The use of stem cells transplanted into the intervertebral disc (IVD) is a promising regenerative approach to treat intervertebral disc degeneration (IDD). The aim of this study was to assess the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) loaded with human mesenchymal stem cells (hMSCs), on IVD extracellular matrix synthesis and nucleus pulposus (NP) marker expression in a whole IVD culture model. HA was blended with batroxobin (BTX), a gelling agent activated in presence of PRP to construct a hydrogel. Bovine IVDs (n=25) were nucleotomised and filled with 1×106 or 2×106 hMSCs suspended in ∼150 mL of the PRP/HA/BTX hydrogel. IVDs harvested at day 0 and nucleotomised IVDs with no hMSCs and/or hydrogel were used as controls. hMSCs alone or encapsulated in the hydrogel were also cultured in well plates to examine the effect of the IVD microenvironment on hMSCs. After 1 week, tissue structure, scaffold integration and gene expression of anabolic (collagen type I, collagen type II and aggrecan), catabolic (matrix metalloproteinase 3 – MMP-3 –, MMP-13 and a disintegrin and metalloproteinase with thrombospondin motifs 4) and NP cell (cytokeratin 19, carbonic anhydrase 12, cluster of differentiation 24) markers were assessed. Histological analysis showed a good integration of the scaffold within the NP area with cell repopulation. At the gene expression level, the hMSC-loaded hydrogels demonstrated to increase disc cell anabolic and catabolic marker expression and promoted hMSC differentiation towards a NP cell phenotype. This study demonstrated that the HA/PRP/BTX may represent a valid carrier for hMSCs being capable of stimulating cell activity and NP marker expression as well as achieving a good integration with the surrounding tissues.
Intervertebral disc (IVD) degeneration is a prominent cause of low back pain. IVD cells expressing angiopoietin-1 receptor Tie2 represent a progenitor cell population which decreases with progression of IVD degeneration. Homing of mesenchymal stem cells (MSCs) is a physiological mechanism aiming to enhance the regenerative capacity of the IVD. The purpose of this study was to assess the effect of MSC homing on the Tie2 positive IVD progenitor cell population, the IVD cell viability, and the proliferative phenotype of the IVD cells. Human MSCs were isolated from bone marrow aspirates and labelled with fluorescent dye. Whole IVDs with endplates were harvested from bovine tails; MSCs were placed on the endplates. Human traumatic, degenerative and healthy IVD tissues were obtained from patients and organ donors. MSCs were added onto tissue samples. After 5 days, IVD cells were isolated. Percentages of Tie2 positive, DAPI positive (dead) and Ki-67 positive (proliferative) IVD cells were determined. MSC homing or co-culture significantly increased the proportion of Tie2 positive progenitor IVD cells in bovine and 7/10 human IVDs, decreased the fraction of dead IVD cells in bovine and 7/10 human IVDs, and induced a proliferative phenotype in bovine and 5/6 human IVDs.Background and Purpose
Methods and Results
Carriers for local delivery of stem cells into degenerative intervertebral discs need to be tested under physiological loading since stem cell viability, density and differentiation, as well as carrier stability are strongly affected by loading. The success of the local delivery of mesenchymal stem cells (MSCs) to degenerative discs relies on three main factors: (i) an appropriate delivery method, (ii) a suitable carrier, (iii) resistance to loading forces. Bioreactors allow the application of loading to whole intervertebral discs and represent a useful tool to screen the potential of new regenerative therapies. We have previously shown that hydrogel delivery through the endplate (EP) leaves the annulus fibrosus (AF) intact (as opposed to an approach through the AF). Furthermore, we have found that the physiological loading needs to be adapted for nucleotomised discs. In this study we compare the behaviour of two MSCs carriers under loading in a whole IVD bioreactor.Summary
Introduction
To test regenerative therapies for the intervertebral disc it is necessary to create a cavity in the nucleus polposus mantaining the annulus fibrosus intact. The transpedicular mechanical nucleotomy represents the best method for this purpose. New cells/hydrogel based treatments for intervertebral disc (IVD) regeneration need to be tested on animal models before clinical translation. Ovine IVD represents a good model but doesn't allow the injection of a significant volume into intact IVD. The objective of the study was to compare different methods to create a cavity into ovine nucleus pulposus (NP) by enzymatic digestion (E), mechanical discectomy (M) and a combination of both (E+M), as a model to study IVD regeneration strategies with intact anulus fibrosus (AF).Summary Statement
Introduction
