the study was conducted in the stem cell facility , all india institute of medical sciences ( aiims ) , new delhi , india , after obtaining prior approval of the study protocol by the institute ethics committee .
preparation of porous 3d scaffolds : chitosan , hydroxyapatite and polycaprolactone were supplied by sigma alderich , usa . the molecular weight and melt index of pcl were specified as 48,000 - 90,000 da and 1.8 ( g/10 min , 80c ) .
the molecular weight and density for cht were 70,000 - 90,000 da and 1.145 g / ml at 25c , respectively .
the molecular weight and particle size of hap were 502.31 da and < 200 nm , respectively .
the pure chitosan , designated as 100cht , was selected as matrix material for the preparation of the composites .
the composites , cht / hap / pcl in the ratio of 60/0/40 , 60/10/30 and 60/15/25 designated as 40cht / hap , 30cht / hap / pcl and 25cht / hap / pcl , respectively , were prepared by freeze drying technique using a lyophilizer ( coolsafe 110 - 4 , labogene , lynge , denmark ) .
the chitosan was dissolved in 2 mm acetic acid and pcl was dissolved in glacial acetic acid ( fisher scientific , mumbai , india ) at 45c using magnetic stirrer .
the gel - like mixture was casted in a glass petri dish and freeze dried at -80c for 24 h. these samples were then neutralized in sodium hydroxide ( naoh , hi - media , mumbai , india ) solution and washed with deionized water to remove any remaining naoh .
the scaffolds were cut into disc of 10 mm diameter and 2 mm thickness for in vitro studies .
morphology of scaffolds - the microstructure of the scaffolds was examined by stereo - scan 360 scanning electron microscope ( sem , cambridge scientific industries , ma , usa ) .
diameter of individual pores in the scaffolds were measured directly from the sem images with 100x magnification using nih image j software ( dr wayne rasband , national institutes of health , bethesda ,
porosity measurement of scaffolds - the porosity of scaffolds was measured by liquid displacement method10 .
the procedure for liquid displacement method was done as follows : the volume ( v0 ) and weight ( w0 ) of the samples were measured .
then , the sample was immersed into absolute ethanol until it was saturated by it .
the porosity of the scaffold was calculated according to the following equation : degree of porosity ( % ) = 100 x ( w1-w0)/ v0 
  (  represents the density of the ethanol ) . in vitro degradation of scaffolds - the ability of scaffolds to degrade in vitro was studied using a lysozyme ( sigma alderich , usa ) degradation test .
lysozyme was used for the degradation study in the concentration similar to the circulatory level in human body ( to mimic in vivo condition ) .
the samples were then placed in 0.1 m pbs ( phosphate buffered saline ) ph 7.4 containing 100 mg / l of lysozyme and incubated at 37c .
the samples were removed at 1 , 3 , 7 , 14 , 21 , 28 , 35 , 42 , 49 and 56 days to evaluate the weight loss . per cent weight loss
was calculated according to the following equation : % weight loss = ( w0-wt ) / w0x100 where , w0 is the starting dry weight and wt is the dry weight at specified time .
mechanical properties of scaffolds - compressive properties of the scaffolds in wet state were evaluated with a universal testing machine model h5ks ( tinius olsen , surrey , england , uk ) with qmat 5.37 professional software .
the specimens were prepared as column of 20 mm in diameter and 10 mm in height .
the scaffolds were immersed in pbs at ph 7.4 and 37c for three days for complete hydration .
the crosshead speed of the machine was set at 1mm / min with a load cell of 1 kilonewton until 50 per cent reduction in specimen height .
culture and seeding of the cells on scaffolds - human bm was collected from the iliac crest of the patients undergoing stem cell transplantation after taking prior consent .
briefly , 1 - 2 ml of bm was mixed with expansion media in 1:1 ratio and plated on the culture flasks .
after incubation at 37c for 24 h non - adherent cells were removed and fresh medium was added . in our experiments , hmscs were cultured and expanded in a non - differentiating growth medium consisting of low glucose dulbecco 's modified eagle medium ( dmem , gibco , life technologies , usa ) supplemented with 10 per cent foetal bovine serum ( fbs ) ( 0.03 m , hyclone , thermo scientific , usa ) , 2 mm of l - glutamine ( gibco , life technologies , usa ) , 100 units / ml of penicillin and 0.1mg / ml of streptomycin ( gibco , life technologies , usa ) .
