Human nucleus pulposus cells react to IL-6: independent actions and amplification of response to IL-1 and TNF-α.

STUDY DESIGN.: Human nucleus pulposus cells were activated with IL-6 plus IL-6 soluble receptor (sR) in the presence or absence of IL-1ß or TNF-α. Cell production of factors modulating the anabolic/catabolic balance of the disc and proteoglycan synthesis were determined. OBJECTIVE.: To evaluate NP cell response to exogenous IL-6, and how IL-6 modulates IL-1 and TNF-α actions in these cells. SUMMARY OF BACKGROUND DATA.: Interleukin-6 (IL-6) is produced by cervical and lumbar herniated discs and is associated with neurological symptoms of intervertebral disc degeneration. It upregulates catabolic gene expression and downregulates matrix protein gene expression in chondrocytes. However, no studies have evaluated the effects of IL-6 on disc nucleus pulposus (NP) cells. METHODS.: NP cells from degenerated human discs were expanded in monolayer, maintained in alginate bead culture, and activated with IL-6 plus IL-6 soluble receptor (sR), in the presence or absence of IL-1ß or TNF-α. Conditioned media was collected and analyzed for nitrite, PGE-2, TIMP-1, MMP-3, VEGF, and IL-8. Proteoglycan synthesis was assayed as S-sulfate incorporation normalized to DNA content and relative gene expression measured by rtPCR. RESULTS.: IL-6 + sR decreased collagen and aggrecan message, proteoglycan synthesis, and exacerbated the downregulation of proteoglycan synthesis effected by IL-1. PGE-2 synthesis was increased by IL-6 + sR, as was the induction of COX-2 mRNA. IL-6 + sR also enhanced IL-1 and TNF-α stimulated synthesis of PGE-2. IL-6 + sR induced MMP-3 approximately twofold and increased gene expression and synthesis in cells exposed to IL-1 and TNF-α. MMP-13 induction by TNF-α was also potentiated by IL-6 + sR. IL-6 + sR induced IL-6 gene expression and increased that stimulated by TNF-α fourfold. CONCLUSION.: The results suggest maneuvers to diminish IL-6 production in the disc could provide some protection against the adverse effects of IL-1 and TNF-α, thus, helping preserve disc composition, structure, and function.

Alterations in gene expression in response to compression of nucleus pulposus cells.

BACKGROUND CONTEXT: It is clear that mechanical forces are involved in initiating disc degeneration but also have the potential to exert beneficial effects. However, the signaling pathways initiated by mechanical stress and thresholds for these responses have not been elucidated. We have developed a metabolically active compression system with the advantages of having the ability to test cells in vitro as well as within their native matrix and control exposure to environmental factors. We hypothesized that nucleus pulposus cells would respond to compressive stress with different thresholds for alterations in catabolic and anabolic gene expression. PURPOSE: The purpose of the study was to establish the utility of a novel compression chamber and examine the effects of various magnitudes and durations of compression on nucleus pulposus inflammatory, catabolic, and anabolic gene expression. STUDY DESIGN: In vitro controlled examination of intervertebral disc cell responses to compression. METHODS: A chamber capable of imparting 0 to 20 MPa of hydrostatic compression onto nucleus pulposus cells was fabricated. Healthy rabbit nucleus pulposus cells were cultured in alginate beads and exposed to static compression at 0.7, 2, and 4 MPa for 4 or 24 hours. Gene expression analysis (real-time polymerase chain reaction) was performed to compare markers of inflammation (inducible nitric oxide synthase, cyclooxygenase-2), matrix catabolism (matrix metalloproteinase-3), and anticatabolic/anabolic metabolism (tissue inhibitor of metalloproteinase-1, aggrecan) in control and compressed cells. RESULTS: Compression resulted in magnitude- and duration-dependent changes in gene expression. Increasing magnitudes showed more anticatabolic gene expression changes, whereas increasing duration resulted in increases in procatabolic gene expression. CONCLUSION: These data demonstrate favorable effects of compression in relation to genes involved in matrix homeostasis and procatabolic gene expression in response to sustained loading levels, consistent with traumatic effects. These data provide an improved understanding of how compression affects cell signaling, which has the potential to be exploited to initiate repair and prevent matrix breakdown.

Prostaglandin E2 and prostaglandin F2α differentially modulate matrix metabolism of human nucleus pulposus cells.

