large bone defects , such as acute injuries , fall fractures in osteoporotic patients , or tumors and congenital malformations of the musculoskeletal system are very common in the clinical cases of orthopedics . it is necessary to resect the affected parts of the bone , which is a major therapeutic challenge for the reconstructive surgery after resection . recently , great progress has been made in bone tissue engineering , which is promising for treatment of bone defects and bone regeneration . scaffold is one of the critical elements , and it is generally acknowledged that appropriate physical structure and good biocompatibility are two important characteristics that are considered ideal for bone tissue engineering . the scaffold , as a temporary template or substrate , formulates the final shape of the new bone , and the architecture of the scaffold , a key property of the scaffolds , determines its interaction with the targeted cells . target cells behave distinctively when they grow on different scaffolds ( hydrophilicity , surface roughness , etc . ) . in the previous study , we had fabricated 3-dimensional porous poly(lactic - co - glycolic acid ) ( plga)/-tricalcium phosphate ( -tcp ) ( plga/-tcp ) scaffold via low - temperature deposition manufacturing ( ldm ) . in vitro and in vivo experiments had proved that the scaffold had favourable mechanical strength , high parity ratio , adjustable biodegradation rate , and facility of process and molding , which satisfied the essential requirements of the scaffold for the bone tissue engineering . however , the hydrophobic surface of plga/-tcp is not adequate for cell adhesion , proliferation , and osteoblastic differentiation [ 6 , 7 ] , which limited the repairing ability of the scaffold . satisfactory hydrophilicity and favourable biocompatibility of the scaffold could guarantee the cells to adhere , proliferate and differentiate , and it also could promote infiltration of oxygen and nutritive material of the body fluid inside the scaffold , which is vital to the successful repair of bone defects . therefore , it is important to modify the surface of the scaffold to achieve satisfied surface characteristics for cell adhesion , proliferation and differentiation . ma reported that water - absorption ratio of the porous scaffold could be improved remarkably by covering the surface of the scaffold with collagen . some studies reported that the adhesion , proliferation , and the differentiation to osteoblast directionally of the bone mesenchymal stem cells ( bmscs ) could be improved when the cells cultured on the type i collagen [ 911 ] . for these reasons , we have fabricated the core - sheath structure composite scaffold composed of plga/-tcp skeleton wrapped with type i collagen on the surface . the core - sheath structure composite scaffold was a 3-dimensional structural bone bracket stuff fabricated by a kind of controllable tachy - forming novel sprayer via ldm . its materials and structure of the scaffold are fairly biomimetic , and the production of the scaffold is based on the bionic principle through simulating human bone actual architecture . the aim of the present study was to analyze the physical properties and the biocompatibility of the core - sheath structure composite scaffold in comparison with plga/-tcp skeleton in vitro . physical properties were evaluated by means of analyzing the pority ratio , aperture , compressive strength , and young 's modulus . the morphology of the scaffolds and the cells on the surfaces of the scaffolds were investigated by scanning electron microscope ( sem ) . the hydrophilicity was assessed by means of water absorption , and the proliferation of the cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay ( mtt ) . the function of the differentiated bmscs was monitored by measuring alkaline phosphates activity ( alp ) of the cells . plga ( la / ga = 75/25 , mw = 104900 , mn = 92210 , pi = 1.14 ) was purchased from department of medical polymers shandong institute , and -tcp ( particle size = 1.9  0.7  m ) was purchased from chemical material factory of wenzhou . plga and -tcp were both dissolved by 1,4-dioxane , and the plga and -tcp solution concentration was 25% . type i collagen was derived from fresh grown rats ' tails following schor 's method   in institute of orthopaedics and traumatology , xijing hospital , the fourth military medical university . type i collagen was dissolved by 0.2  mol / l acetic acid , and the collagen solution concentration was 0.8  wt% . the plga/-tcp skeleton was fabricated by a ldm system as we reported before [ 1316 ] . the core - sheath structure composite scaffold was also fabricated by a ldm system ( designed by the department of mechanical engineering of tsinghua university ) . briefly , plga , -tcp ( plga/-tcp = 7  :  3 ) , and 1,4-dioxane were mixed at the room temperature to make a homogeneous slurry , and the plga/-tcp solution concentration was 25% ; type i collagen