cells were grown in a 5 per cent co2 atmosphere at 37c , and the medium was renewed every three days . before confluence , cells were detached using trypsin - edta ( gibco , life technologies , usa ) and passaged 1:3 into fresh culture flasks .
these cells were characterized using bd - lsr ii ( becton - dickinson , usa ) and for their ability to differentiate into osteogenic , chondrogenic , and adipogenic lineages .
the cells were positive for the cell surface markers cd105 , cd29 , ( ebioscience , usa ) , cd73 and cd90 and negative for hla class ii and cd34/45 ( bd pharmingen , usa ) .
hmscs were grown to 80 per cent confluency , trypsinized , and re - suspended in cell expansion medium .
hmscs ( 5x10/20 l ) were seeded drop wise onto sterilized scaffold and incubated at 37 c for 3 h to allow the cells to adhere , then 2 ml of medium was added to each well .
the seeded scaffolds were cultured for four weeks with medium change thrice a week . for osteogenic differentiation ,
the cell expansion medium was supplemented with 10 nm dexamethasone , 50 g / ml ascorbic acid- 2 phosphate and 10 mm beta - glycerophosphate obtained from sigma , usa . for cytotoxicity studies mouse lung fibroblast l929 cells were procured from national centre for cell science , pune , india .
the cells were grown in dmem ( gibco , life technologies , usa ) supplemented with 10 per cent fbs and 100 units / ml of penicillin and 0.1mg / ml of streptomycin in a humidified incubator .
cytotoxicity evaluation of scaffolds - the cytotoxicity of the scaffolds was assessed by [ 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ] ( mtt ) assay .
for cytotoxicity evaluation , we adopted the procedure from the iso10993 - 5 standard test method21 .
extraction media were prepared by immersing dry specimens in cell culture medium as described in literature21 . l929 and
hmscs were separately cultured in 48 well ( 5x10/cells / well ) plate in 500 l extraction medium . after 24 h , mtt ( sigma , usa ; 5 mg / ml in pbs ) was added to each well and incubated for 3.5 h at 37c .
then , dimethylsulphoxide ( sigma , usa ) was added to each well to dissolve the formazan crystals produced by the activity of live cells and the coloured supernatant was read at 570 nm using plate reader ( el 800 , biotek , usa ) .
in addition , standard calibration curve was made by mtt assay on known number of cells and the absorbance values were plotted against the known cell numbers .
metabolically active cell numbers were then determined using this standard curve based on their mtt absorbance .
cell viability and distribution over scaffold - viability and distribution of the cells seeded in the scaffold were examined after 7 and 14 days using live / dead staining kit ( k501 - 100 , biovision , ca , usa ) .
scaffolds seeded with hmscs ( 5x10 cells / scaffold ) were washed with pbs and incubated with fluorescene di acetate ( fda ) and propidium iodide ( pi ) . after staining
, the cells were visualized using confocal laser scanning microscope ( clsm ) ( tcs sp5 , leica , germany ) .
cell attachment and morphology - cell attachment and morphology of cells seeded on the scaffolds were examined by scanning electron microscopy ( sem ) .
samples were collected at different time points and fixed with 2.5 per cent glutaraldehyde and stored at -80 c .
were mounted over aluminium stubs and sputter - coated with gold prior to imaging with a leo 435 vp scanning electron microscope ( co - operation zeiss , leica , cambridge , uk ) at 5 kva in secondary electron imaging mode .
alkaline phosphatase ( alp ) activity - the differentiation of hmscs into osteoblasts in the scaffolds was evaluated by the measurement of alkaline phosphatase activity22 .
briefly , the samples were harvested and freeze - thawed thrice in lysis buffer ( 1.0% triton x-100 ) for cell lysis .
an aliquot of the lysate was added to the alp substrate buffer ( equal parts of 20 mm p - nitrophenyl phosphate ( 1.5 m 2-amino-2-methyl-1-propanol from sigma , usa , and 10 mm mgcl2 ) , and incubated for 30 min at 37 c . then
hydrolysis of the substrate to p - nitrophenol was measured spectrophotometrically at 405 nm ( el 800 , biotek , usa ) . quantitative reverse transcription - pcr ( qrt - pcr ) analysis for the relative expression of osteoblasts specific genes , in differentiated osteoblasts derieved from hmscs - the expression levels of collagen type 1 ( colia1 ) , alkaline phosphatase ( alp ) , osteocalcin ( oc ) , osteopontin ( op ) ,
briefly , at each time point , total rna was isolated using trizol ( life technologies , usa ) as per the manufacturer 's protocol .