Prostaglandin (PG) actions on disc metabolism are unclear even though certain PGs are highly expressed by disc cells under inflammatory conditions and nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used to block PG production to treat back pain. Hence this study aimed to (1) quantify gene expression of arachidonic acid cascade components responsible for PG synthesis and (2) examine the effects of key PGs on disc matrix homeostasis. Microarray analysis revealed that inflammatory stress increases expression of synthases and receptors for prostaglandin E2 (PGE(2)) and prostaglandin F2α (PGF(2α)), resulting in elevated PGE(2) and PGF(2α) production in conditioned media of disc cells. PGE(2) diminished disc cell proteoglycan synthesis, in a dose-dependent manner. Semiquantitative RT-PCR revealed differential effects of PGE(2) and PGF(2α) on disc cell expression of key matrix structural genes, aggrecan, versican, collagens type I and II. PGE(2) and PGF(2α) also decreased message for the anabolic factor, IGF-1. PGE(2) decreased mRNA expression for the anti-catabolic factor TIMP-1 while PGF(2α) increased mRNAs for catabolic factors MMP-1 and MMP-3. Thus, PGE(2) and PGF(2α) may have an overall negative impact on disc matrix homeostasis, and the use of NSAIDs may impact disc metabolism as well as treat back pain.

Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro.

STUDY DESIGN: We demonstrated the differentiation of notochordal cells by direct observation using a live automated cell imaging system. We also hypothesized that notochordal cells have characteristics of chondrocyte-like cells. OBJECTIVE: To determine characteristics of notochordal cells by matrix protein expression and their differentiation using a live automated cell imager. SUMMARY OF BACKGROUND DATA: Although notochordal cells are critical to homeostasis of intervertebral disc, their fate has not been extensively studied and there is little evidence of notochordal cells as progenitors. METHODS: Notochordal cells purified from rabbit nucleus pulposus were isolated after serial filtration. Notochordal cells in 3-dimensional culture were compared to chondrocyte-like cells by S sulfate incorporation into proteoglycan and reverse transcription polymerase chain reaction for gene expression(collagen II and aggrecan). Notochordal cells in 2-D culture were used for immunocytochemical staining (collagen II, aggrecan, and SOX9) and time-lapsed cell tracking study. RESULTS: Notochordal cells were capable of proteoglycan production at a rate comparable to chondrocyte-like cells (108% +/- 22.6% to chondrocyte-like cells) and expressed collagen II, aggrecan, and SOX9. In time-lapsed cell tracking analysis, notochordal cells were slower in population doubling time than chondrocyte-like cells and differentiated into 3 morphologically distinct cell types: vacuolated cells (area: 2392 +/- 507.1 microm, velocity: 0.09 +/- 0.01 microm/min); giant cells (area: 12678 +/- 1637.0 microm, velocity: 0.08 +/- 0.01 microm/min) which grew rapidly without cell division; polygonal cells (area: 3053 +/- 751.2 microm, 0.14 +/- 0.01 microm/min) morphologically similar to typical differentiation type of chondrocyte-like cells (area: 2671 +/- 235.6 microm, 0.19 +/- 0.01 microm/min). Rarely, notochordal cells formed clusters analogous to that observed in vivo. CONCLUSION: These studies demonstrate a chondrocyte phenotype of notochordal cells and are the first direct evidence of notochordal cell differentiation, suggesting that they may act as progenitor cells, which has the potential to lead to their use in novel approaches to regeneration of degenerative intervertebral disc.

Activated macrophage-like THP-1 cells modulate anulus fibrosus cell production of inflammatory mediators in response to cytokines.

STUDY DESIGN: Anulus fibrosus (AF) cells obtained from patients undergoing surgery were cocultured with macrophage-like cells and production of inflammatory mediators was analyzed by quantitative assay. OBJECTIVE: To investigate the role of macrophages in AF cell production of inflammatory mediators by cytokines stimulation. SUMMARY OF BACKGROUND DATA: Discogenic pain caused by anular disruption is an important cause of low back pain and recent studies show the presence of macrophages in symptomatic discs but not in normal and aging discs. We hypothesize that macrophages play a major role in development of symptomatic disc. METHODS: Human AF cells were cocultured with phorbol myristate acetate stimulated macrophage-like THP-1 cells. The conditioned medium from cells cultured alone or in coculture was assayed for cytokines by Enzyme-linked immunosorbent assay and nitric oxide (NO) by the Greiss method. Using the same outcome measures, comparisons of cell response to cytokines were made among macrophage-like cells, naïve AF cells, and macrophage exposed AF cells. RESULTS.: Tumor necrosis factor (TNF)-alpha, interleukin (IL)-8, IL-6, and NO (TNF-alpha: 1.45 +/- 0.29 ng/mL, IL-8: 97.02 +/- 7.94 ng/mL, IL-6: 33.40 +/- 3.55 ng/mL, NO: 8.42 +/- 0.78 micromol/L) were secreted in much greater amounts by cells maintained in coculture compared to macrophages (TNF-alpha: 0.78 +/- 0.12 ng/mL, IL-8: 58.04 +/- 4.44 ng/mL, IL-6: 0.14 +/- 0.03 ng/mL, NO: 0.30 +/- 0.08 micromol/L) or AF cells cultured alone. In addition, IL-6 secretion from AF cells in response to TNF-alpha was up-regulated by coculture, however, IL-6 secretion in response to IL-1 beta was downregulated in a dose-dependent manner. Coculture with macrophages also up-regulated AF cell secretion of IL-8 dose-dependently and downregulated NO to TNF-alpha or IL-1beta stimulation. CONCLUSION: We conclude that exposure to macrophages, as can be expected after anular injury, can result in enhanced response to local inflammation. Although changes were observed in all inflammatory mediators after macrophage exposure, the most significant change was observed in IL-6 and IL-8, implicating these mediators in development of symptomatic disc.

