A Hyaluronan and Platelet-Rich Plasma Hydrogel for Mesenchymal Stem Cell Delivery in the Intervertebral Disc: An Organ Culture Study.

The purpose of the present pilot study was to evaluate the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) as a carrier for human mesenchymal stem cells (hMSCs) for intervertebral disc (IVD) regeneration using a disc organ culture model. HA was mixed with batroxobin (BTX) and PRP to form a hydrogel encapsulating 1 × 106 or 2 × 106 hMSCs. Bovine IVDs were nucleotomized and filled with hMSCs suspended in ~200 µL of the PRP/HA/BTX hydrogel. IVDs collected at day 0 and nucleotomized IVDs with no hMSCs and/or hydrogel alone were used as controls. hMSCs encapsulated in the hydrogel were also cultured in well plates to evaluate the effect of the IVD environment on hMSCs. After 1 week, tissue structure, scaffold integration, hMSC viability and gene expression of matrix and nucleus pulposus (NP) cell markers were assessed. Histological analysis showed a better preservation of the viability of the IVD tissue adjacent to the gel in the presence of hMSCs (~70%) compared to the hydrogel without hMSCs. Furthermore, disc morphology was maintained, and the hydrogel showed signs of integration with the surrounding tissues. At the gene expression level, the hydrogel loaded with hMSCs preserved the normal metabolism of the tissue. The IVD environment promoted hMSC differentiation towards a NP cell phenotype by increasing cytokeratin-19 (KRT19) gene expression. This study demonstrated that the hydrogel composed of HA/PRP/BTX represents a valid carrier for hMSCs being able to maintain a good cell viability while stimulating cell activity and NP marker expression.

Differential proteomic analysis of fetal and geriatric lumbar nucleus pulposus: immunoinflammation and age-related intervertebral disc degeneration.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a major cause of low back pain. Although the mechanism of degeneration remains unclear, aging has been recognized as a key risk factor for IVDD. Most studies seeking to identify IVDD-associated molecular alterations in the context of human age-related IVDD have focused only on a limited number of proteins. Differential proteomic analysis is an ideal method for comprehensively screening altered protein profiles and identifying the potential pathways related to pathological processes such as disc degeneration. METHODS: In this study, tandem mass tag (TMT) labeling was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for differential proteomic analysis of human fetal and geriatric lumbar disc nucleus pulposus (NP) tissue. Parallel reaction monitoring (PRM) and Western blotting (WB) techniques were used to identify target proteins. Bioinformatic analyses, including Gene Ontology (GO) annotation, domain annotation, pathway annotation, subcellular localization and functional enrichment analyses, were used to interpret the potential significance of the protein alterations in the mechanism of IVDD. Student's t-tests and two-tailed Fisher's exact tests were used for statistical analysis. RESULTS: Six hundred forty five proteins were significantly upregulated and 748 proteins were downregulated in the geriatric group compared with the fetal group. Twelve proteins were verified to have significant differences in abundance between geriatric and fetal NP tissue; most of these have not been previously identified as being associated with human IVDD. The potential significance of the differentially expressed proteins in age-related IVDD was analyzed from multiple perspectives, especially with regard to the association of the immunoinflammatory response with IVDD. CONCLUSIONS: Differential proteomic analysis was used as a comprehensive strategy for elucidating the protein alterations associated with age-related IVDD. The findings of this study will aid in the screening of new biomarkers and molecular targets for the diagnosis and therapy of IVDD. The results may also significantly enhance our understanding of the pathophysiological process and mechanism of age-related IVDD.

Derivation of notochordal cells from human embryonic stem cells reveals unique regulatory networks by single cell-transcriptomics.

Intervertebral disc degeneration (IDD) is a public health dilemma as it is associated with low back and neck pain, a frequent reason for patients to visit the physician. During IDD, nucleus pulposus (NP), the central compartment of intervertebral disc (IVD) undergo degeneration. Stem cells have been adopted as a promising biological source to regenerate the IVD and restore its function. Here, we describe a simple, two-step differentiation strategy using a cocktail of four factors (LDN, AGN, FGF, and CHIR) for efficient derivation of notochordal cells from human embryonic stem cells (hESCs). We employed a CRISPR/Cas9 based genome-editing approach to knock-in the mCherry reporter vector upstream of the 3' untranslated region of the Noto gene in H9-hESCs and monitored notochordal cell differentiation. Our data show that treatment of H9-hESCs with the above-mentioned four factors for 6 days successfully resulted in notochordal cells. These cells were characterized by morphology, immunostaining, and gene and protein expression analyses for established notochordal cell markers including FoxA2, SHH, and Brachyury. Additionally, pan-genomic high-throughput single cell RNA-sequencing revealed an efficient and robust notochordal differentiation. We further identified a key regulatory network consisting of eight candidate genes encoding transcription factors including PAX6, GDF3, FOXD3, TDGF1, and SOX5, which are considered as potential drivers of notochordal differentiation. This is the first single cell transcriptomic analysis of notochordal cells derived from hESCs. The ability to efficiently obtain notochordal cells from pluripotent stem cells provides an additional tool to develop new cell-based therapies for the treatment of IDD.

