Analgesic Effects of Two Types of Spinal Manipulation in Acute Lumbar Radiculopathy Model Rats.

OBJECTIVE: To compare the analgesic effects of two types of spinal manipulation (SM) in acute lumbar radiculopathy (ALR) model rats induced by self-transplantation of autologous nucleus pulposus (ANP), and clarify the therapeutic mechanism. METHODS: Totally 108 male Sprague-Dawley rats were randomly divided into 6 groups by a random number table (18 rats in each group), including a blank group with no interference, a sham operation group with a surgery by making a local soft tissue incision on the left side of L5-6 vertebral segment, a model group with ALR of L5 extraforaminal nerve by ANP self-transplantation without other interference, a sham manipulation (SMA) group with simulating physical rotation, as well as a mobilization (MOB) group with simulating low-velocity and variable-amplitude rotation and a manipulation (MAN) group with simulating high-velocity and low-amplitude rotation. The interventions in SMA, MOB, and MAN groups started 1 day after modeling followed by another 5 treatments at days 3, 5, 8, 10 and 12. Rats in the other 3 groups did not receive any special intervention. Behavioral pain tests of 50% mechanical pain withdrawal threshold (50% PWT) and paw withdrawal latency (PWL) were conducted 1 day before operation followed by another 10 tests on days 1-7, 10, 12 and 14. Immunohistochemical expression of nitric oxide synthase (NOS) was investigated on days 5 and 12 after operation. RESULTS: After 3 experimental SM interventions, 50% PWT and PWL were higher in the MAN group than the SMA group on days 6 and 7, and higher on days 10, 12 and 14 postoperatively (P<0.05 or P<0.01), while the same indices were significantly higher in the MOB group than MAN group on days 1-4 (P<0.05 or P<0.01). The expression of NOS was lower in the MAN and MOB groups than SMA group on day 12 postoperatively (P<0.01). CONCLUSIONS: Both manipulation and mobilization produced better results than sham interference in relieving pain by reducing neuroinflammation possibly. At the early period, compared with manipulation, mobilization presented less sensitive response to pain until later visit. SM may inhibit the overexpression of NOS, thereby alleviating severe radiculopathy.

Analgesic Effects of Two Types of Spinal Manipulation in Acute Lumbar Radiculopathy Model Rats / 中国结合医学杂志

OBJECTIVE@#To compare the analgesic effects of two types of spinal manipulation (SM) in acute lumbar radiculopathy (ALR) model rats induced by self-transplantation of autologous nucleus pulposus (ANP), and clarify the therapeutic mechanism.@*METHODS@#Totally 108 male Sprague-Dawley rats were randomly divided into 6 groups by a random number table (18 rats in each group), including a blank group with no interference, a sham operation group with a surgery by making a local soft tissue incision on the left side of L5-6 vertebral segment, a model group with ALR of L5 extraforaminal nerve by ANP self-transplantation without other interference, a sham manipulation (SMA) group with simulating physical rotation, as well as a mobilization (MOB) group with simulating low-velocity and variable-amplitude rotation and a manipulation (MAN) group with simulating high-velocity and low-amplitude rotation. The interventions in SMA, MOB, and MAN groups started 1 day after modeling followed by another 5 treatments at days 3, 5, 8, 10 and 12. Rats in the other 3 groups did not receive any special intervention. Behavioral pain tests of 50% mechanical pain withdrawal threshold (50% PWT) and paw withdrawal latency (PWL) were conducted 1 day before operation followed by another 10 tests on days 1-7, 10, 12 and 14. Immunohistochemical expression of nitric oxide synthase (NOS) was investigated on days 5 and 12 after operation.@*RESULTS@#After 3 experimental SM interventions, 50% PWT and PWL were higher in the MAN group than the SMA group on days 6 and 7, and higher on days 10, 12 and 14 postoperatively (P<0.05 or P<0.01), while the same indices were significantly higher in the MOB group than MAN group on days 1-4 (P<0.05 or P<0.01). The expression of NOS was lower in the MAN and MOB groups than SMA group on day 12 postoperatively (P<0.01).@*CONCLUSIONS@#Both manipulation and mobilization produced better results than sham interference in relieving pain by reducing neuroinflammation possibly. At the early period, compared with manipulation, mobilization presented less sensitive response to pain until later visit. SM may inhibit the overexpression of NOS, thereby alleviating severe radiculopathy.

