Platelet-rich plasma (PRP) is a fraction of plasma that contains high levels of multiple growth factors. The purpose of this study was to examine the effects of PRP on cell proliferation and matrix ...synthesis by porcine chondrocytes cultured in alginate beads, conditions that promote the retention of the chondrocytic phenotype, in order to determine the plausibility of using this plasma-derived material for engineering cartilage.
PRP and platelet-poor plasma (PPP) were prepared from adult porcine blood. Adult porcine chondrocytes were cultured in the presence of 10% PRP, 10% PPP or 10% fetal bovine serum (FBS) for 3 days. Cell proliferation, proteoglycan (PG) and collagen synthesis were quantified, and the structure of newly synthesized PG and collagen was characterized.
Treatment with 10% PRP resulted in a small but significant increase in DNA content (+11%, vs FBS;
P
<
0.01; vs PPP;
P
<
0.001). PG and collagen syntheses by the PRP-treated chondrocytes were markedly higher than those by chondrocytes treated by FBS or PPP (PG; PRP: +115% vs FBS; +151% vs PPP, both
P
<
0.0001, collagen; PRP: +163% vs FBS; +163% vs PPP, both
P
<
0.0001). Biochemical analyses revealed that treatment with PRP growth factors did not markedly affect the types of PGs and collagens produced by porcine chondrocytes, suggesting that the cells remained phenotypically stable in the presence of PRP.
PRP isolated from autologous blood may be useful as a source of anabolic growth factors for stimulating chondrocytes to engineer cartilage tissue.
While it is known that the degenerated intervertebral disc (IVD) is one of the primary reasons for low-back pain and subsequent need for medical care, there are currently no established effective ...methods for direct treatment. Nuclear factor-κB (NF-κB) is a transcription factor that regulates various genes' expression, among which are inflammatory cytokines, in many tissues including the IVD. NF-κB decoy is an oligodeoxynucleotide containing the NF-κB binding site that entraps NF-κB subunits, resulting in suppression of NF-κB activity. In the present preclinical study, NF-κB decoy was injected into degenerated IVDs using the rabbit anular-puncture model. In terms of distribution, NF-κB decoy persisted in the IVDs up to at least 4 weeks after injection. The remaining amount of NF-κB decoy indicated that it fit a double-exponential-decay equation. Investigation of puncture-caused degeneration of IVDs showed that NF-κB decoy injection recovered, dose-dependently, the reduced disc height that was associated with reparative cell cloning and morphological changes, as assessed through histology. Gene expression, by quantitative real-time polymerase chain reaction (qRT-PCR), showed that NF-κB decoy attenuated inflammatory gene expression, such as that of interleukin-1 and tumor necrosis factor-α, in rabbit degenerated IVDs. NF-κB decoy also reduced the pain response as seen using the "pain sensor" nude rat xenograft-radiculopathy model. This is the first report demonstrating that NF-κB decoy suppresses the inflammatory response in degenerated IVDs and restores IVD disc height loss. Therefore, the intradiscal injection of NF-κB decoy may have the potential as an effective therapeutic strategy for discogenic pain associated with degenerated IVDs.
Nuclear factor-kappa B (NF-κB) has a pivotal role in the progression and distant metastasis of cancers, including malignant bone tumors. To inhibit NF-κB activation, a new molecular therapy using ...synthetic double-stranded oligodeoxynucleotide (ODN) as a 'decoy' cis element against NF-κB has been developed. To determine whether pulmonary metastasis of osteosarcoma is reduced by inhibiting the action of NF-κB, NF-κB decoy ODN was transfected into the nuclei of murine osteosarcoma cells with high pulmonary metastatic potential, the LM8 cell line, using a three-dimensional alginate spheroid culture model. An in vitro study demonstrated the successful transfection of LM8 cells cultured in alginate beads by 'naked' NF-κB decoy ODN and that the activation of NF-κB signaling was significantly suppressed. Tumor growth was not affected by transfection of NF-κB decoy ODN, however, the expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) mRNA was markedly decreased. Furthermore, the transfection of 'naked' NF-κB decoy ODN effectively suppressed pulmonary metastasis in an in vivo alginate bead transplantation model. Our results suggest that NF-κB has a central and specific role in the regulation of tumor metastasis and could be a molecular target for development of anti-metastatic treatments for osteosarcoma.