rna samples were treated with dnase i ( sigma ; 1 u/g rna ) for 25 min at room temperature to remove residual genomic dna .
the concentration of the purified rna was quantified using nanospectrophotometer ( p300 , implen gmbh , germany ) .
cdna was transcribed using 200u of m - mlv reverse transcriptase , 0.5 mm dntps and 12.5 ng / ml oligo - dt and 40 u of rnase inhibitor rnasin ( all from promega , usa ) . the gene specific primers were custom made ( sigma , bangalore , india ) and are summarized in table i. reactions were carried out using sybr green super mix ( kapa biosystems , capetown , south africa ) in a final volume of 10 l with 0.3 m of each primer .
relative gene expression was quantified by 2 method24 in which the accumulated pcr products of each of the genes examined was normalized to the housekeeping gene gapdh in the corresponding samples .
ct was obtained by subtracting the ctgapdh from cttarget ( ct = cttarget  ctgapdh ) and ct was obtained by subtracting ctuninduced cells from ctinduced cells ( ct = ctinduced cells- ctun induced cells ) .
negative controls were used as no template cdna reactions and melting curves were used to confirm the results .
primer sequence used for qpcr amplification the relative quantitative qpcr experiments were performed by using a realplex 4 epgradient mastercycler ( eppendorf , hamburg , germany ) according to the manufacturer 's instructions .
statistical analysis : all statistical analyses were performed using graphpad software ( la jolla , ca , usa ) .
all measurements were collected in triplicates and expressed as means  standard deviation , unless otherwise noted . in cases where statistical comparisons were made between three or more groups of data , a one - way analysis of variance
morphology of scaffolds - the microstructure of the scaffolds was examined by stereo - scan 360 scanning electron microscope ( sem , cambridge scientific industries , ma , usa ) .
diameter of individual pores in the scaffolds were measured directly from the sem images with 100x magnification using nih image j software ( dr wayne rasband , national institutes of health , bethesda ,
porosity measurement of scaffolds - the porosity of scaffolds was measured by liquid displacement method10 . the procedure for liquid displacement method
was done as follows : the volume ( v0 ) and weight ( w0 ) of the samples were measured .
then , the sample was immersed into absolute ethanol until it was saturated by it .
the porosity of the scaffold was calculated according to the following equation : degree of porosity ( % ) = 100 x ( w1-w0)/ v0 
  (  represents the density of the ethanol ) . in vitro degradation of scaffolds -
the ability of scaffolds to degrade in vitro was studied using a lysozyme ( sigma alderich , usa ) degradation test .
lysozyme was used for the degradation study in the concentration similar to the circulatory level in human body ( to mimic in vivo condition ) .
the samples were then placed in 0.1 m pbs ( phosphate buffered saline ) ph 7.4 containing 100 mg / l of lysozyme and incubated at 37c .
the samples were removed at 1 , 3 , 7 , 14 , 21 , 28 , 35 , 42 , 49 and 56 days to evaluate the weight loss . per cent
weight loss was calculated according to the following equation : % weight loss = ( w0-wt ) / w0x100 where , w0 is the starting dry weight and wt is the dry weight at specified time .
mechanical properties of scaffolds - compressive properties of the scaffolds in wet state were evaluated with a universal testing machine model h5ks ( tinius olsen , surrey , england , uk ) with qmat 5.37 professional software .
the specimens were prepared as column of 20 mm in diameter and 10 mm in height .
the scaffolds were immersed in pbs at ph 7.4 and 37c for three days for complete hydration .
the crosshead speed of the machine was set at 1mm / min with a load cell of 1 kilonewton until 50 per cent reduction in specimen height .
culture and seeding of the cells on scaffolds - human bm was collected from the iliac crest of the patients undergoing stem cell transplantation after taking prior consent .
briefly , 1 - 2 ml of bm was mixed with expansion media in 1:1 ratio and plated on the culture flasks .