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.

The research of transgenic human nucleus pulposus cell transplantation in the treatment of lumbar disc degeneration.

The present study determines whether the in vivo injection of TGFß1 and CTGF mediated by AAV2 to transfect nucleus pulposus cells in degenerative lumbar discs can reverse the biological effects of rhesus lumbar disc degeneration. A total of 42 lumbar discs obtained from six rhesus monkeys were classified into three groups: experimental group, control group, and blank group. Degenerative lumbar discs were respectively injected with double gene-transfected human nucleus pulposus cells using minimally invasive techniques. Immumohistochemical staining, RT-PCR, and western blot were performed to observe the biological effects of double gene-transfected human nucleus pulposus cells in degenerative lumbar discs on rhesus lumbar disc degeneration. At 4, 8, and 12 weeks after the transplantation of nucleus pulposus cells, the expression levels of TGF-ß1, CTGF, proteoglycan mRNA, and type-II collagen were detected by RT-PCR. The values of immumohistochemical staining and RT-PCR in the experimental group increased at 8 weeks, decreased with time at 12 weeks, and remained greater than the values in the control group, and the differences were statistically significant (P < .05). The western blot revealed that the values in the experimental group decreased with time, but remained greater than those in the PBS control group and blank control group, and the differences were statistically significant (P < .05). The double gene-transfection of human nucleus pulposus cells in degenerative lumbar discs mediated by rAAV2 can be continuously expressed in vivo after transplantation in lumbar discs of rhesus monkeys, and promotes the synthesis of proteoglycan and type II collagen, achieving the treatment purpose.

Potential of Human Nucleus Pulposus-Like Cells Derived From Umbilical Cord to Treat Degenerative Disc Disease.

BACKGROUND: Degenerative disc disease (DDD) is a common spinal disorder that manifests with neck and lower back pain caused by the degeneration of intervertebral discs (IVDs). Currently, there is no treatment to cure this debilitating ailment. OBJECTIVE: To investigate the potential of nucleus pulposus (NP)-like cells (NPCs) derived from human umbilical cord mesenchymal stem cells (MSCs) to restore degenerated IVDs using a rabbit DDD model. METHODS: NPCs differentiated from MSCs were characterized using quantitative real-time reverse transcription polymerase chain reaction and immunocytochemical analysis. MSCs and NPCs were labeled with fluorescent dye, PKH26, and transplanted into degenerated IVDs of a rabbit model of DDD (n = 9 each). Magnetic resonance imaging of the IVDs was performed before and after IVD degeneration, and following cell transplantation. IVDs were extracted 8 wk post-transplantation and analyzed by various biochemical, immunohistological, and molecular techniques. RESULTS: NPC derivatives of MSCs expressed known NP-specific genes, SOX9, ACAN, COL2, FOXF1, and KRT19. Transplanted cells survived, dispersed, and integrated into the degenerated IVDs. IVDs augmented with NPCs showed significant improvement in the histology, cellularity, sulfated glycosaminoglycan and water contents of the NP. In addition, expression of human genes, SOX9, ACAN, COL2, FOXF1, KRT19, PAX6, CA12, and COMP, as well as proteins, SOX9, ACAN, COL2, and FOXF1, suggest NP biosynthesis due to transplantation of NPCs. Based on these results, a molecular mechanism for NP regeneration was proposed. CONCLUSION: The findings of this study demonstrating feasibility and efficacy of NPCs to regenerate NP should spur interest for clinical studies to treat DDD using cell therapy.