was dissolved by 0.2  mol / l acetic acid at the room temperature to make a homogeneous slurry , and the collagen solution concentration was 0.8  wt% . we prepared a drive pipe constructed with an inner pipe and an outer pipe , the terminals of which were equipped with annular tubes nozzles separately . then the slurry of plga/-tcp and the solution of type i collagen were divided into the inner pipe and outer pipe of the drivepipe separately , and then extruded from a computer - controlled annular tubes nozzle line by line onto a platform or frozen materials in a low - temperature room simultaneously . the slurry and solution would both solidify quickly after being extruded , and , at the same time , the plga/-tcp and the type i collagen would combine tightly when one layer finished , the platform moved down one - layer height and started a new layer . the nozzle moved in x - y plane alternately , and the platform moved in z direction repeatedly until construction of the scaffold was completed . then the frozen scaffold was freeze dried in a freeze dryer , through which the micropores ( less than 10  m ) formed as the phase separation process happened in the low - temperature room . sem ( s-3400n ; hitachi , tokyo , japan ) was used to observe the surface and the microstructure of the scaffolds and the interface configuration of the plga/-tcp and type i collagen . before the observation , each sample from the two groups was freeze dried and coated with a gold layer using a sputter coater . pority ratio was evaluated according to the apparent densities method following vaz et al . . their apparent volumes were calculated after measurement of the apparent dimensions of the cubes , and the samples were freeze dried in a freeze dryer for 24  h , then the weights of the dry samples were measured to an accuracy of 10 g ( n = 3 ) . the apparent density was calculated as follow : the total pority ratio  pt was calculated according to 
 ( 2)pt=(1o)100 , 
 where m is the mass of dry samples , v is the volume of the samples ,  is the apparent density of the samples , and o is the standard density of the samples . scaffolds with the size of 6.5  6.5  13.5  mm were prepared , and material testing was carried out using a computer - controlled electronic universal material testing machine ( shimadzu , kyoto , japan ) to determine the compressive strength and the young 's modulus . the samples were compressed using a standard method with speed - controlled compression force of 1  mm / min . scaffolds with the size 1  1  1  cm were used for water absorption test . samples with the required size were immersed in phosphate - buffered saline ( pbs , ph 7.4 ) , and the weight of each sample was recorded ( wi ) . then the plga/-tcp skeleton and the core - sheath structure composite scaffold were incubated in 500  ml pbs and maintained in a humidified incubator at 37c for 7 days . the samples were then removed from the pbs , gently blotted with filter paper to remove surface water , and immediately weighed ( ws ) . the water - absorption ratios were calculated using the following equation : 
 ( 3)water - absorption ratio=[(wswi)(ws)]100% . each sample from two groups with the size 1  1  0.4  cm was sterilized with ethylene oxide sterilization method . the sterilized scaffolds were immersed into low - glucose dulbecco 's modified eagle media ( lg - dmem , hyclone , utah , usa ) complete medium ( containing 20% fetal bovine serum ( fbs ; hyclone , utah , usa ) and 100  units / ml penicillin and 100  g / ml streptomycin ) for 12  h before cell seeding . bmscs were obtained from the femurs and tibiae of a 1-day - old new zealand white rabbit . the bmscs were incubated at 37c in a humidified environment of 5% co2 , in flasks containing the lg - dmem complete medium . the medium was changed every 2 - 3 days . when the culture flasks became confluent , cells were detached with 0.25% trypsin ( sigma - aldrich , st . osteogenic differentiation was induced by culturing bmscs in osteogenic medium ( dmem supplemented with 10% fbs , 10mol / l dexamethasone , 10  mm -glycerol phosphate , and 50  mg / ml l - ascorbic acid ) ( sigma - aldrich , st . then , the cells were digested from the culture flasks by incubation with 0.25% trypsin , centrifuged for 5  min at 1000  rpm , resuspended with lg - dmem medium seeded onto the top of the scaffolds , and placed in a polystyrene 6-well plate , and incubated at 37c to allow the full adhesion of cells to the scaffolds . briefly , 250  l of the cells suspension were seeded at each scaffold in 6-well plates at a cellular density of 1  10  cells / ml for the cell proliferation assay . after 4  h , 2d , 6d , 10d , and 14d of culturing , the proliferation state of the bmscs on the surfaces was analyzed by mtt at od 490  nm ( n = 3 ) . the morphologies of mscs seeded on the scaffolds after culture for 4h , 2d , 6d , and 14d were observed by sem . the samples were taken out of the culture plates , washed