Bioactive in situ crosslinkable polymer-peptide hydrogel for cell delivery to the intervertebral disc in a rat model

Degeneration of the intervertebral disc (IVD) is associated with significant biochemical and morphological changes that include a loss of disc height, decreased water content and decreased cellularity. Cell delivery has been widely explored as a strategy to supplement the nucleus pulposus (NP) region of the degenerated IVD in both pre-clinical and clinical trials, using progenitor or primary cell sources. We previously demonstrated an ability for a polymer-peptide hydrogel, serving as a culture substrate, to promote adult NP cells to undergo a shift from a degenerative fibroblast-like state to a juvenile-like NP phenotype. In the current study, we evaluate the ability for this peptide-functionalized hydrogel to serve as a bioactive system for cell delivery, retention and preservation of a biosynthetic phenotype for primary IVD cells delivered to the rat caudal disc in an anular puncture degeneration model. Our data suggest that encapsulation of adult degenerative human NP cells in a stiff formulation of the hydrogel functionalized with laminin-mimetic peptides IKVAV and AG73 can promote cell viability and increased biosynthetic activity for this population in 3D culture in vitro. Delivery of the peptide-functionalized biomaterial with primary rat cells to the degenerated IVD supported NP cell retention and NP-specific protein expression in vivo, and promoted improved disc height index (DHI) values and endplate organization compared to untreated degenerated controls. The results of this study suggest the physical cues of this peptide-functionalized hydrogel can serve as a supportive carrier for cell delivery to the IVD. STATEMENT OF SIGNIFICANCE: Cell delivery into the degenerative intervertebral disc has been widely explored as a strategy to supplement the nucleus pulposus. The current work seeks to employ a biomaterial functionalized with laminin-mimetic peptides as a cell delivery scaffold in order to improve cell retention rates within the intradiscal space, while providing the delivered cells with biomimetic cues in order to promote phenotypic expression and increase biosynthetic activity. The use of the in situ crosslinkable material integrated with the native IVD, presenting a system with adequate physical properties to support a degenerative disc

Hydraulic permeability and compressive properties of porcine and human synovium

The synovium is a multilayer connective tissue separating the intra-articular spaces of the diarthrodial joint from the extra-synovial vascular and lymphatic supply. Synovium regulates drug transport into and out of the joint, yet its material properties remain poorly characterized. Here, we measured the compressive properties (aggregate modulus, Young\u27s modulus, and Poisson\u27s ratio) and hydraulic permeability of synovium with a combined experimental-computational approach. A compressive aggregate modulus and Young\u27s modulus for the solid phase of synovium were quantified from linear regression of the equilibrium confined and unconfined compressive stress upon strain, respectively (

Diet-induced obesity differentially regulates behavioral, biomechanical, and molecular risk factors for osteoarthritis in mice

INTRODUCTION: Obesity is a major risk factor for the development of osteoarthritis in both weight-bearing and nonweight-bearing joints. The mechanisms by which obesity influences the structural or symptomatic features of osteoarthritis are not well understood, but may include systemic inflammation associated with increased adiposity. In this study, we examined biomechanical, neurobehavioral, inflammatory, and osteoarthritic changes in C57BL/6J mice fed a high-fat diet. METHODS: Female C57BL/6J mice were fed either a 10% kcal fat or a 45% kcal fat diet from 9 to 54 weeks of age. Longitudinal changes in musculoskeletal function and inflammation were compared with endpoint neurobehavioral and osteoarthritic disease states. Bivariate and multivariate analyses were conducted to determine independent associations with diet, percentage body fat, and knee osteoarthritis severity. We also examined healthy porcine cartilage explants treated with physiologic doses of leptin, alone or in combination with IL-1α and palmitic and oleic fatty acids, to determine the effects of leptin on cartilage extracellular matrix homeostasis. RESULTS: High susceptibility to dietary obesity was associated with increased osteoarthritic changes in the knee and impaired musculoskeletal force generation and motor function compared with controls. A high-fat diet also induced symptomatic characteristics of osteoarthritis, including hyperalgesia and anxiety-like behaviors. Controlling for the effects of diet and percentage body fat with a multivariate model revealed a significant association between knee osteoarthritis severity and serum levels of leptin, adiponectin, and IL-1α. Physiologic doses of leptin, in the presence or absence of IL-1α and fatty acids, did not substantially alter extracellular matrix homeostasis in healthy cartilage explants. CONCLUSIONS: These results indicate that diet-induced obesity increases the risk of symptomatic features of osteoarthritis through changes in musculoskeletal function and pain-related behaviors. Furthermore, the independent association of systemic adipokine levels with knee osteoarthritis severity supports a role for adipose-associated inflammation in the molecular pathogenesis of obesity-induced osteoarthritis. Physiologic levels of leptin do not alter extracellular matrix homeostasis in healthy cartilage, suggesting that leptin may be a secondary mediator of osteoarthritis pathogenesis

Development of a library of laminin-mimetic peptide hydrogels for control of nucleus pulposus cell behaviors

The nucleus pulposus (NP) of the intervertebral disc plays a critical role in distributing mechanical loads to the axial skeleton. Alterations in NP cells and, consequently, NP matrix are some of the earliest changes in the development of disc degeneration. Previous studies demonstrated a role for laminin-presenting biomaterials in promoting a healthy phenotype for human NP cells from degenerated tissue. Here we investigate the use of laminin-mimetic peptides presented individually or in combination on a poly(ethylene) glycol hydrogel as a platform to modulate the behaviors of degenerative human NP cells. Data confirm that NP cells attach to select laminin-mimetic peptides that results in cell signaling downstream of integrin and syndecan binding. Furthermore, the peptide-functionalized hydrogels demonstrate an ability to promote cell behaviors that mimic that of full-length laminins. These results identify a set of peptides that can be used to regulate NP cell behaviors toward a regenerative engineering strategy

Single cell RNA-sequence analyses reveal uniquely expressed genes and heterogeneous immune cell involvement in the rat model of intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is characterized by a loss of cellularity, and changes in cell-mediated activity that drives anatomic changes to IVD structure. In this study, we used single-cell RNA-sequencing analysis of degenerating tissues of the rat IVD following lumbar disc puncture. Two control, uninjured IVDs (L2–3, L3–4) and two degenerated, injured IVDs (L4–5, L5–6) from each animal were examined either at the two- or eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at the single-cell resolution. Unsupervised cluster analysis revealed the presence of four known cell types in both non-degenerative and degenerated IVDs based on previously established gene markers: IVD cells, endothelial cells, myeloid cells, and lymphoid cells. As a majority of cells were associated with the IVD cell cluster, sub-clustering was used to further identify the cell populations of the nucleus pulposus, inner and outer annulus fibrosus. The most notable difference between control and degenerated IVDs was the increase of myeloid and lymphoid cells in degenerated samples at two- and eight-weeks post-surgery. Differential gene expression analysis revealed multiple distinct cell types from the myeloid and lymphoid lineages, most notably macrophages and B lymphocytes, and demonstrated a high degree of immune specificity during degeneration. In addition to the heterogenous infiltrating immune cell populations in the degenerating IVD, the increased number of cells in the AF sub-cluster expressing Ngf and Ngfr, encoding for p75NTR, suggest that NGF signaling may be one of the key mediators of the IVD crosstalk between immune and neuronal cell populations. These findings provide the basis for future work to understand the involvement of select subsets of non-resident cells in IVD degeneration

N-cadherin is key to expression of the nucleus pulposus cell phenotype under selective substrate culture conditions

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