| Summary: | 碩士 === 國立成功大學 === 生物醫學工程學系 === 103 === The Hill-Sachs lesion is usually caused by shoulder dislocation at glenohumeral joint which is a ball and socket structure, and the dimension of humeral head is larger than glenoid fossa which makes it unstable and easily being dislocated. The dislocation causes the superior labrum anterior to posterior lesion and impinges into the bone, making engaging Hill-Sachs defect that is participated for the range of motion (ROM) in the 30% to 40% circumscribed. The Hill-Sachs lesion has been reported in high possibility of recurrent anterior dislocations.
When the instability of the shoulder gets worse caused by Hill-Sachs defect, the patient will suffer severe pain and the defect should consider surgical repair. Several autologous grafts and synthetic materials have been used as graft material; however, their availability and biocompatibility limit their clinical outcome. Thus, this study investigates to treat the Hill-Sachs lesion with injectable alpha Calcium Sulfate Hemihydrate (αCSH).
It is possible to be used inside the human body as bone filler. Virtually, the αCSH has been noticed having the characteristics of the osteo-induction and osteo-conduction in vivo; however, it has never been applied as bone filler to repair the Hill-Sachs defect. This study tests the possibility of using αCSH to repair the Hill-Sachs defect and was divided into three parts including αCSH characterization, in vitro biocompatibility of αCSH and in vivo Hill-Sachs model in rabbits:
Part 1 Material preparation:
The preparation of αCSH was from heat treatment of Calcium Sulfate Dihydrate (CSD, J.T. Baker) and the heat treatment conditions were evaluated by Differential Scanning Calorimetry (DSC) and Thermogravimetry Analysis (TGA). The result of DSC curve had first endothermic peak (△H=713.54 J/g) at 120-140℃ which means 2H2O converts to 1/2H2O, and there is the second endothermic peak (△H=144.54 J/g) at 140-150℃ for 1/2H2O to non-aqueous. Also, the TGA curve of CSD and CSH shows the decreasing in weight around 119.5℃. Combined the DSC and TGA results we defined the heat treatment temperature at 127℃ under 5kg/cm2 pressure for 9 hours. In the results of X-ray Diffraction (XRD) which match with JCPDS card and Jade system were to identify the completeness of phase transformation. The primary crystal planes of CSD were located at (020), (021), and (041). After 9 hours heat treatment the crystal planes of CSD were convert to αCSH at (010), (310), and (202). In spite of that the XRD still cannot specific determine the kind of crystallize (α or β phase), the microstructure from scanning electron microscopy (SEM) was used to further identify the crystal formation. In the SEM images of CSD with wider arrangement which the particles size were 163.06±11μm in length and 28.45±6.53μm in width (N=5). The microstructure of αCSH were with needle-shaped which particles size were 92.02±63μm in length and 17.31±2.4μm in width (N=5), from H2O evaporation.
In addition, the compressive strength of αCSH was tested by Materials Test Systems by following the ASTM 451-99a. The compressive strength of αCSH hardening with DD water was at 5.39±0.42MPa (N=6) and the αCSH with curing agent was enhanced the compressive stress to 9.62±2.05MPa (N=6). However, the curing time of αCSH/DD water and the αCSH/HCl were with no significant difference at 10-14 minutes and with proper hardening time for surgery. (N=6)
Besides, the degradation of materials was with similar degradation rate about 25% weight loss after 49 days and the pH value at 5.5-6.0. However the αCSH/ HCl group has a stronger chemical bounding which can effectively control the release of calcium ions in the results of EDS after 49 days. All of the scaffolds had no significant change for the appearance after 49 days degradation. Therefore, the microstructure of hardening material of αCSH/ DD water has an interlocking structure with 58% of porosity (N=10). However, the material of αCSH/HCl demonstrated the glue-like structure with 47% of porosity (N=10). Both materials have a pore size of 10-100 μm which is adequate dimension for cell migration and attachment.
Part 2 in vitro test:
A human osteoblast-like cell line, MG63 (Homo sapiens bone osteosarcoma), was used to test the biocompatibility of αCSH/DD water, αCSH/HCl, and 15% alginate and followed by ISO-10993-5. The cytotoxicity of αCSH/DD water and αCSH/HCl group which co-cultured with 5×104 cells in 24-well plates in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% Fetal Bovine Serum, 1% penicillin-streptomycin and saturation concentration of material powder. The alginate was coated with 0.1 cc material on the 24-well plates and co-cultured with 5×104 cells. After the incubation of cells with materials for 1, 3, 5, and7 days, absorbance was set at 490 nm to evaluate the cell viability in response to the cytotoxicity of materials. The results of MTS assay shows the co-cultured cells with materials had no significant difference of cytotoxicity compared with control group. (P〉0.05)
The cell attachments of materials were followed by ASTM F813-83. The scaffolds were seeded with 5×104 cells on the surface and incubated in 24 well plates, observed at 1, 3, 5 days. They were dehydrated in a graded series of ethanol, treated with 10% paraformaldehyde, coated with gold, and observed by SEM. In the results of microscopy demonstrated that cells were extended and attached on the scaffolds successfully after 1 day. Cells were spread all over the surface of scaffolds and with plenty of secretion, after 5 days. The SEM shows the cells had migrated into the cut in halve scaffold at day 5.
In bounding test, the humeral heads of pork were used in the push out test. The material without alginate drops out from the bone defect after immersing in Hank’s solution for 5 minutes. However, the material with alginate was effectively attached on the bone defect and the bounding force had reached to 1.73±0.26 MPa (N=6)
Part 3 in vivo test:
The Hill-Sachs model was built by creating a defect on rabbit’s humeral head. The size and location of defect to mimic the Hill-Sachs defect was verified by histology and micro-CT after sacrificed. Defect size was 25% of humeral head and the diameter was 7.5mm and the depth was 5mm. The deflection depth was too deep which destroy the bone marrow and cause angiostasis which lead to a failure of attaching the materials into the bone defects. Therefore, the materials were injected two weeks after the surgery.
The process of mixing αCSH and hardening solution are1:1 ratio at room temperature with stirring for 60 seconds then it was injected with 15% alginate.αCSH hardening reaction took 10 to 11 minutes of curing time at body temperature (37 °C) The functionality of repairing Hill-Sachs defect after αCSH injection were evaluated by radiography and histology at 4 and 12 weeks postoperatively.
CT analysis shows, the bone remodeling had tissue regeneration starting from the edge of defect and the residue of materials were showed on the 2D image after 4 weeks. In contrast, the defect without treatment had no obvious tissue repaired on the 2D image after 4 weeks. However, the material had been completely replaced by the newly bone after 12 weeks. In the result of 3D reorganization, the bone defect had well appearance after 4 and 12weeks (N=6). The value of BV/TV showed that the material groups had obvious difference with defect groups (P〈0.05). Furthermore, there were no significant difference compare with defect group treated with materials after 12 weeks and the control group. (P〉0.05)
The histology applied with HE and Masson trichrome staining by paraffin sections for 4μm was use to confirm the bone regeneration. In the results of histology, the bone repaired started from the edge, the plenty of osteoblasts had migrated into the material and the connective tissue showed the connection between newly bone and the material after 4 weeks. However, the defect group had no tissue regeneration on histology after 4 weeks. Besides, the bone regeneration of material group has been completed, comparing with the defect group which was 100-700μm regenerated after 12 weeks.
The αCSH/HCl/Alginate provided a simple preparation of injectable performance with great biocompatibility in vitro and bone repaired, which has a potential in repairing the Hill-Sachs defect surgery.
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