with pbs three times , and fixed in 3% glutaraldehyde in ph 7.4 pbs for 24  h at 4c , and the cells were then rinsed in pbs twice , postfixed in 1% osmium tetroxide , dehydrated in a graded series of alcohol and subsequently critical point dried , and then sputter coated with a thin layer of gold for observation under the scanning electron microscope operated at 5  kv . the relative alp activity was determined according to colorimetric method with p - nitrophenyl as the substrate . briefly , the cells in both groups were collected as before on 6d , 10d , and 14d . then the cell pellets were rinsed with pbs for three times and lysed with sonication . sample volumes of 0.1  ml were mixed with 0.1  ml p - nitrophenyl phosphate ( pnpp ) in 0.1  m glycine ( ph 13.0 ) , and the mixture was incubated at 37c for 30  min on a bench shaker . the enzymatic reaction was stopped by the addition of 0.3  ml of 0.25  n naoh . enzyme activity was quantified by absorbance measurements at 405  nm for the amount of p - nitrophenol ( sigma - aldrich , st . similarly , nonseeded biomaterials were prepared in an identical manner and analyzed as blank controls . all data are expressed as mean value  standard deviation and analysed by spss 13.0 , and t test was used for hypothesis testing . figures 1(a ) , 1(c ) , and 1(e ) and figures 1(b ) , 1(d ) , and 1(f ) are the magnified image of the side face of the plga/-tcp skeletons and the core - sheath structure composite scaffolds . compared with the plga/-tcp skeleton , the surface of the construction unit of the core - sheath structure composite scaffold is totally wrapped by the type i collagen . figure 1(g ) shows that the interface of the plga/-tcp and type i collagen is combined tightly . table 1 shows that the pority ratio of the plga/-tcp skeleton and the core - sheath structure composite scaffold are 88.1  1.2 and 86.7  3.6 , respectively , and the statistical analysis demonstrated that there was no significant difference in the pority ratio of the two scaffolds . figures 1(a ) and 1(b ) illustrate that the macropore dimensions of the two scaffolds are both 300500  m in size . the compressive strength of the plga/-tcp skeleton and the core - sheath structure composite scaffold are 0.68  0.04 and 0.70  0.07 , and the young 's modulus are 17.91  2.12 and 18.16  3.21 , respectively table 1 . the results indicated that there was no significant difference between the two scaffolds in both the compressive strength and the young 's modulus . the water - absorption capacity of the plga/-tcp skeleton is 2.8  0.2% , and the core - sheath structure composite scaffold is 16.1  0.3% table 1 . the result of mtt indicated that the number of bmscs in the core - sheath structure composite scaffolds was greater significantly than that of the plga/-tcp skeleton at different intervals from day 6 ( p < 0.05 ) , and the number of bmscs in two kinds of scaffolds was both increased from day 2 to day 14 figure 2 . on hour 4 , the differentiated bmscs adhered to the core - sheath structure composite scaffold and the pseudopodium of the cells stretched out figure 3(a ) . at day 2 , the cells stretched onto the core - sheath structure composite scaffold and the pseudopodium of the cells tightly attached to the surface of the scaffold figures 3(c ) and 3(d ) . at day 6 , the cells clustered on the core - sheath structure composite scaffold and were abounded with pseudopodium figures 3(g ) and 3(h ) . at day 14 , the cells covered the surface of the porous core - sheath structure composite scaffold or extended toward the pores figures 3(m ) and 3(n ) , what 's more , the bmscs secreted ecm figures 3(k ) and 3(l ) . obviously , there were fewer cells adhering to the plga/-tcp skeleton proliferation compared with the core - sheath structure composite scaffold figures 3(b ) , 3(e ) , 3(f ) , 3(i ) , 3(j ) , 3(o ) and 3(p ) . figure 4 shows that the alpase expression on each scaffold is increased substantially from day 6 to day 14 , and significantly higher activities on 6 , 10 , and 14 days of cultivation on the core - sheath structure composite scaffolds are observed as compared with the control groups ( p < 0.05 ) . in the present study , we analyzed a core - sheath structure composite scaffold for bone tissue engineering applications . such a scaffold may be particularly suited for implantation into bone defects due to its admirable physical properties and appropriate biocompatibility . ideal scaffolds for bone tissue engineering should have appropriate 3-dimensional porous structure , favourable mechanical strength , adjustable degradation rate , and facility of process and molding . however , it is practically impossible to satisfy the numerous requirements for scaffold materials by using a single material ; therefore , composite systems , which combine the advantages of different materials , are becoming more and more promising . one class of such composites comprises polymers - inorganic materials