Radially Porous Nanocomposite Scaffolds With Enhanced Capability For Guiding Bone Regeneration In Vivo

Objective:To prepare radially porous chitosan(CS)/hydroxyapatite(HAp)nanocomposite scaffolds,and to detect the cytocompatibility,cell activity in vitro and in vivo histocompatibility,and finally to explore the ability to guide bone regeneration in bone defect while excluding interference from non-osteogenic cells and fibrous tissues.Methods:1)Firstly,the porcine femur was obtained from the local market,and the attached muscle,fascia,periosteum and cancellous bone and bone marrow in the medullary cavity were removed clearly to obtain the tubular cortical bone.After removing the remaining fat and collagen by physical and chemical methods,HAp was obtained.CS solution was simply mixed and stirred with HAp to prepare homogeneous CS/HAp slurries,and then the radially porous CS/HAp composite scaffold was constructed by directional freeze-casting.2)The prepared HAp powder was characterized by X-ray diffraction(XRD),Fourier Transform infrared spectroscopy(FTIR)and transmission electron microscopy(TEM).Then,the scaffolds were characterized by photography,optical microscope(OM)and field emission scanning electron microscope(FESEM)equipped with energy dispersive spectrometer(EDS).The morphology,pore structure,pore size and element distribution of the scaffolds were observed.The 3D architecture of the scaffold was scanned by micro-computed tomography(Micro-CT).The porosity and pore size of the scaffold were measured by mercury intrusion method.The mechanical characteristic was tested using an Instron 5565 A instrument equipped with a 500 N load cell,and the compressive stress was imposed on the transversal section with a compression rate of 0.2 mm min^-1.3)Bone marrow mesenchymal stem cells(BMMSCs)were isolated from the femur and tibia of 3-week-old Sprague-Dawley rats(SD)and cultured in vitro.When the cell confluence reached 80%?90%,the cells were digested with trypsin-ethylenediamine tetraacetic acid(EDTA),and the cells in P3?P4 were used for subsequent experiments.The proliferation status and morphological characteristics of BMMSCs in P0,P1,P2 and P3 were observed by inverted microscope and photographed.The specificity antigens on the surface of BMMSCs,such as CD29,CD90,CD45 and CD11b/c,were identified by flow cytometry.4)BMMSCs were co-cultured with axially and radially porous CS/HAp composite scaffolds and porous CS scaffolds,and the cytotoxicity was detected on day 1,3 and7 of culture.BMMSCs were seeded on axially and radially porous CS/HAp composite scaffolds and porous CS scaffolds respectively,and the proliferation,adhesion,activity and morphological changes of BMMSCs on the scaffolds were observed on day 3 and 7 of culture.5)The osteogenic differentiation capacity of BMMSCs were detected by alkaline phosphatase(ALP)staining.The axially and radially porous CS/HAp composite scaffolds and CS porous scaffolds were co-cultured with BMMSCs.ALP staining was performed to observe the ALP production on day 7 and 14 day of culture,respectively.6)Axially and radially porous CS/HAp composite scaffolds and porous CS scaffolds were implanted into the subcutaneous pocket on the back of SD rats.The scaffolds were retrieved on 14 and 28 days after implantation,respectively.The inflammatory response and the invasion of surrounding cells and tissue into scaffolds were detected histologically after samples photography.7)A rabbit bone defect model was established.Thirty-five New Zealand white rabbits of 2.5Kg were randomly divided into 4 groups.After fixation and disinfection under anesthesia,a 3 cm incision was made on both distal femurs to peel off the skin layer,subcutaneous tissue and muscle layer by layer to expose the femoral condyle.Subsequently,a cylindrical defect 6.5 mm in diameter and 7 mm in depth was created on the lateral condyle of the femur with a dental drill,which was penetrated from the cortical bone to the cancellous bone.The resultant defect was rinsed with normal saline to remove bone fragments.Bone defects were repaired in four different ways:Blank group:the created bone defects have not any further treatment,and the wound was directly closed by suturing;Radially porous CS scaffold treatment group:the prepared radially porous CS scaffold was implanted into the bone defect;Axially porous CS/HAp treatment group:the prepared axially