Autologous Mesenchymal Stem Cell Injections for Treatment of Intervertebral Disc Degeneration
J.D. Kang, MD; L.G. Gilbertson, PhD; G. Vadala, MD; J. Larson, III, MD
BACKGROUND CONTEXT: C-fiber and type II A-delta-fiber neurons, normally conducting nociceptive sensations, mediate thermal hyperalgesia. In pathological conditions, they could mediate tactile allodynia via cross-excitation between the A-beta-fiber neurons and the C-fiber and type II A-delta-fiber neurons in the dorsal root ganglia (DRG). The vanilloid receptor 1 (VR1) is expressed only on C-fiber and A-delta-fiber neurons, which involves various noxious stimuli (capsaicin, heat, protons) and acts as a molecular integrator for nociception. Resiniferatoxin (RTX) is a potent and specific VR1 agonist that induces death of VR1-positive neurons. In our pilot study we found VR1 mediated burn-like component of neuropathic pain and involved the development of neuropathic pain.
PURPOSE: Define the role of VR1-positive neurons in the development of mechanical allodynia and thermal hyperalgesia following peripheral nerve injury.
STUDY DESIGN/SETTING: NA
PATIENT SAMPLE: NA
OUTCOME MEASURES: NA
METHODS: Twelve rats were subjected to photochemically induced sciatic nerve injury. The rats that developed tactile allodynia and hyperalgesia were treated by RTX injection in the ipsilateral L3-L6 DRGs. These rats were tested before and after injection for thermal hyperalgesia on hot plate and mechanical allodynia with von Frey filaments for one week. After the behavioral tests were completed, the rats were euthanized and the DRGs were dissected out for immunohistochemical analysis for VR1-positive as well as N52 (myelin producing) neurons. Mechanical allodynia was tested by measuring the paw-withdrawal response to mechanical stimuli (von Frey filaments ranging from 1 to 26 g). If a filament induces 3 paw withdrawals out of 5 stimuli, the value of the filament in gram is considered as the withdrawal threshold of the paw. If the withdrawal value is under 8 grams for a rat, the rat is classified as allodynic. Thermal hyperalgesia was tested by measuring the paw-withdrawal response to noxious heat stimuli (light beam on the hot plate). If the paw withdrawal latency in injured side is 30% shorter than intact side, the rat is taken as thermal hyperalgesia.
RESULTS: Thermal hyperalgesia. The latency from light beam on to paw withdrawal in injured paws was 33% shorter than that in intact paws. After RTX injection the latency in injured paws increased to longer than the intact paws, suggesting thermal hyperalgesia was normalized. Mechanical allodynia. Neuropathic rats exhibited intense mechanical allodynia with average withdrawal threshold 1.53g. After RTX injection, the threshold increased to 5.48g, but was still below 8g. VR1 abolition only suppressed most intensive component of neuropathic pain, leaving moderate allodynia. The results indicate that C-fiber and A-delta type II fiber mediate the most sensitive component of allodynia, while A-beta fiber mediates other components of allodynia. Immunohistochemistry. The number of VR1-positive neurons in allodynic, RTX-injected DRGs was largely reduced, compared to that in allodynic DRGs and in normal DRGs, confirming that VR1 positive neurons carry out the mediation of allodynia.
CONCLUSIONS: Following sciatic nerve injury, VR1-positive (type II A-delta and C-fiber) neurons in the DRG mediate the most sensitive component of tactile allodynia, probably via cross-excitation between the A-beta-fiber and the VR1 positive neurons. Thermal hyperalgesia seems to be mediated exclusively by VR1-positive neurons. Further testing on a larger population is necessary.
C Fiber and Type II A-S Fiber Neurons Mediate Neuropathic Pain
Jian-Guo Cui, MD, PhD; Gabriel Tender
BACKGROUND CONTEXT: Intervertebral disc (IVD) degeneration is a chronic process characterized by progressive loss of cells, proteoglycans, and water content in the nucleus pulposus. Current therapies are aimed at treating the pathologic and disabling conditions arising from IVD degeneration rather than directly treating the disc itself. Our group and others are actively exploring the potential of Stem Cell Therapy to repopulate the disc and prevent loss of proteoglycans in disc degeneration [1,2]. The combination of mesenchymal stem cells (MSC) and nucleus pulposus cells (NPC) has been shown to increase extracellular matrix synthesis—in-vitro as well as in-vivo—though the precise mechanisms remain to be determined.
PURPOSE: The present study tested MSC differentiation towards a chondrogenic phenotype, stimulatory effects exerted by MSCs upon NPCs, and cell fusion after co-culture.
STUDY DESIGN/ SETTING: In-vitro study of adult human cells to study the interaction between NPC and MSC after co-culture.
PATIENT SAMPLE: Human NPC were isolated from femele patients undergoing disc surgery, and human MSC were isolated from male patients undergoing hip surgery.
OUTCOME MEASURES: Fluorescence activated cell sorting (FACS), real-time RT-PCR, and fluorescence in situ hybridization (FISH).
METHODS: NPCs and MSCs were labeled by transduction with Ad/GFP (Green Fluorescent Protein) and Ad/RFP (Red Fluorescent Protein), respectively, and co-cultured at 50:50 ratio in alginate beads and pellets. Alginate beads were dissolved after 1 day (control) and 14 days of co-culture. Cells were then sorted by FACS, mRNA extracted separately from NPCs and MSCs, and collagen-II expression assessed by real-time RT-PCR. Pellets were fixed after 2 weeks of co-culture and FISH was performed visualizing X and Y chromosomes.
RESULTS: The gene expression of collagen-II was increased in both hNPC (4 fold) and hMSC (10.5 fold) after two weeks of co-culture compared with the control. FACS analysis after 14 days of co-culture, showed that 0.5% of cells were double fluorescent for GFP and RFP. However, both male and female cells were independently identified by FISH.
FISH using the Chromosome enumerator probe (CEP) X and Y.
Male MSC and female NPC were identified independently in co-culture.
CONCLUSIONS: The results of this study demonstrate that the interaction between MSC and NPC may derive from a combination of MSC differentiation towards a more chondrogenic phenotype as well as stimulatory paracrine effects exerted by MSCs upon NPCs. The presence of few two-color positive cells by FACS, without any qualitative observation of cells with tetraploid DNA by FISH, leads us to consider cell fusion a rare phenomenon. The ongoing study of mRNA expression of other gene will further clarify the interaction between MSC and NPC. This study further clarifies the mechanism of the interaction between MSCs and NPCs, and improves the understanding of the potential
of MSCs to alter the course of intervertebral disc degeneration.