Thermoreversible hyaluronan-hydrogel and autologous nucleus pulposus cell delivery regenerates human intervertebral discs in an ex vivo, physiological organ culture model.

Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1ρ magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1ρ values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture set-up, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.

Transplantation of allogenic nucleus pulposus cells attenuates intervertebral disc degeneration by inhibiting apoptosis and increasing migration.

Transplantation of nucleus pulposus cells (NPCs) into the intervertebral disc (IVD) has been demonstrated to be an effective treatment of degenerative disc disease (DDD). However, the underlying mechanisms have remained to be sufficiently elucidated. The aim of the present study was to explore the potential cell migration and anti­apoptosis efficacy of NPCs in the treatment of DDD. NPCs cultured from rats expressing green fluorescent protein (GFP­NPCs) were transplanted into the degenerated IVD, and the migration of GFP­NPCs, as well as the degeneration and apoptosis of the IVD were detected to evaluate the therapeutic effect in vivo. In vitro, disc chondrocytes (DCs) and annulus fibrosus cells (AFCs) were co­cultured to explore the underlying mechanism. The results demonstrated that injection of NPCs suppressed DDD by inhibiting apoptosis and increasing extracellular matrix in vivo and in vitro. NPCs migrated into the inner AF in vivo, and NPC migration was observed to be promoted by AFCs and DCs in vitro, particularly by damaged AFCs. These results demonstrated the anti­apoptotic effects and migratory capacity of allogenic NPCs transplanted into the IVD, which evidences the contribution of NPCs to disc regeneration and provide a novel strategy for treating DDD.

Characterisation of porous knitted titanium for replacement of intervertebral disc nucleus pulposus.

Effective restoration of human intervertebral disc degeneration is challenged by numerous limitations of the currently available spinal fusion and arthroplasty treatment strategies. Consequently, use of artificial biomaterial implant is gaining attention as a potential therapeutic strategy. Our study is aimed at investigating and characterizing a novel knitted titanium (Ti6Al4V) implant for the replacement of nucleus pulposus to treat early stages of chronic intervertebral disc degeneration. Specific knitted geometry of the scaffold with a porosity of 67.67 ± 0.824% was used to overcome tissue integration failures. Furthermore, to improve the wear resistance without impairing original mechanical strength, electro-polishing step was employed. Electro-polishing treatment changed a surface roughness from 15.22 ± 3.28 to 4.35 ± 0.87 µm without affecting its wettability which remained at 81.03 ± 8.5°. Subsequently, cellular responses of human mesenchymal stem cells (SCP1 cell line) and human primary chondrocytes were investigated which showed positive responses in terms of adherence and viability. Surface wettability was further enhanced to super hydrophilic nature by oxygen plasma treatment, which eventually caused substantial increase in the proliferation of SCP1 cells and primary chondrocytes. Our study implies that owing to scaffolds physicochemical and biocompatible properties, it could improve the clinical performance of nucleus pulposus replacement.

Src-family kinases activation in spinal microglia contributes to central sensitization and chronic pain after lumbar disc herniation.

Background Lumbar disc herniation is a major cause of radicular pain, but the underlying mechanisms remain largely unknown. Spinal activation of src-family kinases are involved in the development of chronic pain from nerve injury, inflammation, and cancer. In the present study, the role of src-family kinases activation in lumbar disc herniation-induced radicular pain was investigated. Results Lumbar disc herniation was induced by implantation of autologous nucleus pulposus, harvest from tail, in lumbar 4/5 spinal nerve roots of rat. Behavior test and electrophysiologic data showed that nucleus pulposus implantation induced persistent mechanical allodynia and thermal hyperalgesia and increased efficiency of synaptic transmission in spinal dorsal horn which underlies central sensitization of pain sensation. Western blotting and immunohistochemistry staining revealed that the expression of phosphorylated src-family kinases was upregulated mainly in spinal microglia of rats with nucleus pulposus. Intrathecal delivery of src-family kinases inhibitor PP2 alleviated pain behaviors, decreased efficiency of spinal synaptic transmission, and reduced phosphorylated src-family kinases expression. Furthermore, we found that the expression of ionized calcium-binding adapter molecule 1 (marker of microglia), tumor necrosis factor-α, interleukin 1 -ß in spinal dorsal horn was increased in rats with nucleus pulposus. Therapeutic effect of PP2 may be related to its capacity in reducing the expression of these factors. Conclusions These findings suggested that central sensitization was involved in radicular pain from lumbar disc herniation; src-family kinases-mediated inflammatory response may be responsible for central sensitization and chronic pain after lumbar disc herniation.