which come up to those standards . the materials we chose for the composite scaffold were synthetic polymers plga and inorganic material -tcp . but hydrophobicity of the composite material 's surface counts against the adhesion and proliferation of the cells [ 6 , 7 ] . the hydrophilicity of the scaffold 's surface is one of the important factors that facilitate the penetration of nutrients into the scaffold and the exudation of the metabolic products , which thus guaranteeing the nutrition transport and cell growth inside the scaffold . it is acknowledged that material surface will contact and interact with the cells first , so the superficial physicochemical property of the material such as hydrophilicity or hydrophobicity can influence a series of the cells ' responses : adhesion , proliferation , differentiation , and so forth . a lot of studies have authenticated that extracellular matrix ( ecm ) plays a very important part in the adhesion , proliferation , and differentiation of the cells in the surface of the materials , and type i collagen containing tropocollagen molecule is the basis of the ecm and possesses favourable hydrophilicity and low immunogenicity . so it would probably improve the superficial hydrophilicity of the composite material effectively if the surface of the composite material is wrapped with type i collagen . therefore , we chose plga , -tcp , and type i collagen as the raw and processing materials and fabricated the core - sheath structure composite scaffold so as to improve the hydrophilicity of the scaffold . from figure 1 , we can see that the plga/-tcp is totally wrapped toroidally by type i collagen and that the covering layer is well distributed . figure 1(g ) indicates that the contact surface of plga/-tcp and type i collagen is tight . the cementation of the plga/-tcp and type i collagen mainly depends on the diffusion action : the plga/-tcp solution ( plga/-tcp dissolved in the solvent 1,4-dioxane ) and the type i collagen solution ( type i collagen dissolved in the solvent 2% acetic acid ) contacted each other in the process of the molding , and the 1,4-dioxane and 2% acetic acid could mutually be dissolved very well , and the sizes of the two solvents were far less than the two solutes ( macromolecule chain ) , so the two solutions could interdiffuse quickly and smoothly with each other . both of the plga/-tcp molecular chain and the type i collagen polypeptide chain diffused to the contact surface , and then solidified , and the colliquefaction part was shaped , producing the joint part of the plga/-tcp and type i collagen . appropriate pority ratio and pore size are crucial to bone tissue engineering , which can produce a biological environment conducive to adhesion , proliferation of cells , and the smooth transport of nutrients and metabolic waste [ 14 , 20 ] . these properties are also necessary for bone tissue formation because migration and proliferation of osteoblasts and mesenchymal cells as well as vascularization could be allowed . results of this study indicate that both scaffolds have fairly high pority ratio ( over 75% ) , which is considered to be beneficial to cell growth and survival . the pore size should be large enough to support cell migration and bone ingrowth such that cell coverage , pore bridging formation and occlusion can be prevented . the optimal pore size required for bone ingrowth has been suggested by some researchers to be up to 200  m . the macropore dimensions of the novel scaffold are 300500  m in size , which indicates that the scaffolds have the proper pore size for bone tissue engineering . the values of compressive strength and young 's modulus of the two kinds of scaffolds are both lower than those of healthy bone . however , varieties of orthopedic fixations have been found to be widely used in clinic , which could ensure the fastness of the fixation during bone regeneration and degradation of the artificial bone . therefore , as a regenerative scaffold , its basic mechanical strength could be satisfied by the embedded artificial bone if only it can hold its shape when compressed by the surrounding soft tissue . in our experiments , the core - sheath structure composite scaffolds had a compressive modulus of 0.70  0.07  mpa , which meets the above standard very well . on hour 4 , the bmscs had adhered to the novel scaffold and the pseudopodium of the cells had begun to stretch out ; obviously , the bmscs had also adhered to the plga/-tcp skeleton however , there were fewer cells adhering to it figures 3(a ) and 3(b ) ; from day 2 to day 14 , the bmscs stretched onto the core - sheath structure composite scaffold , and the pseudopodium of the cells tightly attached to the surface of the scaffold , and then the cells proliferated and clustered on the scaffold , furthermore , at day 14 , the cells had extended into the pores , in addition , and after the scaffolds and the cells were cultivated together for 14 days , we