| Part Ⅰ Evaluation of optimized decellularization method of IVD and effectsObjective:To evaluate optimized decellularization method of New Zealand White rabbits of IVD and the biochemical propertyies, microstructure & mechanical properties.Methods:IVD samples were collected from the thoracic and lumbar segments of New Zealand White rabbits that were aged approximately four months old (n= 55, of which 45 were used for the in vitro study and ten for in vivoresearch). ICDs were separated randomly as Group A, Group B & Negative Control Group, which were related to different treatment. H&E staining, Alcian Blue staining Immunohistochemistry of Collagen type Ⅱ, Collagen type Ⅰ & Aggrecan of sections of IVP tissues were conducted to evaluate biochemical characteristics in all samples. The content of total genomic DNA of all groups was extracted and isolated, using a DNeasy Blood & Tissue Kit in accordance with the manufacturer’s protocol. The DNA content was measured with a NanoDrop 8000. As a marker amino acid of collagen, the content of HYP can be measured using a spectrophotometric method. The amount of HYP in the samples was determined by a calibration curve with a linear region between 0.2 and 1 mg/100 mL, prepared from HYP standards. The mass changes of normal and decellularized IVDs after freeze-drying were calculated to evaluate the water content. SEM and TEM were performed to examine the micro-architecture of the normal and decellularized IVD. The mechanical properties of AF and NP after decellularization or normal were measured using uniaxial tensile and dynamic strain tests, which performed using a computercontrolled test machine, including Elastic modeulus, ultimate stress & maximum elongation of AF and Compressive modulus of NP.Results:The method used in Group A is the optimized decellularization method of IVD, which is "placed in a 37℃ water bath and liquid nitrogen in rapidly alternating treatments for five cycles.2% Triton X-100 in deionized water was applied to soak the IVDs for 24 h on an orbital shaker at 100 rpm and at 4℃. Second,1% SDS in deionized water was applied for 24 h to further remove the nuclear remnants and cytoplasmic proteins under the above conditions.200 U/ml DNAase in PBS was applied with shaking at 100 rpm and at 37℃ for 12 h. Finally, the IVDs were rinsed with PBS for 6 h to remove the residue reagents effectively. We demonstrated that DID and IVD did not show significant difference in biochemical properties, biomechanical properties & microstructures.Conclusions:Overall, we demonstrated the optimized decellularization method is an ideal method to decellularize IVD, since by this method biochemical properties, biomechanical properties & microstructures of IVD can be preserved perfectly and maximally remove cells in IVD.Part II The Biocompatibility of DIDObjective:To evaluate the biocompatibilyi of DID in vivo & in vitro.Methods:We gathered MSCs form bone marrow of four-month-old New Zealand White rabbits and isolate the MSCs. Then we cultured the MSCs in different concentration of DID leaching solution, using Live-Dead cell staning & CCK-8 kit to infer whether DID leaching soluntion is toxic to the MSC in vitro. We seeded MSCs on DID ECM scaffolds, using Live-Dead cell staning & H&E staining to infer whether DID ECM scaffolds interfere MSC proliferation in vitro. Allograft transplant was used to test the immunogenicity of decellularized IVD. Decellularized IVDs (n= 3) and normal IVDs (n= 3) were separately implanted into corresponding pockets through the incision. One month after implantation, implants were removed and prepared for histological evaluation by submersion in 4% PBS-buffered paraformaldehyde (PFA). H&E staining & immunohistochemistry for MAC387 and CD8 were applied to infer immunoreaction from the hosts to the implants.Results:The histological evaluation indicated a high degree of host cell penetration into the native IVD, whereas only a few host cells were scattered in the decellularized IVD. Neutrophils were observed extensively scattered around the grafts, which suggested a severe inflammatory response. A large number of infiltrating CD8+T-cells and macrophage cells were found in untreated allogenic IVD group, However, the percentage of positive cells of was fewer in decellularization group. Besides, the in vitro cytocompatibility of decellularized IVD was tested by cell counting kit-8 (CCK-8) and Live-Dead cell staining suggesting decellularized IVD has good cytocompatibility in vitro. Live-Dead cell and H&E staining indicated long-term viability of the MSCs after being seeded for 3,7,14 and 21 days. The live cells (green) showed that the majority of the seeded cells migrated into the inside of decellularized disc from day 3 to day 21.Conclusion:DID shows great biocompatibility both in vitro & in vivo. DID will not induce the immunoreaction or immflamation in the hosts, rather than allogenic IVD. Besides, DID will not affect MSC proliferation and even MSC seeding on the surface of DID can migrate to the inner part of DID.Part Ⅲ Decellularzied allogeneic IVD prevented disc degeneration in vivoObjective:To confirm decellularzied allogeneic IVD can induce MSCs differential to IVD-like cells and prevent disc degeneration in vivo.Methods:SEM was performed to examine the shape of the MSCs which were seeded in the decellularized IVD. Gene expression of a panel of IVD cell markers was evaluated (include Col Ⅱ, Col Ⅰ, SOX-9, GPC3 and AGN) by RT-PCR.The experiment in vivo, the model of IVD degeneration was induced by the validated rabbit puncture. L4-L5 and L3-L4 IVDs were sequentially punctured with a 22-gauge needle to a depth of 5 mm, and then, the surgical incisions were closed. L5-L6 was left uninjured as the normal control. L3-L4 received saline water injection and L4-L5 decellularzied allogeneic IVD microparticles. The continuous and dynamic changes of IVD about water content index and disc height in different groups at 0 month,1 month,2 months, and 3 months were demonstrated. H&E staining and Alcian blue staining demonstrated microstructure of IVD.Results:SEM found MSCs seeded in the decellularized IVD, which translated into a column or strip-like shape, and the cell shape was similar to normal intervertebral disc ceils. A group of classic IVD cell markers, Col Ⅱ, Col Ⅰ, SOX-9, GPC3, AGN, were significantly greater than in the non-seeded MSCs at different time points.In the vivo experiment, the water content index showed that decellularized IVD maintained a higher hydration level compared to the saline group. MRI showed that the disc height of the decellularized IVD treated group was also slightly better than the saline group. Moreover, in the decellularized IVD treated group, the histological morphology of collagen staining by H&E did not show obvious changes compared with the control group. However, in the saline group, the inner layer of the AF lost the concentric lamellar structure with cracks, and the NP was also disorganized. Alcian blue staining also shows that the obvious loss of GAGs in the saline group. But this loss was not apparent in the decellularized IVD treated group.Conclusions:Collectively, these data suggested that decellularized IVD could prevent IVD degeneration. |
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