after incubation at 37c for 24 h non - adherent cells were removed and fresh medium was added . in our experiments , hmscs were cultured and expanded in a non - differentiating growth medium consisting of low glucose dulbecco 's modified eagle medium ( dmem , gibco , life technologies , usa ) supplemented with 10 per cent foetal bovine serum ( fbs ) ( 0.03 m , hyclone , thermo scientific , usa ) , 2 mm of l - glutamine ( gibco , life technologies , usa ) , 100 units / ml of penicillin and 0.1mg / ml of streptomycin ( gibco , life technologies , usa ) .
cells were grown in a 5 per cent co2 atmosphere at 37c , and the medium was renewed every three days . before confluence , cells were detached using trypsin - edta ( gibco , life technologies , usa ) and passaged 1:3 into fresh culture flasks .
these cells were characterized using bd - lsr ii ( becton - dickinson , usa ) and for their ability to differentiate into osteogenic , chondrogenic , and adipogenic lineages .
the cells were positive for the cell surface markers cd105 , cd29 , ( ebioscience , usa ) , cd73 and cd90 and negative for hla class ii and cd34/45 ( bd pharmingen , usa ) .
hmscs were grown to 80 per cent confluency , trypsinized , and re - suspended in cell expansion medium .
hmscs ( 5x10/20 l ) were seeded drop wise onto sterilized scaffold and incubated at 37 c for 3 h to allow the cells to adhere , then 2 ml of medium was added to each well .
the seeded scaffolds were cultured for four weeks with medium change thrice a week . for osteogenic differentiation ,
the cell expansion medium was supplemented with 10 nm dexamethasone , 50 g / ml ascorbic acid- 2 phosphate and 10 mm beta - glycerophosphate obtained from sigma , usa . for cytotoxicity studies mouse
lung fibroblast l929 cells were procured from national centre for cell science , pune , india .
the cells were grown in dmem ( gibco , life technologies , usa ) supplemented with 10 per cent fbs and 100 units / ml of penicillin and 0.1mg / ml of streptomycin in a humidified incubator .
cytotoxicity evaluation of scaffolds - the cytotoxicity of the scaffolds was assessed by [ 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide ] ( mtt ) assay . for cytotoxicity evaluation
extraction media were prepared by immersing dry specimens in cell culture medium as described in literature21 . l929 and hmscs were separately cultured in 48 well ( 5x10/cells / well ) plate in 500 l extraction medium .
after 24 h , mtt ( sigma , usa ; 5 mg / ml in pbs ) was added to each well and incubated for 3.5 h at 37c .
then , dimethylsulphoxide ( sigma , usa ) was added to each well to dissolve the formazan crystals produced by the activity of live cells and the coloured supernatant was read at 570 nm using plate reader ( el 800 , biotek , usa ) .
in addition , standard calibration curve was made by mtt assay on known number of cells and the absorbance values were plotted against the known cell numbers .
metabolically active cell numbers were then determined using this standard curve based on their mtt absorbance .
cell viability and distribution over scaffold - viability and distribution of the cells seeded in the scaffold were examined after 7 and 14 days using live / dead staining kit ( k501 - 100 , biovision , ca , usa ) .
scaffolds seeded with hmscs ( 5x10 cells / scaffold ) were washed with pbs and incubated with fluorescene di acetate ( fda ) and propidium iodide ( pi ) . after staining
, the cells were visualized using confocal laser scanning microscope ( clsm ) ( tcs sp5 , leica , germany ) .
cell attachment and morphology - cell attachment and morphology of cells seeded on the scaffolds were examined by scanning electron microscopy ( sem ) .
samples were collected at different time points and fixed with 2.5 per cent glutaraldehyde and stored at -80 c .
were mounted over aluminium stubs and sputter - coated with gold prior to imaging with a leo 435 vp scanning electron microscope ( co - operation zeiss , leica , cambridge , uk ) at 5 kva in secondary electron imaging mode .
alkaline phosphatase ( alp ) activity - the differentiation of hmscs into osteoblasts in the scaffolds was evaluated by the measurement of alkaline phosphatase activity22 . briefly , the samples were harvested and freeze - thawed thrice in lysis buffer ( 1.0% triton x-100 ) for cell lysis .
