Research On Injectable Silk Fibroin/Polyuretane Composite Hydrogel For Nucleus Pulposus Replacement

Background:Degenerative disc disease (DDD) affects the30-to-50-year-old population and contributes to acute and chronic disability. Currently, there is no ideal treatment for degenerated intervertebral discs. The most common therapy is non-operative and consists of physical therapy and analgesics. However, the symptoms may progress with age or other genetic and environmental factors. Many physicians resort to spinal fusion and discectomy to treat the condition. However, spinal fusion is associated with the accelerated degeneration of adjacent discs and an uncertain outcome.According to Tibrewal and Pearcy, following surgery, disc height can decrease, compared with non-operated controls, after a patient undergoes discectomy. The introduction of nucleus replacement has at least theoretically opened a new avenue treating DDD. Several studies have evaluated the biomechanics and clinical outcomes of this new technology.Nucleus replacement offers many potential advantages in the treatment of DDD. It is a less invasive surgical procedure and preserves the remaining disc tissues (i.e., the annulus and endplates). Additionally, the height and mobility of the intervertebral disc are maintained and the overloading of the adjacent levels, which is often a result of spinal fusion, is prevented.The material used to replace a degenerated nucleus pulposus should be a synthetic hydrogel. Numerous synthetic hydrogels are known, most of which are highly biocompatible, and their physical and mechanical properties can be altered to mimic the behaviour of the natural nucleus. Perhaps the most successful nucleus prosthesis is PDN-Solo, developed by RayMedica (Minneapolis, USA). This implant has a hydrogel core packed in a woven polyethylene jacket.One of the drawbacks of the PDN-Solo prosthesis is that it does not completely fill the cavity left by the removed nucleus. Filling the cavity entirely is essential for achieving physiological stress distribution within the disc and minimising implant migration. Moreover, PDN is much stiffer than the original nucleus pulposus, which may eventually result in endplate failure with subsidence and extrusion. PDN is also a highly invasive technique with an anterior approach that involves a lengthy rehabilitation period.Here, we report initial studies on a new hydrogel biomaterial that may be used as a nucleus prosthesis. This material should satisfy four criteria:(1) the hydrogel prosthesis should be radiopaque,(2) the hydrogel prosthesis should be non-cytotoxic,(3) the transformation of the solution gel should allow the entire cavity to be filled completely and minimise the size of the surgical defect to that of a needle, and (4) the swollen hydrogel should exhibit sufficiently strong physical and mechanical properties and fatigue resistance. For example, the prosthesis must resist continuous loading, and the peak loading that occurs upon bending or lifting objects.We report an injectable hydrogel biomaterial that potentially meets these criteria. Filling the entire cavity makes the new implant more biomimetic than the PDN-Solo. In this study, we developed an injectable silk fibroin/polyurethane composite hydrogel, with barium sulphate added to enable monitoring of the hydrogel. Using this material, nucleus prosthesis prototypes were designed and studied. To evaluate the hydrogel with respect to further applications, the study will investigate (1) the synthesis of a silk fibroin polyurethane composite hydrogel,(2) its unconfined compressive behaviour,(3) the impact of height on its biomechanical properties,(4) its rheological properties,(5)a cell proliferation assay,(6) SEM observations of the hydrogel,(7) its implantability, and (8) its visibility via X-rays and magnetic resonance imaging (MRI). Additionally, the steps that must be taken before the prosthesis can be introduced clinically are briefly discussed.PART Ⅰ Synthesis of silk fibroin polyurethane composite hydrogelOBJECTIVE:To systhesis silk fibroin polyurethane composite hydrogel for nucleus pulposus replacement.METHODS:The IPDI, N-220, and