| [Background]Tricortical autogenous iliac crest had been accepted as gold standard for fusion when anterior cervical discectomy and fusion was performed. But it is restricted because of source limited and inevitable complications of harvest. Bone graft substitutes include Polyetheretherketone cage and titanium mesh are excellent in bone fusion. But some shortcomings gain increasing attention, such as higher elastic modulus, subsidence, and postoperative imaging artifacts which interfere with imaging diagnosis. Especially both substitutes could not be absorbed, fusion in biology was irrealizable. The ideal absorbable cage should overcome these defects. Hard enough when implanted, strength reduce as the degradation process, load is gradually transferred to the host bone, which is beneficial to new bone formation and solid fusion. Finally, biology fusion was achieved when implant completely absorbed, and high-quality postoperative MRI imaging was granted. Three-dimensional printing is a kind of rapid prototyping technology, individual implants could be made according to the characteristics of CT imaging by three-dimensional printing. If a material has proper mechanical strength just like autologous bone, and degradation rate could be controlled in vivo, individual cage could be designed and fabricated according to CT imaging preoperatively by 3 d printing, and implanted in body when perform operation.[Objectives]1 Measurement of the degenerative cervical vertebra anatomy parameters to analyse why individual cage was needed in cervical spine;2 Fabricate a novel absorbable ND/PLGA materials; evaluated mechanics strength, biocompatibility and toxicity, to assess the feasibility of using in fabricating cervical cage;3 To explore the feasibility of fabricating individual cervical cage by three-dimensional printing, verify the accuracy and endplate matching of cage.[Materials and Methods]1 A total of 80 patients who were diagnosed of cervical spondylosis from June 2013 to June 2014 were enrolled. Including 40 males and 40 females. All patients accepted routine CT scan and 3d reconstruction preoperative. Various cervical spine parameters of CT were measured in the PACS system. Evaluated sagittal diameter, transverse diameter of vertebral and disc, as well as Luschka Joint diameter in CT axial plane. Cervical vertebral body, disc height and screw spacing were evaluated in sagittal plane.2 Hydrophilic ND particles were modified by amphipathic phospholipid with physical absorption for enhancing of dispersivity in dichloromethane containing PLGA. Evaluated the optimized ratio of ND and phospholipids. PLGA films loaded with NDPC to fabricate NDPC-PFs, and assessed the mechanical properties of ND/PLGA, evaluated the optimized proportion of NDPC and PLGA.3 For NDPC cytotoxicity test, the 1×105 of hFOB1.19 osteoblast cells were cultured in 24 h, with NDPC50 powers at 1 mg/ml,10 mg/ml,100 mg/ml and 1000 mg/ml, and pure ND powers at 1000 mg/ml, respectively. And the cytotoxicity of powers was assessed by cell viability with CCK-8 assay. Similarly, cell viability and proliferation on NDPC50-PFs were also assessed with same cells by CCK-8 assay in 7 days. Using the Quantitect SYBR Green PCR Kit quantitative detection of 3 kinds of osteogenic gene markers for cell differentiation, alkaline phosphatase assay(ALP), collagen type-1 (COL-1) and osteocalcin (OCN), were studied by Real-time RT-qPCR, respectively.to assess the biocompatibility of ND/PLGA;4 Healthy Kunming mice were employed for animal experiments for investigating the histological response and in vivo degradation of NDPC-PFs. All film sterilized by 75% (v/v) ethyl alcohol were implanted subcutaneously on the back in the haunch. For each film, every 3 male and 3 female mice were used for in vivo experiments for 2 weeks,4 weeks and 8 weeks, respectively. At expected observation time points, the films and around tissue were retrieved observed under optical microscopy for immunological evaluation.5 Ten adult cervical specimens were selected to take CT-scan. After original Dicom data imported into the Mimics software, the fifth and sixth cervical vertebra and disc model was created, which was used to design and generate the individual cervical cage based on a three-dimensional printing forming technique. The specimens accepted anterior cervical discectomy and fusion in C5/6. CT-scan was taken postoperatively to access the accuracy and endplate matching of cage.