A retrospective study.
We have encountered several cases of complete sensorimotor paralysis in which patellar tendon reflex (PTR) was demonstrated approximately 3 days after injury and improvement of ...motor paralysis was subsequently achieved. We considered that PTR apparent in the early stage after injury may offer an index to predict improvements in motor paralysis.
A total of 142 patients assessed as ASIA Impairment Scale A on admission from 1979 to 1998 were included in the study. The patients who demonstrated PTR within 72 h after injury were classified as the PTR(+) group and those who did not constituted the PTR(-) group. With regard to the method of motor paralysis assessment at about 6 months after injury, patients assessed as ASIA Impairment Scale A or B (that is, complete motor paralysis) were classified as 'Non-recovered', whereas those assessed as ASIA Impairment Scale C, D or E (that is, showing obvious improvement of motor paralysis) were considered as 'Recovered'.
A significant difference was noted between groups, with the Recovered group including 16 of the 17 PTR(+) patients (94.1%) and 11 of the 115 PTR(-) patients (9.6%) (P<0.0001).
The results obtained indicate that motor paralysis recovery could be expected at a very high rate among patients demonstrating PTR within 72 h of injury. As all physicians should be familiar with the PTR, this seems to represent a simple and highly useful sign to predict improvements in motor paralysis during the acute stage of cervical cord injury.
Low back pain (LBP) is now regarded as the first cause of disability worldwide and should be a priority for future research on prevention and therapy. Intervertebral disc (IVD) degeneration is an ...important pathogenesis of LBP. Platelet-rich plasma (PRP) is an autologous blood concentrate that contains a natural concentration of autologous growth factors and cytokines and is currently widely used in the clinical setting for tissue regeneration and repair. PRP has great potential to stimulate cell proliferation and metabolic activity of IVD cells in vitro. Several animal studies have shown that the injection of PRP into degenerated IVDs is effective in restoring structural changes (IVD height) and improving the matrix integrity of degenerated IVDs as evaluated by magnetic resonance imaging (MRI) and histology. The results of this basic research have shown the great possibility that PRP has significant biological effects for tissue repair to counteract IVD degeneration. Clinical studies for evaluating the effects of the injection of PRP into degenerated IVDs for patients with discogenic LBP have been reviewed. Although there was only one double-blind randomized controlled trial, all the studies reported that PRP was safe and effective in reducing back pain. While the clinical evidence of tissue repair of IVDs by PRP treatment is currently lacking, there is a great possibility that the application of PRP has the potential to lead to a feasible intradiscal therapy for the treatment of degenerative disc diseases. Further large-scale studies may be required to confirm the clinical evidence of PRP for the treatment of discogenic LBP.
Central sensitization (CS) is defined as the increased responsiveness of nociceptive neurons in the central nervous system to normal or subthreshold afferent input. CS has been proposed as an ...underlying mechanism of chronic pain in musculoskeletal disorders including low back pain (LBP). A Central Sensitization Inventory (CSI) has recently been developed for screening participants with CS. However, the association of CS with chronic LBP (cLBP) in the general population remains unknown. The purpose of this study was to investigate the association of CS with cLBP using the CSI in a population-based cohort of a Japanese mountain village.
Participants aged more than 50 years were recruited from the inhabitants of a mountain village in Japan. Participants completed the following patient-reported outcome measures. Severity of CS was assessed by the CSI. LBP intensity was measured on a numerical rating scale (NRS). Health-related quality of life (QOL) was measured using the EuroQol 5-dimension (EQ-5D), EuroQol-visual analogue scales (EQ-VAS), and the Oswestry Disability Index (ODI). The association of CS and each parameter was statistically evaluated.
