The Study On Multi-specific Customized 3D Printing Of Autogenous Skull For Bone Regeneration Of Rabbit Critical Size Skull Defects Repair

At present,three-dimensional(3D)printing has been gradually applied to the manufacture of cranioplasty implants such as titanium,polymethyl methacrylate and polyetheretherketone,making the processing of such shape-specific implants more convenient,efficient and accurate.However,with the goal of cranioplasty has begun to progress from shape reconstruction to bone regeneration,the current 3D printing methods are still mostly focused on the shape of the skull implants to match the defects and are short of optimal materials and cells,which obviously does not meet the bone regeneration requirements for skull defects repair.To date,among the biomaterials that have been proven to promote bone regeneration,there are still some shortcomings in most of them,such as high cost,exogenous and limited bone regeneration capacity.Long-term exogenous material implantations and the use of allogenous cells are likely to lead to foreign body reactions and immunological rejections.Autologous skull flaps are truly patient-specific in terms of biological origin and are considered to be the optimal cranioplasty materials.However,aseptic osteonecrosis-induced bone flap resorption always occurs in some cases,which is believed to be related to traumatic brain injury,fragmented skull flaps,large-scale defect size and delayed reimplantation.In addition,children in critical skull growth stages are more susceptible to bone flap resorption due to active bone remodeling.In this work,we propose a multi-specific customized 3D printing strategy,including shape specificity(shape),material specificity(autogenous skull matrices,Auto-BM)and cell specificity(autogenous cells).We plan to fabricate material-specific 3D printed autogenous bone implants(3DP-Auto-Bone)applying autogenous skull flaps,and to construct multi-specific autogenous bone implants(Multi-Auto-Bone)by combining 3DP-Auto-Bone with autogenous bone mesenchymal stem cells(Auto-BMSCs).Furthermore,the osteogenic capacity of Multi-Auto-Bone will be evaluated in vitro and in vivo.It is expected to provide a potential true patient-specific solution for skull defects repair and bone regeneration.Part I The Realization of Specific Shapes and Structures for 3DP-AutoBones and the Evaluation of Physicochemical Properties for these SamplesObjective: To explore the feasibility of establishment for an Auto-BM based 3D printing system;To test the stability,utility and diversity of this 3D printing system in terms of specific shapes and structures;To evaluate the physicochemical properties of 3DP-AutoBones under this 3D printing system.Methods: Lyophilized Auto-BM(autologous bone flaps of rabbits)was cyro-ground into particles,the microstructure of particles were observed under scanning electron microscope(SEM)and the particle size distribution was measured with a laser particle size analyzer.The Auto-BM particles were mixed with poly(ε-caprolactone)(PCL)ink prepared from various solvents to obtain Auto-BM slurry.The printability of bone slurry based on different solvents was evaluated.Under optimized print ing parameters,simple models and imagingbased human scale models were printed in different filling rates and interlayer offset.The microscopic morphology of 3DP-Auto-Bones was observed under SEM,and the mechanical strength test was performed with a Universal Testing System.L929 cells were incubated with sample extracts to evaluate the cytotoxicity of samples.Results: The particle yield of the Auto-BM was about 47%,and the particle size was in micron scale.The Auto-BM slurry with glacial acetic acid(GAA)as a solvent was extruded smoothly and was easy to shape,22 G was the minimum size of the nozzle through which the slurry can pass.The models can be solidified in water alone.The GAA-based Auto-BM slurry can be used to print samples in various structures and specifications.There were sponge-like microstructures under SEM.Young’s modulus of 3DP-Auto-Bones was 3.50 ± 0.73 MPa,and the cytotoxicity reached 0 after 72 h washing by water.Conclusions: 1)It is feasible to establish a 3D printing system with Auto-BM as raw materials.2)There are considerable printing stability,utility and diversity of Auto-BM slurry system based on GAA/PCL ink.3)There are certain mechanical strength and biosafety of 3DP-Auto-Bone.Part II The Isolation,Culture and Multilineage Potential Identification of Auto-BMSCsObjective: To Isolate and culture Auto-BMSCs using bone marrow blood in vitro,and confirm their multilineage potential by performing differentiation of multicellular lineages.Methods: Bone marrow blood was harvested from the upper parts of the femur of rabbits.Mononuclear cells were collected by density gradient centrifugation and cultured by adherent screening method,and proliferation of passage 3(P3)Auto-BMSCs was detected by Alamar Blue assay.P3 cells in exponential phase growth stage were induced to differentiate exclusively into the adipocytic,chondrocytic,and osteocytic lineages to confirm their multilineage potential.The cells were labeled to ensure that their origin and corresponding skull flaps are the same individual.Results: Auto-BMSCs were spindle shaped,arranged in bundles and more homogeneous in morphology when subcultured to passage 3(P3).P3 Auto-BMSCs came into exponential phase growth stage on day 3,and reached the plateau stage on day 13.nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate(NBT/BCIP)staining of alkaline phosphatase(ALP)and Alizarin Red S staining of extracellular calcium were positive after osteogenic induction,Oil Red O staining of intracellular lipid droplets was positive after adipogenic induction,and after chondrogenic