an aliquot of the lysate was added to the alp substrate buffer ( equal parts of 20 mm p - nitrophenyl phosphate ( 1.5 m 2-amino-2-methyl-1-propanol from sigma , usa , and 10 mm mgcl2 ) , and incubated for 30 min at 37 c . then
hydrolysis of the substrate to p - nitrophenol was measured spectrophotometrically at 405 nm ( el 800 , biotek , usa ) .
quantitative reverse transcription - pcr ( qrt - pcr ) analysis for the relative expression of osteoblasts specific genes , in differentiated osteoblasts derieved from hmscs - the expression levels of collagen type 1 ( colia1 ) , alkaline phosphatase ( alp ) , osteocalcin ( oc ) , osteopontin ( op ) , transcription factor cbfa 1 and osterix genes were quantitated23 . briefly , at each time point , total rna was isolated using trizol ( life technologies , usa ) as per the manufacturer 's protocol .
rna samples were treated with dnase i ( sigma ; 1 u/g rna ) for 25 min at room temperature to remove residual genomic dna .
the concentration of the purified rna was quantified using nanospectrophotometer ( p300 , implen gmbh , germany ) .
cdna was transcribed using 200u of m - mlv reverse transcriptase , 0.5 mm dntps and 12.5 ng / ml oligo - dt and 40 u of rnase inhibitor rnasin ( all from promega , usa ) . the gene specific primers were custom made ( sigma , bangalore , india ) and are summarized in table i. reactions were carried out using sybr green super mix ( kapa biosystems , capetown , south africa ) in a final volume of 10 l with 0.3 m of each primer .
relative gene expression was quantified by 2 method24 in which the accumulated pcr products of each of the genes examined was normalized to the housekeeping gene gapdh in the corresponding samples .
ct was obtained by subtracting the ctgapdh from cttarget ( ct = cttarget  ctgapdh ) and ct was obtained by subtracting ctuninduced cells from ctinduced cells ( ct = ctinduced cells- ctun induced cells ) .
negative controls were used as no template cdna reactions and melting curves were used to confirm the results .
primer sequence used for qpcr amplification the relative quantitative qpcr experiments were performed by using a realplex 4 epgradient mastercycler ( eppendorf , hamburg , germany ) according to the manufacturer 's instructions .
statistical analysis : all statistical analyses were performed using graphpad software ( la jolla , ca , usa ) .
all measurements were collected in triplicates and expressed as means  standard deviation , unless otherwise noted . in cases
where statistical comparisons were made between three or more groups of data , a one - way analysis of variance was performed . in cases where only two sets of data were compared ,
microstructure of scaffolds : the external morphology of scaffolds was studied by sem ( fig .
scaffolds were seen to be highly porous with open and interconnected pores surrounded by thin polymer walls .
the microstructure of the scaffolds did not seem to vary from one another since the treatment given to each was similar .
the composition did not affect the pore size and porosity of the scaffolds . pore size and porosity data of the scaffolds are given in table ii .
representative scanning electron microscope ( sem ) photomicrographs illustrating the open porous structure of the scaffolds with homogenous distribution of pores .
( a ) 100cht ( b ) 40cht / hap ( c ) 30cht / hap / pcl and ( d ) 25cht / hap / pcl ( scale bar 100m ) .
pore parameters of scaffolds , determined by scanning electron microscopy and liquid displacement methods in vitro degradation of scaffolds : fig .
2a shows the weight loss of scaffolds as a function of soaking time in pbs .
the 100cht showed maximum weight loss ( 91% ) , followed by 40cht / hap ( 70% ) , 30cht / hap / pcl ( 34% ) and 25cht / hap / pcl ( 30% ) at the end of 56 days .
after enzymatic degradation for 56 days , change in morphology of the 100cht and 40cht / hap scaffolds was observed ( fig .
the scaffold 30cht / hap / pcl and 25cht / hap / pcl remained in the original shape , indicating that these scaffolds had good structural stability .
( a ) degradation kinetics of 100cht , 40cht / hap , 30cht / hap / pcl and 25cht / hap / pcl scaffolds .
data represent mean value of five independent experiments , ( b ) digital images showing the morphology of scaffolds after in vitro enzymatic treatment at days 1 and 56 . mechanical properties of scaffolds : the compressive stress - strain curves of the pure and composite scaffolds are shown in fig .