A total of 272 participants (average age: 72.1 years-old) were analyzed in this study, and 28.3% had cLBP. Average NRS, ODI and CSI scores were significantly higher in the cLBP group than in the without LBP (LBP-) group. There was a significant correlation between CSI and NRS scores (
=0.34, P<0.0001), ODI (
=0.60, P<0.0001), EQ5D (
=-0.55, P<0.0001) and EQ-VAS (
=-0.52, P<0.0001). A multiple regression analysis identified that ODI, EQ-VAS and age were factors significantly associated with CSI.
The results of this study suggest that CS is involved in the pathological condition of cLBP in the local residents of a Japanese mountain village.
In vivo study of the effect of injection of osteogenic protein-1 (OP-1) on a rabbit anular needle puncture model of intervertebral disc (IVD) degeneration.
To study radiographic, magnetic resonance ...imaging (MRI), biochemical, and histologic changes in the rabbit IVD after injection of OP-1 into the nucleus pulposus in a needle puncture disc degeneration model.
Growth factors, such as OP-1, have the ability to stimulate synthesis of proteoglycans and collagen in vitro. The in vivo injection of OP-1 into the normal rabbit IVD has increased disc height and proteoglycan content in the anulus fibrosus and nucleus pulposus. However, to our knowledge, no attempts have yet been made to determine the effects of these growth factors in an in vivo model of disc degeneration.
New Zealand adolescent white rabbits (n = 90, 8 for baseline evaluation, 82 at 8 times) received an anular puncture in 2 noncontiguous discs with an 18-gauge needle to induce disc degeneration. Four weeks later, either 5% lactose (10 microL) or OP-1 (100 microg in 10 microL 5% lactose) was injected into the center of the nucleus pulposus. The disc height was followed radiographically for up to 24 weeks after the injections. At the 2, 4, 8, 12, and 24-week times after the injection, rabbits were euthanized, and MRI of the harvested spinal columns was obtained to grade the degeneration. The discs injected with OP-1 or lactose and noninjected discs were subjected to biochemical and histologic analysis. The specimens at the 24-week time were limited to histologic evaluation.
The anular puncture with a needle induced a consistent disc narrowing within 4 weeks. The injection of OP-1 induced a restoration of disc height at 6 weeks, which was sustained for the entire experimental period, up to 24 weeks after the injection. The injection of lactose alone did not change the course of disc narrowing over the same time. MRI grading score showed significant differences between the OP-1 and lactose groups at the 8, 12, and 24-week times, suggesting an increase in water content in the nucleus pulposus of the OP-1 group. The proteoglycan content of the nucleus pulposus and anulus fibrosus was significantly higher in the OP-1 group than in the control group. The degeneration grades of the punctured discs in the OP-1 group were significantly lower than those in the lactose group.
The results of this study show the feasibility of restoring degenerative rabbit discs by a single injection of OP-1 into the nucleus pulposus. Importantly, the effects of the OP-1 injection on disc height were sustained for up to 24 weeks. The metabolic changes in the cells, following a single injection, might be sustained and, thus, induce long-term changes in disc structure. An efficacy study in large animals is required to show further that the intradiscal injection of OP-1, or bone morphogenetic proteins or growth factors with similar properties would be useful for the structural restoration of the IVD in humans.
In vitro studies of the effects of proinflammatory cytokines on the production of nerve growth factor (NGF) by human intervertebral disc (IVD) cells.
To determine the constitutive expression and ...production of NGF and the effect of cytokines on the expression of NGF by human IVD cells.
NGF may play a role in the collateral sprouting of sensory axons, neural survival, and regulation of nociceptive sensory neurons. NGF is known to be up-regulated by proinflammatory cytokines.
The presence of NGF protein was analyzed by immunohistochemistry using human IVD cells obtained from cadaveric human spines with no known disc disease (MRI Thompson grades 2-4). The effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on NGF production and mRNA expression of NGF by IVD cells were examined. The expression of NGF receptors, trkA and p75, was also assessed immunohistochemically.
Cadaveric anulus fibrosus (AF) and nucleus pulposus (NP) cells cultured in vitro in monolayer and in alginate beads positively stained with an anti-NGF antibody. The constitutive production of NGF protein in IVD cells was low (NP) or not detectable (AF). The expression of NGF mRNA was detectable in both cell types. IL-1beta and TNF-alpha up-regulated the NGF mRNA expression and the secretion of NGF protein into the media. TrkA was immunolocalized in AF and NP cells.
