Study On Repairing Mandible Defect With Application Of Computer Aided Surgical Technique And 3D Printing Technique In Dogs

BackgroundAutogenous bone transplantation is widely used in clinic to repair oral and maxillofacial bone defects.Such repair can induce trauma of the tissue of the implant at donor site,and the operator needs to shape the defect according to the size of the defect,which is greatly influenced by the subjective factors,and the postoperative appearance is often unable to meet the requirements of the patient.Simultaneously,in traditional operation,there is a possibility of excessive excision or limited resection.Furthermore,traditional repair requires a long operation time and high technical level for the surgeon,which will exert great impact on the physiology and psychology of patients.The rapid development of computer-assisted surgical technique,3D printing technology and tissue engineering repair technology has brought new possibilities for lesion resection and defect repair in the oral and maxillofacial region.Autogenic bone marrow mesenchymal stem cells(Auto-BMSCs)has been well developed to construct tissue engineeredbone(TEB)for bone defects repair.Besides,computer-assisted surgical technique and 3D printing technology have been preliminarily applied to the clinical practice of bone defect repair.However,there are fewer devices for 3D printing biological scaffold materials that is also characterized by high cost,which brings difficulties for laboratory research and future clinical application.Furthermore,the common complex method of constructing TEB is to implant the seed cells in the body after they are amplified and co-cultured with scaffolds for 1~2 weeks,which increase the time for the implantation and the risk of TEB contamination greatly.ObjectiveAiming at designing animal mandibular defect model,surgical guide plate and scaffold mold with computer-assisted surgical technique,surgical guide plates and scaffold molds were printed quickly by using 3D printing technology,and nano-hydroxyapatite scaffolds were fabricated by high-temperature sintering.Furthermore,TEB was constructed on the basis of the combination of scaffolds with auto-BMSCs after osteoinduction.Subsequently,mandibular defect model of canine was established by using surgical guide plates,and TEB was implanted into the site of canine mandibular defect.The study was performed with the expectation of exploring the feasibility of using computer-assisted surgical technique and low-cost 3D printing technique to remove and repair mandibular defects of large mammals,and evaluating the difference with traditional surgicalmethod.This study was designed to understand the difference in situ osteogenesis and different co-culture time of TEB constructed by autogenous osteogenesis induced BMSCs combined with nano-hydroxyapatite scaffolds,so as to provide more available approaches and reference for the construction of TEB.Methods1.Bone marrow was extracted from beagle dogs by ilium puncture.BMSCs were separated with Ficoll separating medium via density gradient centrifugation,cooperate with amplification by adherence screening method and osteoinduction for BMSCs culture.Simultaneously,the second generation of BMSCs were selected for adipogenic differentiation.Furthermore,Alizarin red staining and Oil red O staining were used to identify the osteoinduction and adipogenic differentiation of BMSCs,respectively.2.Data related to the canine maxillofacial bones were obtained by CT scanning.Animal model was designed by computer-assisted surgical technique to simulate the removed whole bone tissue of bilateral mandibular body in the canine,which was about 18 mm in length,9mm in width and 5mm in height.The surgical guide plate and the scaffold mold were designed.According to the preoperative design,3D printing technology was used to fabricate surgical guide plates and scaffold molds.Besides,nano-hydroxyapatite scaffolds were made by high-temperaturesintering.3.The second generation of BMSCs generated by osteogenic differentiation was seeded on the nano-hydroxyapatite scaffold to construct TEB.TEB was then divided into 2 groups: TEB8D(control group),with co-culture of TEB in vitro for 8 days;and TEB2D(experimental group),with co-culture of TEB in vitro for 2 days.The growth of cells on scaffolds was observed by scanning electron microscopy.4.A mandibular defect model was constructed by using surgical guide plate to remove bilateral partial mandible of the experimental canine.The experimental canine mandibles were randomly divided into 3 groups: group1(blank control),with the implantation of nano-hydroxyapatite scaffolds;group 2(control),with the implantation of co-cultured TEB for 8 days;and group 3(experimental group),with the implantation of co-cultured TEB for2 days.Among them,in group 2,one side of the mandible was randomly selected,with the treatment with traditional resection and repair as control.Then,the difference in surgical difficulty and repair accuracy by applying computer-assisted surgical technique and 3D printing technique was compared with those of the traditional mandibular resection and repair.CT scanning was performed at 3 and 6 months after operation to observe the osteogenesis and degradation of materials.Furthermore,9 months after operation,the implants were removed for gross observation,Micro-CT scanning,scanning electron microscopy examination,hard tissue slicingand methylene blue/acid fuchsin staining.Results1.BMSCs extracted from canine bone marrow could proliferate and differentiate rapidly after osteoinduction and culture in vitro.Osteogenic induction of the second generation of BMSCs overexpressed osteoblast related markers,and appeared specific