions and other impurities .
human gingival fibroblasts were obtained from biopsies of the attached gingival of sound permanent molar teeth of healthy persons . informed consent based on an appropriate protocol was obtained from the donors .
the biopsies were stored at 4c for at most 24 hours in collection medium ( rpmi 1640 supplemented with penicillin 100  u / ml , streptomycin 100  mg / ml , and amphotericin 2.5  mg / ml ) prior to amplification .  
the gingival tissues were cut into 1 to 2  mm pieces then washed three times by rpmi 1640 .
after that , the cut biopsies were placed into 25  cm tissue culture flasks .
the explants were incubated with culture medium consisting of dmem 90% , 10  mm hepes , glucose ( 4.5 
g / l ) , penicillin ( 100  u / ml ) , streptomycin ( 100  mg / ml ) , and amphotericin ( 2.5  mg / ml ) , supplemented with 10% heat - inactivated fetal calf serum ( fcs ) .
the tissue samples were grown at 37c in a humidified atmosphere of 10% carbon dioxide in the air .
when outgrowth of cells was observed , the medium was replaced twice weekly until cells reached confluence .
cells were detached from the monolayer by a brief treatment with trypsin - edta ( 0.25% trypsin , 0.02% edta ) and recultured in 75  cm tissue flasks until confluent monolayer was reobtained .  
the mtt cytotoxicity test is based on iso 10993 - 5   of biological evaluation of medical devices , part 5 : tests for in vitro cytotoxicity .
cells ( 1  10  cells / ml ) in dmem of 50  l were seeded into 96-well plates and maintained in culture for 24 hours to form a semiconfluent monolayer .
they were then exposed to the cricula triphenestrata cocoon extracts ( 50  l ) over a range of 0.01100  g / ml concentration .
after 24-hour exposure , the formazan formations were determined for each treatment concentration by elisa reader at a wavelength of 570  nm .
the relative viability of the treated cells as compared to the control cells were expressed as the % cytoviability , using the following formula : 
 ( 1)%cytoviability=100%od570  treatedod570  control , 
 where od570  treated is mean value of the measured optical density of the treated cells ; od570  control is mean value of the measured optical density of the control cells .
statistical analysis was performed using analysis of variance ( anova ) to determine the effect of cocoon extract concentration on the fibroblast cells cytoviability .
a standard curve was performed based on the extract concentrations numbers versus the cytoviability percentage value .
the extract concentrations used were 100 , 50 , 25 , 12.5 , 6.25 , 3.125 , 1.563 , 0.781 , and 0.010  g / ml . each concentration was performed in four replicates .
this research used the cytoviability values that only fall in the linear range of the standard curve to reduce error .
a linear regression analysis was performed regarding to the corresponding concentration numbers and cytoviability values .
the regression formula was y = ax + b. the ic50 value was estimated using the fitted line of the linear regression as ic50 = ( 0.5  b)/a .
the result of relative viability of the fibroblast treated cells as compared to the control cells was expressed in table 1 .  

table 1 showed that increased concentration of the cricula triphenestrata cocoon extract exposure on the cells resulted in the decreasing of cytoviability percentage of human gingival fibroblast cells .
further statistical analysis by anova was shown in table 2 , which described that there was not any significant influence of the treated extracts concentration on the cytoviability of human gingival fibroblast ( p > 0.05 ) .  
the ic50 value of cricula triphenestrata cocoon extract for a battery of fibroblast cells was obtained by regression analysis of the corresponding dose - response curve .
the regression formula for the dose - response curve was y = 67.630  0.390x . by the formula ,
in the field of biomaterials , it is necessary to consider aspects of security , such as elimination of cytotoxicity and other harmful effects of the material to be used . by definition
, the cytotoxicity of an agent means the toxicological risks caused by a material or its extract in a cell culture .
the interactions of the materials and their components with the cells at a molecular level are responsible for tissue reactions , such as inflammation , necrosis , immunological alterations , genotoxicity , and apoptosis . during the last years
, the interest of in vitro systems as an alternative to animal experiments in toxicological research has been steadily increasing .
we used indirect test , in which the rate of cell growth ( cell number ) and the metabolic activity ( mtt ) have indicated the degree of cytotoxicity of cricula triphenestrata cocoon extract . 

