Abstract
BACKGROUND CONTEXT Degenerative disc disease (DDD) involves the loss of viable cells and degradation of the extracellular matrix (ECM) within the avascular, ischemic and hypoxic intervertebral disc (IVD) nucleus pulposus (NP). We have previously demonstrated that injection of a molecular therapeutic (NTG-101) containing TGF-β1 and CTGF can mediate the progression of DDD in a rat and canine model. However, patients suffering from more advanced degenerative disc may require cellular replacement. Bone marrow derived mesenchymal stem cells (MSCs) originate from a highly vascularized and immune competent niche but are incompatible with the harsh IVD NP environment. Therefore, we harvested a novel source of articular cartilage derived stem cells (CDSCs) from the hypoxic, ischemic and avascular cartilage niche that may be compatible with the severe NP tissue compartment and transplanted them into the NP alone and in combination with NTG-101. PURPOSE To identify and characterize a novel source of stem cells (CDSCs) and determine the efficacy of CDSC intradiscal transplantation alone or in combination with a molecular therapeutic 'NTG-101′ in our validated rat-tail model of DDD. STUDY DESIGN/SETTING In vivo laboratory experimentation involving female Wistar rats aged 12 weeks. PATIENT SAMPLE No human subjects. Animal Care approved study involving female Wistar rats using validated rat-tail injury and injection model. OUTCOME MEASURES Histology, immunohistochemistry, Western blotting. METHODS Cells sourced from the tibial and femoral plateaus of Wistar rats, were expanded in free-floating culture and sphere forming cells collected and passaged under clonal conditions. Stemness was confirmed by differentiation into chondrogenic, osteogenic, adipogenic and neurogenic lineages. DDD was induced within five rat-tail IVDs/animal in three groups of seven animals using image-guided needle puncture injury. Ten weeks later, 8ul/IVD phosphate buffered saline (PBS) was injected into the IVD NPs of five discs/animal (7 animals/group). Other groups of animals with identical disc injuries received injections of 150,000 CDSCs suspended in 8ul PBS, or 150,000 CDSCs suspended within 8ul NTG-101. The animals were humanely sacrificed 10 weeks later, and normal and treated IVDs were assayed by histological, immunohistochemical and Western blotting to determine levels of aggrecan, collagen type 2, Oct4, MMP-3, MMP-13 and COX-2. RESULTS CDSCs have stemness characteristics as defined by their differentiation into multiple cellular lineages. PBS-injected IVDs develop a degenerative phenotype whereas CDSC and CDSC+NTG-101 injected discs retain robust cellularity, maintenance of disc height and size of the NP. Immunohistochemical analyses demonstrated that PBS-injected IVDs become deficient in collagen type 2, aggrecan and Oct4 but show increased levels of matrix degrading (MMP-3 and -13) and pro-inflammatory (COX2) markers. IVD's that received CDSCs or CDSCs+NTG-101 showed undetectable levels of COX-2, MMP-3 and MMP-13 however Oct4 was expressed at levels like healthy controls. Western blotting supported the immunohistochemical results with PBS-injected discs showing low levels of Oct4, but markedly increased levels of COX-2, MMP-3 and MMP-13. CONCLUSIONS These results clearly support the use of CDSCs as a source of cellular therapy that is enhanced by suspension within a molecular therapeutic (NTG-101) for the treatment of advanced DDD. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.
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