Abstract
Introduction
Nucleus pulposus (NP) replacement offers an alternative minimally invasive treatment for traditional spinal fusion or total disc replacement for degenerative disc disease (DDD). Recently, a novel polyurethane scaffold (PUS) with swelling capability in situ was developed to restore the mechanical functions of the intervertebral disc (IVD). In addition, a fibrinogen–hyaluronic acid (FBG–HA) conjugate-based hydrogel was manufactured to mimic the native NP extracellular matrix. The aim of this study is to evaluate the PUS and the FBG–HA hydrogel, with/without transglutaminase crosslinked bone morphogenetic protein (TG-BMP) 2/7 heterodimer in an organ culture system under dynamic load.
Materials and Methods
Discoid/ravioli-shaped polyurethane scaffolds were manufactured with a PU hydrogel-based core with swelling capacity in between two electrospun nanofiber envelope sheets. In addition, FBG–HA conjugate solution was synthesized with 235 kDa HA at FBG/HA w/w ratio of 17:1. FBG–HA hydrogels were prepared by mixing ⅔ volume of FBG–HA conjugate solution with ⅓ volume of thrombin solution (5.2 U/mL). Bovine IVDs with endplates were nucleotomized by incision through the endplate and refilled with either (1) PUS, (2) PUS surrounded by 50 to 80 µL FBG–HA or (3) PUS surrounded by 50 to 80µL FBG–HA containing 5,000 ng/mL TG–BMP 2/7. The endplate defect was closed with an endogenous endplate stopper and sealed with polymethyl methacrylate. Empty discs served as negative controls. To assess the mechanical compatibility of the different implants, dynamic compressive stiffness modulus (DCSM) was measured for each disc at different time points: intact, after nucleotomy, after refilling with biomaterial and free swelling recovery, after 3-hour dynamic load at 0 to 0.1 MPa, 0.1 Hz within a bioreactor system, and after free swelling recovery overnight. Moreover, biomaterials were evaluated in an organ culture system under dynamic load for 14 days at 0 to 0.1 MPa, 0.1 Hz for 3 hours/d. Disc height was recorded after load and recovery at day 1, 7, and 14. After 14 days, disc tissue was harvested and gene expression analyzed using real-time PCR. Glycosaminoglycan- (GAG) and collagen-content of the disc tissue was assessed and proteoglycan synthesis was analyzed by Sulfur-35 (35S) incorporation measurement. Histology was performed using Safranin O/Fast Green staining. One-way ANOVA was used to determine the statistical significance.
Results
After dynamic load, all three implant groups maintained their disc height, while it dropped by 7% in the empty controls (p < 0.001 vs. implant groups). The PUS and addition of FBG–HA hydrogel caused immediate restoration of DCSM (PUS: 73 ± 21%; p < 0.05 vs. empty control: 25 ± 5%). While addition of FBG–HA hydrogel surrounding the PUS delayed the stiffness restoring effect, stiffness also increased to 69 ± 16% after dynamic load and free swelling recovery (p < 0.05 vs. empty control: 27 ± 5%). GAG-/collagen-content was maintained in all three implant groups. Aggrecan and collagen-2 gene expression were upregulated in NP cells by TG-BMP2/7 and FBG–HA hydrogel, respectively, although not significantly.
Conclusion
Results indicate that PUS is able to restore the mechanical functions of nucleotomized discs. Addition of FBG–HA hydrogel does not affect the swelling capacity of PUS in situ. FBG–HA hydrogel and TG-BMP2/7 may further support the biological repair of NP tissue.
Acknowledgment
Funded by the European Commission under the FP7–NMP Project NPMimetic.