table 1 shows the effect of cricula triphenestrata cocoon extract on human gingival fibroblast cells viability measured by mtt test .
mtt is a yellow water - soluble tetrazolium dye which is reduced by live cells to a purple formazan product insoluble in aqueous solutions .
as can be seen from table 1 , the cocoon extract exposure of 0.01100  g / ml during 24 hour of incubation induces the cytoviability of the fibroblast to be 71.18464.486% in comparison to the control .
the highest inhibition effect is induced by 100  g / ml extract concentration and the weakest is on 0.01  g / ml one . although further statistical analysis shows no significant difference among the concentrations , but it is proved that there is an increasing cytoviability on the decreasing of the extract concentration exposure .
the half maximal inhibitory concentration ( ic50 ) is a measure of the effectiveness of a compound in inhibiting biological or biochemical function .
this quantitative measure indicates how much a particular substance is needed to inhibit a given biological process by half . in other words ,
it is the half maximal ( 50% ) inhibitory concentration ( ic ) of a substance ( 50% ic , or ic50 ) .
the score of ic50 represents the concentration of a drug that is required for 50% inhibition in vitro . in this study ,
the determination of ic50 by the formula of y = 67.630  0.390x is 172  g / ml . it means that the highest treated concentration of 100  g / ml cocoon extract reveals no significant cytotoxicity against human fibroblasts ( less than 172  g / ml ) .  
previous study reported that the ic50 of bombyx mori silkworm cocoon extract on vero cells was 230  g / ml .
comparing to the ic50 of cricula triphenestrata cocoon extract in this study , the cricula triphenestrata cocoon extract is lower than the bombyx mori .
it means that the cricula triphenestrata cocoon extract is more cytotoxic than the bombyx mori .
previous study reported that there was a correlation between environmental condition and the protein production of cocoon .
the difference between the environmental condition and food consumption may influence the cocoon characteristic including the cytotoxicity . in the present study , the cytotoxicity of cricula triphenestrata cocoon extract which is aimed to be used as bone substitute
like other tissues , normal fibroblast function is critical to maintain the periodontal tissue function for optimal healing .
based on the research of the cytotoxicity of indonesian silkworm cocoon extract of cricula triphenestrata on human fibroblasts , it was concluded that cricula triphenestrata cocoon extract up to 100  g / ml was not cytotoxic on human gingival fibroblast cells . by this finding
, further researches are proposed to develop the cricula triphenestrata cocoon extract as an alternative biomaterial for bone substitute .
 
 objectives . the aim of this paper was to evaluate the cytotoxicity of indonesian silkworm cocoon extract of cricula triphenestrata on human fibroblasts . methods and materials . 
 the cocoon shells of the silkworm cricula triphenestrata were degumming . 
 the shells were mixed with an aqueous solution of 0.3% na2co3 at 98c for 1 hour . 
 the solution was then dialyzed in cellulose membranes against deionized water for 3 days . 
 the cocoon shells extract powder was collected via rotary evaporation and dried under freeze dryer . 
 cell culture medium was exposed to cricula triphenestrata cocoon extract ( 0.01100  g / ml ) for 24 hours . 
 the primary human gingival fibroblasts were exposed to the treated cell culture medium for 24 hours . 
 cytotoxicity evaluation was done by mtt method . 
 the data were analyzed by one - way anova . 
 result . 
 the result revealed no significant cytotoxicity of cricula triphenestrata cocoon extract against human fibroblasts at a concentration up to 100  g / ml ( p > 0.05 ) . 
 conclusion . 
 cricula triphenestrata cocoon extract was not cytotoxic on human gingival fibroblast cells . 
1. Introduction
2. Materials and Method
3. Results
4. Discussion
5. Conclusion