3 , and the data of compressive strength and compressive modulus are given in table iii . as expected , 100cht scaffold showed low compressive strength and modulus .
the mixing of hap with cht significantly improved the compressive strength and compressive modulus of 40cht / hap .
further addition of pcl ( 10% ) to the 40cht / hap showed no significant difference in the compressive strength and modulus of the 30cht / hap / pcl composite scaffold . increasing the pcl content to 15 per cent dramatically increased the compressive strength ( p<0.01 ) and compressive modulus ( p<0.001 ) of 25cht / hap / pcl composite scaffold .
mechanical properties of porous scaffolds cytotoxicity of the scaffolds : the cytotoxicity of 3d composite scaffolds was tested by quantitative analysis using the mtt assay .
the absorbance values of test samples after exposure to the extraction medium for 24 h were similar to the negative control ( table iv ) .
absorbance values correspond to the number of metabolically active cells . thus our results ( fig .
4 ) indicated that the metabolic activity of l929 and hmscs cells was not inhibited by the extraction medium . from the quantitative scores
, it was concluded that the extracts of the scaffolds demonstrated no cytotoxic reactivity in this test .
the viability of mouse lung fibroblast cell line l929 and hmscs was evaluated that had been cultured for 24 h with the extraction media from the specimens .
viability and distribution of hmscs in scaffolds : the viability and distribution of hmscs on scaffolds were visualized using clsm after 7 and 14 days of cells seeding , which depicted a high ratio of living cells on all scaffold compositions . as shown in fig . 5 ,
> 90 per cent cells were live , randomly distributed within the porous 3d scaffolds .
hmscs were connected to each other and formed a cell network across the surface of the scaffolds .
the cells proliferated and their number increased from day 7 to day 14 ( fig .
up to 14 days of cultivation , no large difference in the viability and morphology was observed .
cells were more evenly distributed over 30cht / hap / pcl and 25cht / hap / pcl scaffolds compared to 100cht and 40cht / hap scaffolds .
confocal laser microscopic images of hmscs grown on scaffolds after seven days ( a ) cells were randomly distributed and forming patch on 100cht , ( b ) hmscs adhered and attained fibroblastic morphology on 40cht / hap , ( c , d ) cells were more evenly distributed over 30cht / hap / pcl and 25cht / hap / pcl , covered whole surface of the scaffold .
viability and distribution of cells were detected using fluorescence markers that allowed life / death stain ( fda / pi ) : dead cells were stained red whereas viable cells were stained green ( magnification 10x ) .
( a ) cells proliferated and form a large patch on 100cht , ( b ) cells increased in number and evenly distributed on 40cht / hap , ( c , d ) cells proliferated and their number increased from day 7 to 14 .
cells were connected to each other and formed a cell network across the surface of 30cht / hap / pcl and 25cht / hap / pcl scaffolds . viability and distribution of cells
were detected using fluorescence markers that allowed life / death stain ( fda / pi ) : dead cells were stained red whereas viable cells were stained green .
the qualitative image analysis revealed that a larger number of cells stayed viable throughout the entire culture period .
attachment and morphology of hmscs on scaffolds : after seven days of cell seeding , cells adhered and spreaded over the porous scaffold surface and merged completely with each other so that intercellular connections were not visible ( fig .
after 14 days it was found that cells were merged and formed a complete cellular layer on the surface of the scaffolds , so that the surface was almost covered and only a few pores were visible and a few cells migrated inside the pores ( fig .
scanning electron microscope ( sem ) images of human mesenchymal stem cells ( hmscs ) grown on 100cht , 40cht / hap , 30cht / hap / pcl and 25cht / hap / pcl scaffolds after seven days .
sem images of hmscs grown on 100cht , 40cht / hap , 30cht / hap / pcl and 25cht / hap / pcl scaffolds after 14 days . at 14 days
, cells grew and merged to form a cell sheet , got enlarged and migrated into the pores of scaffolds .
( scale bar 30 m ) . specific alkaline phosphatase activity of hmscs differentiated into osteoblasts on scaffolds : alp is an enzyme produced by differentiating osteoblasts .
alp activity can serve as a marker indicating differentiation of hmscs toward the osteoblastic lineage . in un - induced culture condition ( fig .