observed the cells attached with the scaffold had secreted ecm ; obviously , there were fewer proliferation of the cells on the plga/-tcp skeleton figures 3(c)3(p ) . from the sem observation , we can conclude that the core - sheath structure composite scaffold had better biocompatibility , which is an important principle property of scaffolds and is considered ideal for bone tissue engineering . the results of mtt also indicated that the bmscs showed better adhesion and proliferation activity on the surface of the core - sheath structure composite scaffold than on the plga/-tcp skeleton , which were consistent with the results of the sem observation . there are two important pillars in the bone tissue engineering field , biomaterials and cells . the former must permit the rapid grow and proliferation of the seeded cells while keep their potential for further differentiation , which is one of the key processes for bone regeneration . alp is a kind of cell surface glycoprotein , which is involved in mineralization   and is the most widely recognized marker of osteoblastic differentiation . according to the results , the core - sheath structure composite scaffolds possessed a greater ability to promote the osteoblastic differentiation of bmscs than that of the plga/-tcp skeletons , which also demonstrate that the core - sheath structure composite scaffold has comparatively good biocompatibility . adhesion of cells on the scaffolds and cells is very complex and is influenced by various factors . surface properties of the scaffold such as electrical characteristics , free energy , hydrophilicity , or hydrophobicity are crucial to the cell adhesion , because the real touch between the scaffold and cells occurs between the material surface and the cell . the results of our study demonstrated that the hydrophilicity of the core - sheath structure composite scaffold was improved significantly , and it is acknowledged that favourable hydrophilicity is beneficial to cell adhesion . cells contacting the surface of the scaffolds were the first to attach and adhere ; the quality of this adhesion would influence their morphology , and their capacity for proliferation and differentiation . the core - sheath structure composite scaffold 's surface was covered with type i collagen , which could improve the hydrophilicity of the scaffold , and was probably the reason for the better cell adhesion , proliferation activity , and differentiation . the results of our present study indicated that the novel scaffold was more suitable for adhesion , proliferation , and differentiation of the bmscs than the plga/-tcp skeleton , which that demonstrated that the core - sheath structure composite scaffold possessed good biocompatibility . in this study , we created a core - sheath structure composite scaffold with an annular tubes nozzle by the ldm system . the scaffold has an open pore structure with the pore size up to 300  m , and over 75% of the appropriate pority ratios have been achieved . moreover , the scaffold has achieved a better hydrophilicity , and bmscs have adhered , spread , proliferated , and osteogenically differentiated well on the scaffolds . so , we may conclude that such a scaffold may act as an ideal implant into bone defects thanks to its favourable physical properties and biocompatibility . all these data may provide further proofs that the core - sheath structure composite scaffold has potential in bone tissue engineering .
scaffolds play a critical role in the practical realization of bone tissue engineering . 
 the purpose of this study was to assess whether a core - sheath structure composite scaffold possesses admirable physical properties and biocompatibility in vitro . 
 a novel scaffold composed of poly(lactic - co - glycolic acid)/-tricalcium phosphate ( plga/-tcp ) skeleton wrapped with type i collagen via low - temperature deposition manufacturing ( ldm ) was prepared , and bone mesenchymal stem cells ( bmscs ) were used to evaluate cell behavior on the scaffold . 
 plga/-tcp skeleton was chosen as the control group . 
 physical properties were evaluated by pority ratio , compressive strength , and young 's modulus . 
 scanning electron microscope ( sem ) was used to study morphology of cells . 
 hydrophilicity was evaluated by water absorption ratio . 
 cell proliferation was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay ( mtt ) . 
 osteogenic differentiation of bmscs was evaluated by alkaline phosphates activity ( alp ) . 
 the results indicated that physical properties of the novel scaffold were as good as those of the control group , hydrophilicity was observably better ( p < 0.01 ) than that of control group , and abilities of proliferation and osteogenic differentiation of bmscs on novel scaffold were significantly greater ( p < 0.05 ) than those of control group , which suggests that the novel scaffold possesses preferable characteristics and have high value in bone tissue engineering .