Our results demonstrate, for the first time, that human AF and NP cells constitutively express NGF protein and mRNA, and that the proinflammatory cytokines IL-1beta and TNF-alpha stimulate the production of NGF. The precise role of NGF produced by IVD cells in the generation of discogenic pain or on the metabolism of IVD cells, especially under certain physiologic conditions in which cytokines are up-regulated, needs to be clarified in future experimentation.
In vitro assessment of the effects of platelet-rich plasma on the extracellular matrix metabolism of porcine intervertebral disc cells.
To determine whether platelet-rich plasma is effective in ...stimulating cell proliferation and extracellular matrix metabolism by porcine disc cells cultured in alginate beads.
Platelet-rich plasma is used to accelerate wound healing and tissue regeneration. Activated platelets release multiple growth factors that regulate cell proliferation, differentiation, and morphogenesis. Individual growth factors present in platelet-rich plasma have been demonstrated to affect the metabolism of intervertebral disc cells.
Platelet-poor and platelet-rich plasma was isolated from fresh porcine blood using a commercially available platelet concentration system. After preculture for 7 days and serum starvation for 24 hours, the beads containing nucleus pulposus and anulus fibrosus cells were then cultured for another 72 hours in serum-free medium, 10% fetal bovine serum, 10% platelet-poor plasma, or 10% platelet-rich plasma. The synthesis of proteoglycans and collagen, the accumulation of proteoglycans, and the DNA content were biochemically assessed.
Platelet-rich plasma had a mild stimulatory effect on cell proliferation of intervertebral disc cells. Platelet-rich plasma treatment significantly upregulated proteoglycan and collagen synthesis and proteoglycan accumulation when compared with platelet-poor plasma.
Platelet-rich plasma was effective in stimulating cell proliferation and extracellular matrix metabolism. The response to platelet-rich plasma was greater in the case of anulus fibrosus cells than of nucleuspulposus cells. The local administration of platelet-rich plasma might stimulate intervertebral disc repair. In addition, given the risks of using animal serum for tissue engineering, autologous blood may gain favor as a source of growth factors and serum supplements needed for stimulating cells to engineer intervertebral disc tissues.
In vitro study on the effects of pulsed low intensity ultrasound on the cellular metabolism of bovine intervertebral disc cells.
To determine whether pulsed low intensity ultrasound has effects on ...cell proliferation and extracellular matrix metabolism by bovine intervertebral disc cells.
The application of pulsed low intensity ultrasound is known to be effective in stimulating fracture and cartilage repair. However, the effects of pulsed low intensity ultrasound on intervertebral disc cells are not known.
Cells of the nucleus pulposus and inner and outer anulus fibrosus were enzymatically isolated from bovine coccygeal tissue and precultured in alginate beads for 14 days. In the ultrasound group, pulsed low intensity ultrasound was administered to the culture for 20 minutes daily for an additional 20 days. The control group was cultured in the same way but without administration of ultrasound. Cell viability, DNA content, proteoglycan and collagen synthesis, and proteoglycan content at days 10 and 20 after the initiation of treatment were evaluated. Characterization of newly synthesized collagen and proteoglycan was performed.
No significant differences in cell viability and DNA content were observed between the two groups. On day 20, proteoglycan synthesis was increased by the application of pulsed low intensity ultrasound in nucleus pulposus and inner and outer anulus fibrosus cells (24%-26% increase, P < 0.001). The application of pulsed low intensity ultrasound increased proteoglycan content in alginate beads containing inner and outer anulus fibrosus cells (P < 0.05). Collagen synthesis by cells isolated from all three zones of the intervertebral disc was increased by the application of pulsed low intensity ultrasound (16%-19% increase, P < 0.05-0.0001).
The application of pulsed low intensity ultrasound stimulated extracellular matrix metabolism in intervertebral disc cells. Pulsed low intensity ultrasound may prove useful for the physical stimulation of cell metabolism for tissue engineering of intervertebral disc tissue.
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