Upregulation of nuclear factor­κB and acid sensing ion channel 3 in dorsal root ganglion following application of nucleus pulposus onto the nerve root in rats.

The nucleus pulposus (NP) is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. To determine the mechanisms by which intervertebral disc degeneration is caused by the nucleus pulposus, the expression and regulation of nuclear factor (NF)­κB and acid sensing ion channel 3 (ASIC3) were examined. For the intervertebral disc degeneration model, NP was harvested from the tail of rats and applied to the L5 dorsal root ganglion (DRG). The mechanical pain withdrawal threshold (PWT) in NP model rats was assessed. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to examine NF­κB and ASIC3 expression levels in DRG. Finally, the effect of the NF­κB inhibitor pyrrolidine dithiocarbamate (PDTC) and the ASIC3 signaling pathway blocker amiloride were examined. Rats exposed to NP exhibited decreased PWT for 12 days, and NF­κB and ASIC3 was upregulated in DRG induced by NP 14 days after surgery. After administration of amiloride and PDTC to DRG affected by NP, the levels of nitric oxide (NO), tumor necrosis factor­α (TNF­α), interleukin­6 (IL­6), NF­κB and ASIC3 were downregulated, and the levels of aquaporin (AQP) 1 and AQP3 were significantly increased for 14 days. In conclusion, these results suggested that NF­κB and ASIC3 may serve an important role in intervertebral disc degeneration caused by NP.

Sciatic nerve regeneration by transplantation of in vitro differentiated nucleus pulposus progenitor cells.

AIM: To assess the applicability of mouse intervertebral disc-derived nucleus pulposus (NP) progenitor cells as a cell source for sciatic nerve regeneration. MATERIALS & METHODS: P0-Cre/Floxed-EGFP-transgenic mouse-derived NP progenitor cells were differentiated to Schwann-like cells in conventional induction medium. Schwann-like cells were subsequently transplanted into a mouse model of sciatic nerve transection, and nerve regeneration assessed by immunohistochemistry, electron microscopy and functional walking track analysis and heat stimulus reflex. RESULTS & CONCLUSION: NP progenitor cells differentiated into Schwann-like cells. Transplantation of these cells promoted myelinated axon formation, morphology restoration and nerve function improvement. NP progenitor cells have the capacity to differentiate into neuronal cells and are candidates for peripheral nerve regeneration therapy.

Characteristics and potentials of stem cells derived from human degenerated nucleus pulposus: potential for regeneration of the intervertebral disc.

BACKGROUND: Eliminating the symptoms during treatment of intervertebral disc degeneration (IVDD) is only a temporary solution that does not cure the underlying cause. A biological method to treat this disorder may be possible by the newly discovered nucleus pulposus derived stem cells (NPDCs). However, the uncertain characteristics and potential of NPDCs calls for a comprehensive study. METHODS: In the present study, nucleus pulposus samples were obtained from 5 patients with IVDD undergoing discectomy procedure and NPDCs were harvested using fluorescence activated cell sorting (FACS) by the co-expression of GD2+ and Tie2+. After in vitro expansion, the properties of NPDCs were compared with those of bone marrow mesenchyme stem cells (BMSCs) from the same subjects. RESULTS: NPDCs performed similar properties in cell colony-forming ability, cell proliferation rate, cell cycle and stem cell gene expression similar to those of BMSCs. In addition, NPDCs could be differentiated into osteoblasts, adipocytes, and chondrocytes, and are found to be superior in chondrogenesis but inferior in adipocyte differentiation. CONCLUSIONS: NPDCs derived from the degenerated intervertebral disc still keep the regeneration ability similar to BMSCs. Besides, the superior capacity in chondrogenesis may provide a promising cell candidate for cell-based regenerative medicine and tissue engineering in IVDD.