porous CS/HAp composite scaffold was implanted into the bone defect;Radially porous CS/HAp treatment group:the prepared radially porous CS/HAp composite scaffold was implanted into the bone defect.The animals were sacrificed after 6 and 12 weeks respectively,and knee joint specimens were obtained.The knee joint specimens were observed and photographed.Micro-CT was used to evaluate and compare the repair efficacy of bone defects in each group.The repair efficacy of bone defect was further observed and evaluated by hematoxylin-eosin staining(HE)and Masson trichromatic staining(MT).Results:1.The successful preparation of nanoscale HAp was confirmed by relevant detection means.Subsequently,the radially and axially porous CS/HAp composite scaffolds were successfully fabricated by freezing-casting method.2.The average porosity and pore size of the radially porous CS/HAp scaffold were97.63%,the pore width was 84?m while the pore length was 219?m.The average porosity and pore size of the axially porous CS/HAp scaffold were 90.23%,the pore width was 98?m while the pore length was 230?m.The vertical compression test showed that the mechanical strength and elastic modulus of the porous CS/HAp composite scaffold were significantly higher than that of the porous CS scaffold,and the yield strength of the axially porous CS/HAp composite scaffold was higher than that of the axially porous CS scaffold.In addition,the axially porous CS/HAp composite scaffold could support a larger compressive load under the same strain(60%),but began to plastically deform at a much lower strain(approximate 10%),while the radially porous CS/HAp composite scaffold had a larger elastic region on the stress-strain curve.3.BMMSCs were successfully isolated from SD rats,and observed under inverted microscope after adhering proliferation,the cells in P0 and P1 generations showed spindle shape and polygonal shape,while there were also many impure cells.When the cells passed to the P2 generation,they showed a uniform distribution and morphed into a long spindle,arranging as fibers and reticulated forms;The P3 generation showed long spindle and fibrous morphology,and the cell pseudopodia were gradually extended.4.For in vitro biocompatibility assessment,CCK-8 assay confirmed that both CS and CS/HAp scaffold groups had significant cell proliferation after 7 days of culture,which was comparable to that of blank control group(without scaffold).On days 3and 7 of culture,fluorescein diacetate(FDA)fluorescence staining further confirmed the non-toxicity of CS and CS/HAp scaffolds,and showed the distribution and migration behavior of the BMMSCs in different scaffold structures.The observation of cytoskeleton and nuclear fluorescence staining by confocal laser scanning microscope(CLSM)revealed that the BMMSCs exhibited good adhesion and proliferation on the channel walls in both kinds of CS/HAp scaffolds after 3 days of culture.After 7 days of culture,the BMMSCs exhibited well-developed cytoskeleton and fusiform morphology distributed along the pore channels in these scaffolds.Further observation by SEM showed that the BMMSCs spread relatively finite filopodia on the channel walls of both kinds of CS/HAp scaffolds after 3 days of incubation.After 7 days of incubation,cell density increased significantly,and the BMMSCs extended more filopodia and completely spread onto the channel walls of both kinds of CS/HAp scaffolds.5.In order to determine the effect of the CS/HAp composite scaffolds on BMMSC osteogenic differentiation,in vitro alkaline phosphatase(ALP)assay was performed on the cell-seeded CS/HAp composite scaffolds with different porous structures and different contents of HAp.The ALP activity on the CS/HAp composite scaffolds(1:2)with higher content of HAp was evidently higher than that of the CS/HAp(1:1)and pure CS scaffolds after 7 and 14 days of culture,respectively.Furthermore,the radially porous scaffolds did not show obvious difference in ALP production compared to the axially porous scaffolds during the co-culturing time.Notably,ALP expression in both the composite scaffolds was enhanced greatly with induction time.6.In order to evaluate in vivo biocompatibility and tissue responses of the CS/HAp composite scaffolds with different structures,these CS/HAp composite scaffolds(1:2)were implanted subcutaneously in rat for a certain time.Photograph images of the harvested samples