Injectable laminin-functionalized hydrogel for nucleus pulposus regeneration

Abstract

Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration. The development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the nucleus pulposus (NP) region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into carriers for cell delivery may be beneficial for promoting NP cell survival and phenotype. Here, an injectable, laminin-111 functionalized poly(ethylene glycol) (PEG-LM111) hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. We evaluated the mechanical properties of the PEG-LM111 hydrogel, and its ability to retain delivered cells in the IVD space. Gelation occurred in approximately 20 min without an initiator, with dynamic shear moduli in the range of 0.9-1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 carrier, as compared to cells in liquid suspension. Together, these results suggest this injectable laminin-functionalized biomaterial may be an easy to use carrier for delivering cells to the IVD.

Keywords: Injectable; Intervertebral disc; Laminin; Nucleus pulposus; Organ culture; Polyethylene glycol.

Figure 1. PEG-LM111 conjugate synthesis and degree of modification (%)

(A) Conjugation of PEG molecules to laminin via amine reactive NHS groups. (B) Degree of modification of LM111 calculated from LM111 and PEG-LM111 conjugate absorbance at 340nm using TNBS assay. Degree of modification increased with increasing molar excess of Ac-PEG-NHS in the PEGylation reaction (mean ± SEM, n=3, conditions labeled with different letters significantly different, p<0.01).

Figure 2. Bioactivity of PEG-LM111 conjugates

(A) Porcine NP cells were allowed to attach to LM111 and PEG-LM111 conjugates for 2 hours. NP cell attachment to PEG-LM111 conjugates decreased with increasing molar excess Ac-PEG-NHS in the PEGylation reaction (cell attachment levels normalized to NP cells on 25 μg/ml LM111, mean ± SEM, n=3, conditions labeled with different letters significantly different, p<0.04). (B) LM111 induced ERK activation in a lung epithelial cell line. Cells from a lung epithelial cell line (WI26VA4) were seeded onto LM111 and PEG-LM111 conjugate coated surfaces, or cultured in suspension. After 30 and 60 minutes, cells were lysed and levels of phosphorylated ERK in cell lysates were compared using ELISA.

Figure 3. Rheometric characterization of PEG-LM111 hydrogels

(A) Gel point occurred in less than 25 minutes for hydrogels containing different concentrations of PEG-LM111 conjugate (mean ± SEM, n=5, conditions labeled with different letters significantly different, p<0.04). (B) Final gel stiffness (∣G*∣) of PEG-LM111 hydrogels increased with increasing concentration of PEG-LM111 conjugate in the precursor solution.

Figure 4. Luciferase activity in cultured porcine NP cells

NP cells transduced with lentivirus encoding for firefly luciferase were cultured for 35 days post transduction. At each time point, photons per second per cell was calculated from measured bioluminescent intensity 10 minutes after addition of luciferin to the media (mean ± SEM, n=3, conditions labeled with different letters significantly different, p<0.03).

Figure 5. Cell retention in IVD motion segments

(A) NP-luc cells were delivered to rat IVD motion segments and explants were cultured for 14 days. Cell retention, as measured by total photons per motion segment, was higher for cells delivered in PEG-LM111 biomaterial as compared to cells delivered in PBS (mean ± SEM, n=6, conditions labeled with different letters significantly different, p<0.001). (B) Region of interest (ROI) defined on photographic image of motion segment (top) used quantify total photons per motion segment in overlay image (bottom). (C) Representative images of IVD motion segments 30 minutes and 7 days post cell injection within PEG-LM111 biomaterial carrier (top) or PBS (bottom).

Figure 6

In vivo bioluminescent imaging 15 hours after 1×10⁶ NP-luc cells were injected into the rat tail disc space. Luciferin (150 mg/kg) was injected intraperitoneally 15 min before imaging. Rat was anesthetized with 2% isoflurane during imaging (5 min exposure time).