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 minutes 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.

Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to low back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Previous studies have shown that NP cell-laminin interactions promote cell adhesion and biosynthesis, which suggests a laminin-functionalized biomaterial may be useful for promoting or maintaining the NP cell phenotype. Here, a photocrosslinkable poly(ethylene glycol)-laminin 111 (PEG-LM111) hydrogel was developed. PEG-LM111 hydrogel mechanical properties could be tuned within the range of dynamic shear moduli values previously reported for human NP. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, LM111 presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111 presenting and PEG-only gels. When cells were encapsulated in 3D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1 kPa) PEG-LM111 hydrogels. Overall, these findings suggest that soft, LM111 functionalized hydrogels may promote or maintain the expression of specific markers characteristic of an immature NP cell phenotype.

Hyaluronic acid (HA) poly(ethylene glycol) (PEG) composite hydrogels have been widely studied for both cell delivery and soft tissue regeneration applications. A very broad range of physical and biological properties have been engineered into HA-PEG hydrogels that may differentially affect cellular “outcomes” of survival, synthesis and metabolism. The objective of this study was to rapidly screen multiple HA-PEG composite hydrogel formulations for an effect on matrix synthesis and behaviors of nucleus pulposus (NP) and anulus fibrosus (AF) cells of the intervertebral disc (IVD). A secondary objective was to apply artificial neural network (ANN) analysis to identify relationships between HA-PEG composite hydrogel formulation parameters and biological outcome measures for each cell type of the IVD. Eight different hydrogels were developed from preparations of thiolated HA (HA-SH) and PEG vinylsulfone (PEG-VS) macromers, and used as substrates for NP and AF cell culture in vitro. Hydrogel mechanical properties ranged from 70-489 kPa depending on HA molecular weight, and measures of matrix synthesis, metabolite consumption and production, and cell morphology were obtained to study relationships to hydrogel parameters. Results showed that NP and AF cell numbers were highest upon the HA-PEG hydrogels formed from the lower molecular weight HA, with evidence of higher sGAG production also upon lower HA molecular weight composite gels. All cells formed more multi-cell clusters upon any HA-PEG composite hydrogel as compared to gelatin substrates. Formulations were clustered into neurons based largely on their HA molecular weight, with few effects of PEG molecular weight observed on any measured parameters.

Biological and anatomical changes of intervertebral disc (IVD) degeneration frequently occur in the nucleus pulposus (NP) [1]. Changes in NP matrix composition coincide with the loss of a distinct notochord derived cell population [2],[3], which may have the potential to generate or maintain a functional NP-like matrix. Immature NP cells reside in an environment rich in laminin and express specific laminin-binding receptors [4],[5]. Additionally, NP cells attach in higher numbers to laminins as compared to cells isolated from other regions of the IVD [6]. Our initial work demonstrated that matrix protein and stiffness modulate NP cell-cell interactions upon surfaces [7], with results that suggest soft, laminin-functionalized hydrogels may be useful for promoting an NP-like cell phenotype.

Introduction Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration1-4; however, few viable sources of cells have been identified. Induced pluripotent stem cells (iPSCs) are an attractive cell source since they are derived from the patient's somatic cells,5 thereby providing an autologous source of cells for IVD regeneration. We have previously demonstrated that mouse and human iPSCs can differentiate into nucleus pulposus (NP)-like cells in vitro when cultured in a laminin-rich (Matrigel) system.6 To provide for a more controlled microenvironment, we have developed an injectable, laminin-111 functionalized poly (ethylene glycol) (PEG-LM111) hydrogel for IVD cell-mediated regeneration.6 The stiffness and laminin density of this PEG-LM111 hydrogel was previously optimized to promote or maintain the expression of immature NP-specific markers for primary NP cells in vitro.7 The goal of this study is to evaluate if human iPSCs can be promoted to similarly express markers specific to the immature NP cell when cultured upon this PEG-LM111 hydrogel, toward the goal of promoting generation of native NP-like tissue from stem cells in vitro. Materials and Methods Cell Generation iPSCs were generated from human embryonic dermal fibroblasts through transient inducible overexpression of transcription factors (OCT4, SOX2, KLF4, and MYC) by a lentiviral-based gene delivery system (a polycystronic vector with the reverse tetracycline transactivator (M2rtTA) and doxycycline). Selected iPSC colonies were maintained in culture upon a primary mouse embryo fibroblasts (PMEF) feeder layer. As we reported previously, iPSC colonies expressed a subset of human pluripotent cell markers (i.e., OCT4, SOX2, SSEA-4, TRA1-60, TRA1-81, and alkaline phosphatase), NP-associated integrins (α3, α6, and β1 subunit), NP-specific molecular markers (CD24 and Brachyury), as well as MSC markers (CD29 and CD90).9 Cell Differentiation “Soft” PEG-LM111 (2% PEG-diacrylate prepared with chemically coupled 1 mg/mL laminin-111/PEG; ∼ 200 Pa) hydrogels were UV-crosslinked in Transwell inserts. Undifferentiated iPSCs were seeded (106 cells/insert) on PEG-LM111 substrates in in each Transwell and cultured in notochordal cell conditioned medium (NCCM) from porcine NP tissue (DMEM based, 1% ITS10) under 2% hypoxia condition;11 undifferentiated iPSCs cultured under equivalent conditions but with NP differentiation medium (DMEM/F12, ITS, NEAA9) was used as a control. Immunohistochemistry Differentiated cells were harvested for cryosectioning at 10 or 21 days of culture. Cell morphology and proteoglycan synthesis was assessed by histological staining (H&E and Safranin O). Expression of NP markers was evaluated by immunostaining for matrix proteins (type II collagen [COL II], laminin 10 [LM511]), laminin related receptors (CD239, integrin subunits α6, β4), a subset of NP-specific markers (cytokeratin 8 [KRT8], N-cadherin, CD24), and some non-NP-markers (type I collagen, E-cadherin). Results O...