[Results]1 The sagittal diameter increased gradually from superior edge to inferior edge in each vertebral body. The sagittal diameter also increased from cranial to caudal in cervical spine, 16.75±1.16mm in inferior edge of C2,19.28±2.08mm in inferior edge of C6. The transverse diameter increased gradually from middle point to inferior edge in each vertebral body. The transverse diameter also increased from cranial to caudal in cervical spine,31.01±2.28mm in superior edge of C7. Transverse diameter of Luschka Joint also increased gradually from C3 to C7. Three was not tendency of increasing of the height of disc and vertebral body from cranial to caudal of cervical spine. The screw spacing was 16.96±1.95mm to 17.51±1.59mm based on current plate, higher than 12.84±1.53mm to 14.50±1.69mm of true screw spacing which needed in operation.2 With increase of phospholipid in the mixture, the precipitate of ND reduced signally. When the ratio of ND:PL (wt/wt) was less than 100:50 (NDPC50), the precipitate of ND disappeared. NDPC50 should be an optimal ratio of ND:PL. NDPC-PFs had a gradual change with adding of NDPC. NDPC50-PF10 had packaged more and bigger NDPC particles and aggregates in PLGA matrix. And the interconnected NDPC particle chains were formed in NDPC50-PF10. Addition of NDPC into the PLGA film enhanced the mechanical properties at all NDPC50-PLGA film samples. NDPC50-PF10 had highest value of Young’s modulus of 5.74 Gpa, and it was had approximate 100% increasing of Young’s modulus and approximate 550% increasing of hardness, in contrasted with pure PLGA.3 The cells viability of hFOB1.19 osteoblasts cultured on NDPC50-PFs was detected in 7 days. The result showed hFOB1.19 osteoblasts quickly proliferate on NDPC, closed to that on pure PLGA film and TCP as controls. At day 7, most cells inter-contacted to form cell population, and some pseudopodiums of cells were found tightly adhered on PLGA films. The expression of OCN, an important osteogenesis marker, was up-regulated significantly with decrease of ALP and COL in all samples, NDPC50-PF10 (10%wt NDPC50) had highest OCN expression, closed to that on TCP.4 Inflammatory cells and fibrocyte were visualized around implanted NDPC-PFs and PFO at week 4, implying severe level tissue inflammatory responses for implants. Only few inflammatory cells surrounded PFO or NDPC50-PF1, NDPC50-PF20 with high ND content showed worst inflammatory and fibrocyte areas, but it was acceptable. At week 8 of post-operation, the amount of inflammatory cells decreased signally for each sample. In addition, adding of NDPC could impede the fast degradation of PLGA, which is benefit to use in bone tissue engineering.5 Smooth surface was observed in entities Cage, the longitudinal pore can be clearly seen. The cage was smaller than digital cage, print accuracy higher than 95%, There was no statistically significant difference between STL format data and cage. Gross observation, the cage was affiliated to veterbral disc, and stability was good. No cage was mobility or too large to implant into veterbral disc.6 cases (60%) of specimens had excellent matching, 14 points (3.89%) match good in 3 cases (30%) of specimens. A total of 343 points (95.28%) had excellent match.[Conclusions]1 There were some individual differences among degenerative cervical vertebra, especially the difference the height of disc. The uniform cage cannot meet the clinical needs completely, it is necessary to design and fabricate individual cage for degenerative cervical spine.2 We have prepared a novel high mechanical property PLGA composite loaded with 10%wt of uniform nanodiamond-phospholipid compound (NDPC) particles. Based on phospholipid modification, NDPC particles can disperse in PLGA matrix and the act as nanofillers to enhance 100% of Young’s modulus and 550% increasing of hardness, and also change the PLGA film’s surface morphology and hydrophilcity. For human osteoblasts, the detections of biocompatibility and osteogenic gene markers explained NDPC and NDPC-PFs are nontoxic in vitro, and also support cell proliferation and osteogenic differentiation. Specially, we focused on in vivo behaviors in mice model, and verified NDPC-PFs with phospholipid-modified ND particles had acceptable immune response and can reduce fast-biodegradation of PLGA matrix. Due to combination of these good properties, we believe the PLGA composite with NDPC is a promising material for bone tissue engineering and regenerative medicine.3 The anatomical structures of cervical vertebral body and intervertebral disc can well present by mimics software, individual cervical cage could be designed based on the model. The rapid prototyping individual cage could be completed through three-dimensional printing, and the printing model matched host endplate accurately. |
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