induction Alcian Blue(p H 1.0)staining of extracellular sulfated glycosaminoglycans was also positive.Conclusions: 1)There are considerable growth activity and multilineage potential of AutoBMSCs.2)Auto-BMSCs are expected to be combined with 3DP-Auto-Bone to construct Multi-Auto-Bone.Part III The Assessment for Auto-BMSCs Affinity of 3DP-Auto-BonesObjective: To investigate the initial adhesion rate,the proliferation and the viability of Auto-BMSCs on 3DP-Auto-Bone.Methods: The CCK-8 assay was performed after P3 Auto-BMSCs in exponential phase growth stage were seeded in 3DP-Auto-Bone for 24 hours.The relative effective adhesion rate of BMSCs was normalized to the corresponding cell number of BMSCs cultured in 2D.The Alamar Blue assay was performed every other day from day 1 to detect the proliferation of Auto-BMSCs in 3DP-Auto-Bone.Live/Dead staining was performed at different time points to calculate the cell viability of Auto-BMSCs.The morphology of Auto-BMSCs in 3DP-Auto-Bone was observed under SEM.3D printed n HA bone(3DP-n HA-Bone)and 3D printed allogenous bone(3DP-Allo-Bone)with n HA and allogenous bone particles as printing materials,respectively,were set as controls.Results: There was the highest cell adhesion rate of 3DP-Auto-Bone to Auto-BMSCs.The cell number in the three 3D printed bones peaked on day 15 and remained for 4 days.The cell viability of BMSCs in 3DP-Auto-Bone and 3DP-n HA-Bone was significantly better than that of 3DP-Allo-Bone throughout the culture.On day 14,cells in 3DP-Auto-Bone under SEM were observed to be similar in morphology to osteoblasts,while cells in 3DPn HA-Bone still retained the cell morphology of BMSCs,and cells in 3DP-Allo-Bone showed signs of aging.Conclusions: 1)There are considerable cell affinity of 3DP-Auto-Bone to Auto-BMSCs.2)Auto-BMSCs may spontaneously differentiate into osteoblasts in 3DP-Auto-Bone.3)The combination of Auto-BMSCs and 3DP-Auto-Bone is expected to construct a Multi-AutoBone with bone regeneration capacity.Part IV In Vitro Osteogenic Capacity Evaluation of Auto-BMSCs in 3DP-Auto-BonesObjective: To explore the ability of Auto-BMSCs to spontaneously differentiate into osteoblasts in 3DP-Auto-Bone.To provide molecular biology support for the construction of Multi-Auto-Bone with bone regeneration capacity.Methods: ALP activity of BMSCs in 3DP-Auto-Bone was detected at different time points.Quantitative real-time polymerase chain reaction was applied to detect the expression of osteogenic differentiation-related genes such as runt-related transcription factor 2(RUNX2),ALP,type I collagen ALPHA 1(COL1A1),osteopontin(OPN)and osteocalcin(OCN)on day7,14,and 21 d.3DP-n HA-Bone and 3DP-Allo-Bone were set as controls.Results: The ALP activity of Auto-BMSCs in 3DP-Auto-Bone increased earlier and more significantly without exogenous osteogenic factors.All the trend of expression of osteogenic differentiation-related genes(RUNX2,ALP,COL1A1,OPN and OCN)suggestted that Auto-BMSCs in 3DP-Auto-Bone were more likely to spontaneously differentiate into osteoblasts.Conclusions: 1)Auto-BMSCs in 3DP-Auto-Bone can spontaneously differentiate into osteoblasts better.2)Auto-BMSCs and 3DP-Auto-Bone provides a good molecular biology basis for Multi-Auto-Bone construction.Part V The Study on Bone Regeneration of Multi-Auto-Bones in Rabbit Critical Size Skull Defects RepairObjective: To construct the Multi-Auto-Bone combining 3DP-Auto-Bone and AutoBMSCs.To validate the in vivo bone regeneration capacity of this multi-specific implant in a rabbit critical-sized skull defect model.Methods: Rabbit critical size skull defect models were created.3DP-Auto-Bone was fabricated and Auto-BMSCs were extracted as described above.Auto-BMSCs were seeded in 3DP-Auto-Bones to obtain Multi-Auto-Bones.One month after the models were established,each Multi-Auto-Bone was re-implanted,and each specimen was harvested after 3 months.The new bone mineralization were analyzed by micro computed tomography(Micro-CT).The new bone formation was further evaluated by HE staining,Masson trichrome staining,collagen I and OCN immunohistochemical analyses.The new bone angiogenesis was evaluated by von Willebrand factor(v WF)immunofluorescence analysis.The same method was also applied to 3DP-Auto-Bone(cell-free)and 3DP-n HABone(both cell-seeded and cell-free)to assess their in vivo osteogenic capacity.Results: Micro-CT analysis results suggested that Multi-Auto-Bone promoted mineral formation and deposition to form mineralized new bone.HE staining showed that the interior new bone was formed around the filaments of Multi-Auto-Bone and significant active osteoblasts were observed at the edge of the new bone.Most of the new bone was dyed red by Masson trichrome staining,suggesting that the new bone was mature bone.The immunohistocheical staining of collagen I in new bone was positive,indicating that the new bone was still active in osteogenesis.v WF immunofluorescence staining showed that there were endogenetic blood vessels in some new mature bones.New bone was also generated around the filaments of 3DP-Auto-Bone(cell-free).However,its bone maturity and osteogenic activity were both weaker than the Multi-Auto-Bone.Only a little initial formation of osteoid tissue was observed in 3DP-n HA-Bone,and a very small amount of new bone was generated when BMSCs were bound.Conclusions: 1)There is considerable inherent bone regeneration capacity of Multi-AutoBone,which is the prototype of an osteoregenerative patient-specific skull repair implant.2)The fabrication of this multi-specific skull implant provides potential feasibility for true patient-specific skull defects repair.3)With weaker in vivo bone regeneration capacity,3DP-Auto-Bone(cell-free)are available as an alternative for Multi-Auto-Bone.