Gene therapy for the treatment of degenerative disk disease.

Recent biologic and biochemical advances have furthered our understanding of the complex environment of the intervertebral disk. This new understanding has allowed researchers to pursue novel treatments of intervertebral disk degeneration, targeting the biochemical pathways involved in the degenerative cascade. Gene therapy has shown much promise in this regard. Many new targets for gene therapy in the intervertebral disk have been identified, such as TGF-beta1, TIMP-1, and LMP-1. In addition, new vectors, such as the adeno-associated virus, are being investigated for use in intervertebral disk applications. Cell-based therapy has also shown significant promise in the biologic treatment of intervertebral disk degeneration. With continued efforts, gene therapy may prove to be an extremely powerful tool in the treatment of intervertebral disk degeneration.

The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells.

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are potential therapeutic factors for degenerative discs, and BMP-12 does not have the osteogenic potential of BMP-2, making it better suited for intradiscal injection. However, no reports have compared the actions of BMP-2 and -12 on human annulus fibrosus (AF) and nucleus pulposus (NP) cells nor evaluated adenoviral-mediated gene therapy in human AF cells. PURPOSE: To evaluate and compare the effects of recombinant human (rh) BMP-2, rhBMP-12, and adenoviral BMP-12 (Ad-BMP-12) on nucleus pulposus and annulus fibrosis cell matrix protein synthesis. STUDY DESIGN: In vitro study using rhBMP-2 and -12 and adenoviral BMP-12 with human intervertebral disc (IVD) cells. METHODS: Human NP and AF IVD cells were isolated, maintained in monolayer, and incubated with BMP-2 or -12 for 2 days. AF and NP cells were transduced with Ad-BMP-12, pellets formed, and incubated for 6 days. Growth factor-treated cells were labelled with either 35-S or 3H-proline to assay matrix protein synthesis. RESULTS: rhBMP-2 increased NP proteoglycan, collagen, and noncollagen protein synthesis to 355%, 388%, and 234% of control. RhBMP-12 increased the same NP matrix proteins' synthesis to 140%, 143%, and 160% of control. Effects on AF matrix protein synthesis were minimal. Ad-BMP-12 significantly increased matrix protein synthesis and DNA content of AF and NP cells in pellet culture. NP synthesis of all matrix proteins and AF synthesis of proteoglycans was increased when the data were normalized to pellet DNA. AF synthesis of noncollagen protein and collagen was not modulated by Ad-BMP-12 if the data are normalized to pellet DNA content. CONCLUSIONS: Both rhBMP-2 and -12 increase human NP cell matrix protein synthesis while having minimal effects on AF cells. However, Ad-BMP-12 did increase matrix protein synthesis in both NP and AF cells, making it a potential therapy for enhancing matrix production in the IVD. These responses plus the proliferative action of Ad-BMP-12 seen in the current studies, and the lack of an osteogenic action noted in other studies justifies future studies to determine if gene therapy with BMP-12 could provide protective and/or reparative actions in degenerating discs.

Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion.