PEG-1000were weighed into a dry three-neck flask equipped with a magnetic stirrer, nitrogen inlet, and air condenser attached to the flask through a distillation head. The reaction mixture was stirred and heated to90℃for2h under a nitrogen atmosphere. Then, the DMPA, BDO, acetone, and tin caprylate were added to the flask; the temperature of the flask was decreased to70℃for approximately3h. The polyurethane prepolymer was then transferred to a single-neck flask.Silk fibroin solution (5wt%) was added to the flask in a1:1ratio with polyurethane prepolymer by volume. The reaction mixture was stirred at room temperature (25℃) and atmospheric conditions.RESULTS:silk fibroin polyurethane composite hydrogel was synthesised in room temperature.CONCLUSIONS:silk fibroin polyurethane composite hydrogel was synthesised in room temperature.PART Ⅱ Unconfined compression tests OBJECTIVE:To investigate unconfined compression mechanical property Of silk fibroin polyurethane composite hydrogel for nucleus pulposus replacement.METHODS:Hydrogel samples were made using silk fibroin and polyurethane, as described above. Samples were immersed in phosphate buffered saline (PBS) solution (pH=7.4) at25℃and unconfined compression tests were performed after1and14days of immersion. The hydrogel samples were loaded into a Zwick Z2.5compression bench with a2500-N load cell (Zwick/Roell Z2.5, Zwick GmbH&Co., Ulm, Germany). The initial diameter and height of the cylindrical hydrogel were measured using a Vernier calliper. Swollen cylindrical samples (15.09±0.06mm in diameter and16.07±0.13mm in height, after equilibrium swelling) of the hydrogel were compressed at a strain rate of100%strain/min. Load and displacement data were recorded at10Hz using testXpert II software. Both stress-strain values and a tangent compressive modulus for each hydrogel sample were calculated at15%,20%, and25%of the compressive strain.RESULTS:Hydrogels can be compressed to by more than70%.The hydrogels did not break, even at80%compressive strain. The tangent modulus values at15%,20%and25%strain were0.30,0.34and0.39MPa, respectively.CONCLUSIONS:the hydrogels was more suitable mechanical properties for nucleus pulposus replacement, showing its potential in clinical application.PART Ⅲ correlating the height features with their biomechanical propertiesOBJECTIVE:To correlate the height features with their biomechanical properties.METHODS:specimens (n=3for the16.60mm group with cylindrical specimens measuring15.12mm in diameter and16.60mm in height, n=3for the14.60mm group with cylindrical specimens of15.12mm diameter and14.60mm height) were subjected to mechanical testing using the Zwick Z2.5compression system, as described above. The two groups had the same diameter and different heights.RESULTS:In all cases (Fig.7), the tangent modulus values at15%,20%and25%strain (3/3of the16.60mm group,3/3of the14.60mm group) observed in the16.60mm group were higher than those of the14.60mm group. The tangent modulus values of the16.60mm group at15%,20%and25%strain were0.2-fold,0.22-fold, and0.21-fold higher than the corresponding values of the14.60mm group(0.31±0.01MPa vs.0.25±0.00MPa, p<0.05;0.36±0.00MPa vs.0.30±0.00MPa, p<0.05;0.41±0.01MPa vs.0.33±0.00MPa, p<0.05).CONCLUSIONS:Our preliminary findings on the impact of height indicated that the tangent modulus values increased with an increase in cylinder height.PART IV Rheological propertiesOBJECTIVE:To study the visco-elastic behaviour of the hydrogels.METHODS:To evaluate the rheological properties of the formed hydrogels, rheological tests were performed on swollen discs (diameter25of mm, thickness of2mm) using a parallel plate viscometer (ARES3LSLC1, Rheometric Scientific) at37℃.The samples (n=4) were compressed to a standard force of approximately0.5N. Dynamic frequency tests were carried out on all samples at a controlled strain of0.01%.RESULTS:The frequency-dependent behaviour of|G*|and G’of the hydrogel is shown. Furthermore, the loss modulus G" is always smaller than the storage modulus G’, indicating that the hydrogels show mostly elastic behaviour. The phase shift angle was less than45℃, which indicates that the