Longitudinal Comparison of Enzyme- and Laser-Treated Intervertebral Disc by MRI, X-Ray, and Histological Analyses Reveals Discrepancies in the Progression of Disc Degeneration: A Rabbit Study.

Regenerative medicine is considered an attractive prospect for the treatment of intervertebral disc (IVD) degeneration. To assess the efficacy of the regenerative approach, animal models of IVD degeneration are needed. Among these animal models, chemonucleolysis based on the enzymatic degradation of the Nucleus Pulposus (NP) is often used, but this technique remains far from the natural physiopathological process of IVD degeneration. Recently, we developed an innovative animal model of IVD degeneration based on the use of a laser beam. In the present study, this laser model was compared with the chemonucleolysis model in a longitudinal study in rabbits. The effects of the treatments were studied by MRI (T2-weighted signal intensity (T2wsi)), radiography (IVD height index), and histology (NP area and Boos' scoring). The results showed that both treatments induced a degeneration of the IVD with a decrease in IVD height and T2wsi as well as NP area and an increase in Boos' scoring. The enzyme treatment leads to a rapid and acute process of IVD degeneration. Conversely, laser radiation induced more progressive and less pronounced degeneration. It can be concluded that laser treatment provides an instrumental in vivo model of slowly evolving IVD degenerative disease that can be of preclinical relevance for assessing new prophylactic biological treatments of disc degeneration.

Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells.

During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells. Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured as a micromass or in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I, and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR and western blotting. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility, and cell proliferation increased after collagen type II treatment. Expressions of SOX9, aggrecan, and collagen type II were increased in a collagen type II dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type II concentration. Only hADMSCs highly expressed NP cell marker KRT19 in collagen type II culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type II-stimulation. The concentration and type of collagen affect hADMSC differentiation into NP cells. Collagen type II significantly ameliorates hADMSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type II may attenuate the differentiation and biosynthesis of transplanted stem cells.

TGF-ß1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

Degenerative disc disease (DDD) primarily affects the central part of the intervertebral disc namely the nucleus pulposus (NP). DDD explains about 40% of low back pain and is characterized by massive cellular alterations that ultimately result in the disappearance of resident NP cells. Thus, repopulating the NP with regenerative cells is a promising therapeutic approach and remains a great challenge. The objectives of this study were to evaluate the potential of growth factor-driven protocols to commit human adipose stromal cells (hASCs) toward NP-like cell phenotype and the involvement of Smad proteins in this differentiation process. Here, we demonstrate that the transforming growth factor-ß1 and the growth differentiation factor 5 synergistically drive the nucleopulpogenic differentiation process. The commitment of the hASCs was robust and highly specific as attested by the expression of NP-related genes characteristic of young healthy human NP cells. In addition, the engineered NP-like cells secreted an abundant aggrecan and type II collagen rich extracellular matrix comparable with that of native NP. Furthermore, we demonstrate that these in vitro engineered cells survived, maintained their specialized phenotype and secretory activity after in vivo transplantation in nude mice subcutis. Finally, we provide evidence suggesting that the Smad 2/3 pathway mainly governed the acquisition of the NP cell molecular identity while the Smad1/5/8 pathway controlled the NP cell morphology. This study offers valuable insights for the development of biologically-inspired treatments for DDD by generating adapted and exhaustively characterized autologous regenerative cells.