9a ) , the alp activity of hmscs on scaffolds and tissue culture plastic surface ( tcps ) slightly increased over the whole culture period of 28 days .
9b ) the alp activity of the hmscs , increased progressively over time from day 3 to 28 .
alp activity of hmscs in osteogenic condition was significantly higher on all scaffolds compared to tcps ( p<0.01 ) . among the scaffolds ,
determination of alkaline phosphatase ( alp ) activity of hmscs over 100cht , 40cht / hap , 30cht / hap / pcl , 25cht / hap / pcl scaffolds and tissue culture plastic surface ( tcps ) under
( a ) un - induced culture condition and ( b ) induced culture condition .
alp activity of hmscs in the cell - scaffold constructs was assessed during four weeks of culture .
qpcr analysis for relative expression of osteoblasts specific genes , in osteoblasts derived from differentiation of hmscs : results obtained from qrt - pcr were quantitative and presented as a  fold change in mrna levels in differentiated hmscs when compared to undifferentiated hmscs ( fig . 10 ) .
differentiation of osteogenic - induced hmsc on scaffolds and tcps was further demonstrated by an increase in the relative expression of the osteoblasts specific genes .
the relative expression of osteoblasts specific genes on scaffolds was several fold upregulated compared to tcps
. a slightly higher maximal expression of genes was detected on scaffold 25cht / hap / pcl compared to 30cht / hap / pcl , 40cht / hap and 100cht scaffolds ( found on day 28 ) .
real - time quantitative reverse transcriptase pcr analysis of the expression levels of osteogenic markers .
expression levels of cbfa-1 , osterix , alkaline phosphatase , collagen type i , osteocalcin , and osteopontin genes were determined in hmscs cultured over 100cht , 40cht / hap , 30cht / hap / pcl and 25cht / hap / pcl scaffolds and tissue culture plastic surface ( tcps ) under osteogenic and non - osteogenic culture conditions for 28 days .
in this study we prepared porous foam like scaffolds with average pore size of 140 m , which could support cell penetration , migration , tissue in - growth and vascularisation as reported in the literature2526 and the average porosity of the scaffold was 82 per cent , which was in the range of the porosity of the trabecular bone ( 50 - 90% ) .
mean pore sizes increase , mechanical strength is decreased . in this study , by fabricating scaffolds with optimal pore size and porosity it was possible to maintain the mechanical strength of scaffold .
the porous 100cht scaffolds were tissue biocompatible but mechanically weak and unable to maintain structure integrity as also reported in literature11 .
absorbs a lot of water and swells resulting in poor structural stability and low mechanical strength . to overcome this problem we doped cht with pcl and hap .
pcl being hydrophobic decreased the total water absorption and swelling and hap as a filler strengthened the scaffold .
the resulting composite 25cht / hap / pcl scaffold showed better compressive strength compared to pure 100cht scaffold , which was very close to the lower limit of compressive strength of trabecular bone ( 2 - 10 mpa)6 .
thus , incorporation of hap and pcl into composite scaffolds in specific ratios could improve their mechanical properties . in in vitro degradation study , it was found that the weight loss of the scaffolds increased gradually .
the degradation rate of 100cht and 40cht / hap scaffold was high compared to the composite scaffolds , 30cht / hap / pcl and 25cht / hap / pcl .
the result suggested that presence of pcl decreased degradation of the composite scaffold because of its being hydrophobic and semi - crystalline material with a slow in vitro hydrolytic degradation rate27 .
pure 100cht scaffolds were found to degrade > 90 per cent and disintegrated upon handling .
in contrast , the composite 30cht / hap / pcl and 25cht / hap / pcl scaffolds retained their original shape , despite evidence of degradation from weight loss measurements .
the mtt assay showed that all the scaffolds prepared in this study were nontoxic and biocompatible .
the polymer composition and the method of fabrication also play an important role in cell biocompatibility .
our results showed overall good viability of the cells indicating that these scaffolds were free of toxic chemicals and suitable for in vitro and in vivo studies .
the ability of hmscs to differentiate into osteogenic lineage is the key to the use of these cells to improve bone formation .
sem images showed that the large surface area of the porous scaffolds allowed hmscs to adhere , spread and grow on the scaffolds .
it has been shown that chitosan coating on 2-d polymeric films or 3-d polymer scaffolds may affect the attachment , proliferation and differentiation of cells28 .