Expression and effects of leukemia inhibitory factor on nucleus pulposus degeneration.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine. The present study aimed to determine the expression and effects of LIF on nucleus pulposus generation. Degenerated nucleus pulposus samples were obtained from animal models and patients with lumbar intervertebral disc herniation. Degradation scores of intervertebral discs were evaluated via magnetic resonance imaging (MRI) and histology, and the protein expression levels of LIF were detected. Furthermore, cultured primary human degenerated nucleus pulposus cells (DNPCs) were stimulated with various concentrations of recombinant human LIF protein (rhLIF), and aggrecan and collagen type II α1 (COL2α1) protein expression levels were detected by western blotting. In addition, aggrecan expression was determined by toluidine blue staining. The effects of rhLIF on proliferation and apoptosis of DNPCs were evaluated by Cell Counting Kit­8 and flow cytometry, respectively. The results revealed that the degradation scores of intervertebral discs were significantly associated with modeling time, as determined by MRI and histology. In addition, the protein expression levels of LIF were initially increased in patients with lumbar disc herniation and in rabbit models, particularly in the 2­week modeling group; however, its expression decreased with the progression of disc degeneration. Notably, LIF expression in each modeling group was higher than that in the control and 0 week modeling group. The in vitro study revealed that the protein expression levels of aggrecan and COL2α1 were significantly increased in response to rhLIF, in a dose­dependent manner, and statistical differences were identified between the treatment groups and control group. The results of toluidine blue staining were consistent with this finding. Although rhLIF had no effect on proliferation, it inhibited apoptosis of DNPCs in a concentration­dependent manner. In conclusion, LIF was upregulated during the process of intervertebral disc degeneration, and may promote the expression of extracellular matrix components. It may also be hypothesized that LIF acts as a potential protective factor by inhibiting apoptosis of DNPCs without affecting cell proliferation.

Meniscus, articular cartilage and nucleus pulposus: a comparative review of cartilage-like tissues in anatomy, development and function.

The degradation of cartilage in the human body is impacted by aging, disease, genetic predisposition and continued insults resulting from daily activity. The burden of cartilage defects (osteoarthritis, rheumatoid arthritis, intervertebral disc damage, knee replacement surgeries, etc.) is daunting in light of substantial economic and social stresses. This review strives to broaden the scope of regenerative medicine and tissue engineering approaches used for cartilage repair by comparing and contrasting the anatomical and functional nature of the meniscus, articular cartilage (AC) and nucleus pulposus (NP). Many review papers have provided detailed evaluations of these cartilages and cartilage-like tissues individually but none have comprehensively examined the parallels and inconsistencies in signaling, genetic expression and extracellular matrix composition between tissues. For the first time, this review outlines the importance of understanding these three tissues as unique entities, providing a comparative analysis of anatomy, ultrastructure, biochemistry and function for each tissue. This novel approach highlights the similarities and differences between tissues, progressing research toward an understanding of what defines each tissue as distinctive. The goal of this paper is to provide researchers with the fundamental knowledge to correctly engineer the meniscus, AC and NP without inadvertently developing the wrong tissue function or biochemistry.

Unique glycosignature for intervertebral disc and articular cartilage cells and tissues in immaturity and maturity.

In this study, on/off markers for intervertebral disc (IVD) and articular cartilage (AC) cells (chondrocytes) and distinct glycoprofiles of cell and tissue-types were identified from immaturity to maturity. Three and eleven month-old ovine IVD and AC tissues were histochemically profiled with a panel of lectins and antibodies. Relationships between tissue and cell types were analysed by hierarchical clustering. Chondroitin sulfate (CS) composition of annulus fibrosus (AF), nucleus pulposus (NP) and AC tissues was determined by HPLC analysis. Clear on/off cell type markers were identified, which enabled the discrimination of chondrocytes, AF and NP cells. AF and NP cells were distinguishable using MAA, SNA-I, SBA and WFA lectins, which bound to both NP cells and chondrocytes but not AF cells. Chondrocytes were distinguished from NP and AF cells with a specific binding of LTA and PNA lectins to chondrocytes. Each tissue showed a unique CS composition with a distinct switch in sulfation pattern in AF and NP tissues upon disc maturity while cartilage maintained the same sulfation pattern over time. In conclusion, distinct glycoprofiles for cell and tissue-types across age groups were identified in addition to altered CS composition and sulfation patterns for tissue types upon maturity.