calcium nodule deposition.Furthermore,a large number of vacuolar lipid droplets appeared in the cytoplasm of the second generation of BMSCs after adipogenic differentiation,indicating that BMSCs could differentiate into adipocytes after adipogenic differentiation.2.Data of experimental canine mandible were obtained by CT scanning,and were imported into Geomagic Foundation 2013 software to generate 3D digital images.Using the MIMICS software,the animal model,the surgical guide plate and the implant mold were designed,besides,the3 D printer was used to print out the surgical guide plate and the material mold quickly.The implant material of nano-hydroxyapatite scaffolds was made by using the printed mold by high-temperature sintering.3.The osteogenic differentiated auto-BMSCs was seeded on nano-hydroxyapatite scaffolds to construct TEB.After scanning electron microscope,it was found that after the transplantation of the second generation of BMSCs into scaffolds in TEB8 D group and TEB2 D group,cells in the outer layer of the scaffold grew well and proliferated rapidly inboth groups.In addition,the number of adherent cells in the middle layer of the scaffold was greater than that in TEB8 D group,and the number of cells in TEB2 D group was less than that in TEB8 D group.4.By using the preoperative design of computer-assisted surgical technique,a canine’s bilateral mandibular defect model was established using surgical guide plates,and implanted into the scaffold accurately.In the traditional surgical group,the appearance of the defect was poor after resection that required temporary shaping of the implant material,the operation time was prolonged obviously,and the implant was basically anastomosed with the defect after implantation with naked eyes.The postoperative gross observation,as well as CT and Micro-CT scan showed that the operation group with computer-assisted surgical technique and 3D printing was in accordance with the original normal shape of the mandible.However,in the traditional surgical group,there was a mismatch between the material and the bone shape.The above results suggested that although the material shape was similar to the defect in the operation,there was still a deviation after the actual implantation,which might lead to the abnormal appearance of the material after osteogenesis.5.After implantation,bone mineral density of the implant was increased continuously in group 2(control group)and group 3(experimental group)at 3,6,and 9 months after operation,and eventually similar to the surrounding bone density.But there was no significantdifference in bone mineral density between the two experimental groups(P>0.05).Bone mineral density of TEB in group 1(blank control)was gradually decreased with time,and was significantly lower than that in group 2 and group 3 at 6 and 9 months(P<0.0001).At 9 months after operation,gross observation and Micro-CT scan showed that the TEB in group 2 and group 3 was healed with surrounding bone tissue,which could maintain the integrity of mandible.However,the implant material did not heal with the surrounding bone tissue in group 1,and the defect region was filled by soft tissues.Scanning electron microscopy revealed that there were large amounts of ordered collagen fibers similar to the structure of Haversian tube and mineral crystals in TEB of group 2 and group 3.Meanwhile,there were few spindle-shaped and polygonal cells on the surface of materials in group 1,and no signs of osteogenesis were found.In addition,hard tissue slicing and methylene blue/acid fuchsin staining showed that in TEB of group 2 and group 3,the structure of the Haversian system could be observed,associated with orderly arranged bone cells,red stained bone tissues and incompletely degraded scaffolds,and osseous connection was formed between the edge of the defect and the normal bone.In group 1,there was only tiny amount of bone tissue formation,and the scaffolds were degraded in varying degrees.Conclusion:1.Compared with traditional surgical resection,computer-assisted surgical technique can individualize the range of surgical excision before surgery.Besides,the combination of 3D printed surgical guide plate can ensure the rapid and accurate resection of the diseased bone tissues according to the preoperative design.2.The computer-assisted surgical technique is utilized for the designation of individualized implants.The 3D printer is used for rapid printing and the traditional high-temperature sintering method for scaffolds fabrication.The processing is convenient,fast,low-cost and high accuracy.The operation time of the present method is shorter with reduced difficulty,and the appearance is more in line with the normal bone morphology compared with traditional bone defect repair technology.It is quite suitable for the generalization and popularization of TEB research and clinical application in the future.3.Our experiments confirm that BMSCs seed cells play an important role in TEB formation,which is necessary to construct TEB and repair large-range bone tissue defects.Nano-hydroxyapatite scaffolds has good biocompatibility in vitro and in vivo,which can promote the growth and osteogenesis of seed cells,and can be used as a scaffold material of TEB for the repair of large-range bone tissue defects.4.When BMSCs combined with nano-hydroxyapatite scaffolds are used to construct TEB,there is obvious difference in the number of cells at the center of the scaffold following 8 days and 2 days in vitro culture,butno significant difference is observed between the two groups after in vivo implantation.It is confirmed that during TEB fabrication,the co-culture time of seed cells and scaffold materials can be shortened to 2 days.