the flat morphology , and excellent spreading in and around the interconnected porous structure , indicated strong cellular adhesion and growth of cells .
the 3d structure of the scaffold might have been responsible for the attachment of hmscs and differentiation into osteoblasts29 .
the parallel investigations of cell viability and distribution carried out by clsm revealed proliferation of the cells on the scaffolds with clear spreading and good morphology .
live dead staining and clsm showed that > 90 per cent hmscs were live and distributed throughout the scaffolds .
this indicates that the pores of the scaffolds are interconnected and large enough for cells to penetrate and colonize the porous structure .
alp is an enzyme expressed by mscs during osteogenesis and is a well - defined marker of osteoblastic lineage30 .
thein - han et al16 has demonstrated that presence of hap nanoparticles in cht / nhap nanocomposite scaffolds influence cytoskeleton organization , which regulates cellular signal transduction , cell differentiation and gene expression . in this study
, we also found that hmscs cultured over composite scaffolds showed higher specific alp activity than those cultured over pure cht scaffolds and tcps .
the expression of osteoblasts specific genes was multiple folds in induced hmscs compared to the un - induced hmscs . however , in non - osteogenic conditions the cells over 3d scaffolds showed enhanced osteoblastic genes expression compared to cells cultured on tcps .
it might be because of the inherent osteoinductivity and osteoconductivity of the cht and hap present in the scaffold .
in addition , results of a cell culture study showed both enhanced proliferation and differentiation of rat calvarial osteoblasts on bone cement scaffold32 . after comparing all the properties of scaffolds
it was found that the novel 25cht / hap / pcl biomedical composite scaffold had good mechanical strength , optimum pore size and porosity , required degradability , cell biocompatibility and osteo - inductivity . in conclusion ,
the present study demonstrated the feasibility of using the freeze drying technique to fabricate 3d biocomposite scaffolds .
the addition of hap and pcl to cht improved the compressive strength while maintaining high porosity with interconnected pores .
the in vitro biocompatibility study of the scaffolds showed that the polycaprolactone did not affect attachment , proliferation and differentiation on the scaffolds . the 25cht / hap / pcl composite scaffold possessed the inherent physiochemical and efficient mechanical and biocompatible characteristics .
the 25cht / hap / pcl biocomposite appears to be potential scaffolds as an off the shelf , synthetic , resorbable bone graft substitute for bone regeneration .
 background & objectives : there is a significant bone tissue loss in patients from diseases and traumatic injury . 
 the current autograft transplantation gold standard treatment has drawbacks , namely donor site morbidity and limited supply . 
 the field of tissue engineering has emerged with a goal to provide alternative sources for transplantations to bridge this gap between the need and lack of bone graft . 
 the aim of this study was to prepare biocomposite scaffolds based on chitosan ( cht ) , polycaprolactone ( pcl ) and hydroxyapatite ( hap ) by freeze drying method and to assess the role of scaffolds in spatial organization , proliferation , and osteogenic differentiation of human mesenchymal stem cells ( hmscs ) in vitro , in order to achieve bone graft substitutes with improved physical - chemical and biological properties.methods:pure chitosan ( 100cht ) and composites ( 40cht / hap , 30cht / hap / pcl and 25cht / hap / pcl scaffolds containing 40 , 30 , 25 parts per hundred resin ( phr ) filler , respectively ) in acetic acid were freeze dried and the porous foams were studied for physicochemical and in vitro biological properties.results:scanning electron microscope ( sem ) images of the scaffolds showed porous microstructure ( 20 - 300 m ) with uniform pore distribution in all compositions . 
 materials were tested under compressive load in wet condition ( using phosphate buffered saline at ph 7.4 ) . 
 the in vitro studies showed that all the scaffold compositions supported mesenchymal stem cell attachment , proliferation and differentiation as visible from sem images , [ 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide ] ( mtt ) assay , alkaline phosphatase ( alp ) assay and quantitative reverse transcription ( qrt)-pcr.interpretation & conclusions : scaffold composition 25cht / hap / pcl showed better biomechanical and osteoinductive properties as evident by mechanical test and alkaline phosphatase activity and osteoblast specific gene expression studies . 
 this study suggests that this novel degradable 3d composite may have great potential to be used as scaffold in bone tissue engineering . 