showed that both kinds of CS/HAp composite scaffolds were coated with a thick layer of fibrous capsule after 14 days in vivo,while the thickness of the fibrous capsule reduced obviously after 28 days in vivo.Histological evaluation via HE staining of the harvested samples showed the obvious aggregation and infiltration of inflammatory cells around the samples on day 14,which was reduced on day 28,respectively.We could see the obvious invasion of fibrous tissue into the radially porous scaffold from its periphery,and into the axially porous scaffold from its both ends.Notably,the invasion depth and area of fibrous tissue were remarkably enhanced after 28 days of implantation.However,cells and fibrous tissue were not easy to penetrate into the scaffolds perpendicular to the channel direction due to the absence of open pores.The HE staining of the implanted CS/HAp composite scaffolds indicated their superior in vivo biocompatibility.Additionally,both kinds of the porous structures were conducive to the ingrowth of cells and fibrous tissue along the pore channels.7.To demonstrate the superiority of the CS/HAp composite scaffolds with radial structure relative to that with axial structure in terms of regeneration efficacy of bone defects,we performed in vivo evaluation using the models of rabbit femoral defects.The bone repair effects were first evaluated by micro-CT analysis with 3D reconstructions and 2D cross section slices,which indicated that all the scaffold treated groups showed larger new bone formation at the edge of the defects after 6weeks,in contrast to the blank group.Furthermore,the newly formed bone could effectively penetrate into the radially structured CS/HAp composite scaffolds along the peripheral pore channels.Nevertheless,the newly formed bone was mainly distributed around the periphery of axially structured CS/HAp composite scaffold.The quantitative analysis of the micro-CT results showed that the radially structured CS/HAp composite scaffold group had the largest value of new bone volume/total defect volume(BV/TV)among all groups.The value of bone volume(BV)was also highest in the radially structured CS/HAp composite scaffold group.Moreover,the radially structured CS/HAp composite scaffold group had significantly higher trabecular number(Tb.N)value than other groups,while the trabecular separation(Tb.Sp)value was the lowest among all groups.8.The microscopic details of the bone defect areas were further validated via histological examinations.The HE and MT staining were performed to assess the penetration of new bone at 6 and 12 weeks after implantation.As we expected,after6 weeks of implantation,the fibrous tissue was found to be blocked outside of the top surface of the radially structured CS/HAp composite scaffold due to the absence of open pores,while the fibrous tissue could penetrate into the axially structured CS/HAp composite scaffold along the open pore channels from its top surface.Notably,the radially structured CS scaffold was found to suffer from obvious degradation.After 12 weeks of implantation,all groups showed increased formation of new bone.As indicated in HE and MT staining images,newly formed bone could be obviously observed to penetrate into the radially structured CS/HAp composite scaffold along the pore channels.In contrast,newly formed bone was still distributed around the axially structured CS/HAp composite scaffold,and its internal pore channels were covered by a large amount of fibrous tissue.Conclusions:1.CS/HAp nanocomposite scaffolds with radially porous structure were successfully constructed by modified freeze-casting method.2.The radially porous structure of the CS/HAp composite scaffold showed structural and mechanical anisotropy,and the compressive deformation resistance was enhanced,which could avoid structural collapse.3.The CS/HAp nanocomposite scaffold had good biocompatibility and was beneficial to cell adhesion and migration along the pores in vitro.In addition,in vivo animal studies had shown that CS/HAp composite scaffolds could induce a low inflammatory response,and the radially porous structure could promote the migration and infiltration of resident bone repair related cells into the scaffolds along the pores,while blocking the non-osteogenic cells and fibrous tissues invading into the scaffolds from the longitudinal direction,ensuring the normal bone remodeling process.