Injectable laminin-functionalized hydrogel for cell delivery to the intervertebral disc

Abstract

Purpose: Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration. The importance of a biomaterial carrier for maintaining cell localization and promoting cell survival in the IVD has been demonstrated using collagen and other carriers. Our work has shown that specific laminin isoforms and receptors may be important in regulating cell-laminin interactions in the nucleus pulposus (NP) regions of the IVD, such that incorporating these ligands into carriers for cell delivery may be desirable for promoting NP cell survival and phenotype. Here we describe our work with synthesis of an injectable PEGylated laminin-111 (PEG-LM111) hydrogel that can be engineered to mechanical specifications, and describe use of the hydrogel for an ability to retain NP cells in the IVD space. Methods: Hydrogel synthesis: Conjugates of LM111 and PEG (PEG-LM111) were synthesized with reactive NHS groups as described previously (25:1 molar ratio of acrylate-PEG-NHS to LM111) Eight-arm PEG-acrylate (20kDa, Creative PEGworks) and PEG-dithiol (3.4 kDa, Creative PEGworks) were dissolved separately in PEG-LM111 conjugate solutions to final concentrations of 10% (w/v) PEG and 0, 100 or 500 μg/ml PEG-LM111 conjugate. Solutions were mixed, and gelation behavior (|G*|) measured by oscillatory shear testing (ω = 0.5Hz, shear strain =0.05, 37°C). Cell delivery to IVD motion segments: Primary porcine NP cells were transduced with a lentivirus encoding firefly luciferase under control of the constitutive EF1α promoter. PEG-dithiol and PEG-acrylate were dissolved separately in a PEG-LM111 conjugate solution and mixed to obtain PEG-LM111 solutions for gelation. NP cells were suspended in PEG-LM111 solution (10% PEG, 500 μg/ml PEG-LM111 conjugate) or PBS (106 cells/ml) and 10 μl was delivered to the NP of individual rat caudal motion segments. Motion segments receiving sham injection and PEG-LM111 solution only were used as negative controls. All motion segments were cultured out to 14 days at 37C and 21% O2. Cell retention was evaluated at multiple time points by imaging luminescence within motion segments in wells supplemented with 300 μg/ml luciferin (IVIS Kinetic, Caliper Life Sciences). Results: PEG-LM111 hydrogels gelled in less than 25 minutes, and gel stiffness (|G*|) continued to increase over one hour (Figure 1). Hydrogel stiffness increased with increasing concentration of PEG-LM111 conjugate in precursor solutions. Cell number as measured by total photons per motion segment was significantly higher over 14 days in culture when cells were delivered within a PEG-LM111 carrier, as compared to cells alone (Figure 2). For both groups, signal rapidly decayed over the first 24 hours of culture. Conclusions: An injectable laminin-functionalized biomaterial was developed for cell delivery to the IVD, and shown to undergo gelation within minutes under physiological conditions. Luciferase expressing NP cells were delivered with and without PEG-LM111 carrier to motion segments, with results demonstrating significantly greater cell retention for cells delivered with the PEG-LM111 hydrogel. These findings suggest this injectable laminin-functionalized biomaterial may be a useful carrier for cell delivery to the IVD.Figure 2Total photons per motion segment over 14 days in culture (mean ± SEM, n=6, conditions labeled with different letters significantly different, p<0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)