Abstract
Introduction
Nucleus pulposus (NP) replacement by the application of injectable hydrogels seems a straightforward approach for tissue engineering. We investigate a thermo-reversible hydrogel (TH-RHG), based on a modified polysaccharide with a thermo-reversible polyamide (poly(N-isopropylacrylamide = pNIPAM). Using “click chemistry” and reversible addition fragmentation transfer (RAFT) polymerization,2,3 the gel is made to behave as a liquid at room temperature and hardens at >32°C. To inject the hydrogel, a mild papain disk degeneration model (PDDM) is employed that creates a cavity in the intervertebral disk. Here, we investigate the performance of the TH-RHG in situ in bovine IVD with preconditioned human mesenchymal stem cells (hMSC) with rhGDF-5.
Materials and Methods
IVDs were harvested from six calf tails, aged 6 to 9 months, which were received from a local abattoir within 3 to 5 hour of slaughter. The bovine IVDs of intermediate size (max. height of 2 cm and diameter of 1.5 cm) were used included the bony endplates and cultured in vitro for 16 days. Utilizing our previously established PDDM was necessary to create a cavity in the NP. For the papain injection, a 25 G needle was used to inject centrally the enzyme into the bovine IVD. Simultaneously, primary hMSCs expanded from bone marrow obtained from five patients undergoing spine surgery (ethically approved) were seeded at 4M cells/mL in 3-D in the TH-RHG and preconditioned with 100 ng/mL rhGDF-5 for 7 days. Afterwards, the TH-RHG was reversed to a fluid and injected into the IVD organ culture and kept under static loading of 0.1 MPa for 7 days using custom-designed specimen chambers.1 Experimental designs are as follows (i) fresh d0 control PBS injection (ii) PDDM + PBS injection (iii) PDDM + TH-RHG + bovine NPCs (Cell control) (iv) PDDM + TH-RHG + hMSC.4 Magnetic resonance imaging (MRI) was employed to image the hydrogel-filled cavity before and after loading at day 9 and day 16, and RT-PCR comparison of the hMSCs at day 1, 8, and 15 investigated the changes in gene expression. On the same days of cell sampling, cell viability (trypan blue) was determined. Sulfated glycosaminoglycan synthesis (DMMB assay) and DNA content (Picogreen assay) from anterior and posterior annulus fibrosus (AF) segments of each disk at day 16 were performed.
Results
MRI images confirmed a central positioning of the TH-RHG into the PDDM. A considerable drop in volume across all groups in the NP region and consequently of disk height was observed between day 9 and day 16. The RT-PCR results of injected hMSCs showed significant differences between day 8 and day 15 for versican and Sox9 relative to day 1 (Fig. 1). The results of bovine AF gene expression relative to control AF showed significant differences between groups for ACAN, Col1, Col2, IL-b1, versican, ADAMTS-4, Casp-8, MMP13, and MMP3. Cell viability of injected cells dropped from around ∼100% down to ∼87% for bovine cells and ∼86% for hMSC after 7 days in 3D culture. It further dropped to ∼72% after organ culture. GAG/DNA ratio showed significant difference across groups irrespective of posterior or anterior location.
Conclusion
MRI imaging of TH-RHG seems a promising protocol for noninvasively observing the gel performance in vitro. 10% TH-RHG is a feasible scaffold for in vitro 3D preconditioning of hMSCs and can be used at the same time for direct injection into the IVD. However, the TH-RHG has been shown to be unable to bare static load leading to a drop of cell viability. Relative gene expression of the hMSC phenotype after “co-culture”, however, points towards “discogenic” differentiation.5
No
None declared
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