hydrogels exhibit more elastic than fluid-like behaviour.CONCLUSIONS:the hydrogels show mostly elastic behaviour. we found that the hydrogels studied herein were stiffer and more elastic than natural NP tissue. PART V Cell proliferation assayOBJECTIVE:To evaluate the cytotoxicity of the silk fibroin polyurethane composite materials.METHODS:The cytotoxicity of the materials was evaluated. Bone marrow stromal cells (R-BMSC) were seeded on the material surface at3×105cells/sample/well (thickness0.2cm/sample). The cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium (L-DMEM) supplemented with10%oetal bovine serum (FBS) at37℃for1,3,5, or7days (n=4per time point) in24-well plates. The absorbance was measured at570nm. The cell number was correlated with the optical density (OD).RESULTS:The possible cytotoxicity of our materials (after washing) was assessed using a methyl thiazolyl tetrazolium (MTT) assay and SEM analysis, all of the surfaces showed R-BMSC proliferation, indicating excellent biocompatibility of the hydrogels in direct contact with cells. Nearly all of the cells on the surfaces of our materials were viable. SEM images of showed the cell growth and proliferation after3days on the surface of a hydrogel.CONCLUSIONS:all of the surfaces showed R-BMSC proliferation, indicating excellent biocompatibility of the hydrogels in direct contact with cells.PART VI SEM observationsOBJECTIVE:To observe cross-sectional morphology of the swollen composite hydrogel.METHODS:Scanning electron microscopy (SEM; Hitachi S-3000N, Japan) was used to observe the cross-sectional morphology of the swollen composite hydrogel.RESULTS:The surfaces of the hydrogels contains many pores, which vary in size, although most were smaller than10μm in diameter.CONCLUSIONS:silk fibroin and polyurethane have good compatibility based on SEM assays.PART VII Hydrogel injectionOBJECTIVE:To access the silk fibroin polyurethane composite hydrogel injection.METHODS:The annulus of the disc was cut anterolaterally using a surgical blade. A small incision was made through the annulus to reach the nucleus cavity. As much of the gel-like nuclear material as possible was removed through the small hole. Then, our injectable hydrogel, made from silk fibroin and polyurethane, was injected into the area of degeneration or treated site through a small-gauge needle.The native hydrogel was carefully injected into the nucleus cavity.RESULTS:The hydrogel was injected into degenerated or treated sites easily using a small-gauge needle through a small incision in the annulus fibrosus. The implant was observed to swell in situ to almost1.3times the volume of the dry hydrogel, which should impede the extrusion of the hydrogel.CONCLUSIONS:The hydrogel was injected into degenerated or treated sites easily using a small-gauge needle through a small incision in the annulus fibrosus in room temperature. PART Ⅷ Hydrogel VisualisationOBJECTIVE:To access the silk fibroin polyurethane composite hydrogel visualization.METHODS:To verify the position of the native hydrogel inside the nucleus cavity, CT scans and MRI were used. CT scans were performed with a64multidetector CT scanner (Brilliance64, Philips). For the purposes of the study, CT images were digitally stored and analysed at the workstation (Extended Brilliance Workspace V3.5.0.2254). the implants can also be visualized clearly using a T2-weighted MRI sequence. This study used1.5T MR units (Signa HD Excite, General Electric Healthcare, Milwaukee, USA) at room temperature..After the scans, the disc was sectioned to expose the implant, which was photographed.RESULTS:Both techniques can clearly visualise the hydrogel implant in the nucleus cavity. CT provides three-dimensional information-. Additionally, due to the high water content, the hydrogel can be observed clearly via MRI. The hydrogel shows adequate X-ray visibility between two vertebrae. The intervertebral discs were then sectioned to reveal the hydrogel within the nucleus cavity. This confirmed the imaging results, i.e., the injectable hydrogel completely filled the nucleus cavity.CONCLUSIONS:The silk fibroin polyurethane composite hydrogel have good visualization.