STUDY DESIGN: Changes in gene expression profile and cell fusion of mesenchymal stem cells (MSC) and nucleus pulposus cells (NPC) after coculture were analyzed. OBJECTIVE: To investigate the mechanisms of the interaction between NPC and MSC such us differentiation, stimulatory effect, and cell fusion. SUMMARY OF BACKGROUND DATA: Introduction of exogenous cells to supplement and replenish intervertebral disc cell population offers a potential approach to treat intervertebral disc degeneration (IDD). Recent evidences showed that intradiscal injection of MSC effectively alter the course of IDD in vivo, and the regenerative potential may result from up-regulated extracellular matrix protein synthesis mediated by MSC and NPC interaction. METHODS: Using a double labeling cell system and flow activated cell sorting, we quantitatively analyzed changes in the gene expression profile of human male MSC and female NPC after coculture in a 3-dimensional system that allows short distance paracrine interactions typical of the nucleus pulposus. Furthermore, we analyzed for cell fusion in the cell interaction by fluorescence in situ hybridization (FISH) for X and Y chromosomes, using a 3-dimensional culture system to allow cell-to-cell interactions conducive to cell fusion. RESULTS: Two weeks of coculture cell interaction in a 3-dimensional environment induces a change in MSCs towards a more chondrogenic gene expression profile indicating MSC differentiation, and NPC gene expression changes in matrix and chondrogenic genes demonstrating only a modest trophic effect of MSC on NPC. Moreover, FISH analysis demonstrated that cell fusion is not responsible for MSC plasticity in the interaction with NPCs. CONCLUSION: This study clarifies the mechanism of MSCs and NPCs interaction in a 3-dimensional environment, excluding cell fusion. These data support the use of undifferentiated MSC for stem cell therapy for IDD treatment.

p38 MAPK inhibition modulates rabbit nucleus pulposus cell response to IL-1.

Analysis of disc gene expression implicated IL-1 in the development of intervertebral disc degeneration (IDD) in a rabbit stab model. The purpose of these studies is to determine the role of p38 Mitogen Activated Protein Kinase (p38 MAPK) signaling in nucleus pulposus cell response to IL-1, and to compare rabbit nucleus pulposus (rNP) cell responses to IL-1 activation with those in a stab model of disc degeneration. NP cells maintained in alginate bead culture were exposed to IL-1, with or without p38 MAPK inhibition. RNA was isolated for reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression, conditioned media analyzed for accumulation of nitric oxide (NO) and prostaglandin E-2 (PGE-2), and proteoglycan synthesis measured after 10 days. IL-1 upregulation of mRNA for cycloxygenase-2 (COX-2), matrix metalloproteinase-3 (MMP-3), IL-1, and IL-6, was blunted by p38 inhibition while downregulation of matrix proteins (collagen I, collagen II, aggrecan) and insulin-like-growth-factor I (IFG-1) was also reversed. mRNA for tissue inhibitor of matrixmetalloproteinase-1 (TIMP-1) was modestly increased by IL-1, while those for Transforming Growth Factor-beta (TGF-beta) SOX-9, and versican remained unchanged. Blocking p38 MAPK reduced IL-1 induced NO and PGE-2 accumulation and partially restored proteoglycan synthesis. p38 MAPK inhibition in control cells increased mRNA for matrix proteins (aggrecan, collagen II, versican, collagen I) and anabolic factors (IGF-1, TGF, and SOX-9) from 50% to 120%, decreased basal PGE-2 accumulation, but had no effect on message for TIMP-1, MMP-3, or COX-2. Inhibition of p38 MAPK in cytokine-activated disc cells blunts gene expression and production of factors associated with inflammation, pain, and disc matrix catabolism while reversing IL-1 downregulation of matrix protein gene expression and proteoglycan synthesis. The results support the hypothesis that IL-1 could be responsible for many of the mRNA changes seen in rabbit NP in the stab model of disc degeneration, and uphold the concept that development of molecular techniques to block p38 MAPK could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

Clinical diagnosis of potentially treatable early articular cartilage degeneration using optical coherence tomography.

A series of bench to operating room studies was conducted to determine whether it is feasible to use optical coherence tomography (OCT) clinically to diagnose potentially reversible early cartilage degeneration. A human cadaver study was performed to confirm the reproducibility of OCT imaging and grading based on identification of changes to cartilage OCT form birefringence using a polarized OCT system approved for clinical use. Segregation of grossly normal appearing human articular cartilage into two groups based on the presence or absence of OCT form birefringence showed that cartilage without OCT form birefringence had reduced ability to increase proteoglycan synthetic activity in response to the anabolic growth factor IGF-1. The bench data further show that IGF-1 insensitivity in cartilage without OCT form birefringence was reversible. To show clinical feasibility, OCT was then used arthroscopically in 19 human subjects. Clinical results confirmed that differences to OCT form birefringence observed in ex vivo study were detectable during arthroscopic surgery. More prevalent loss of cartilage OCT form birefringence was observed in cartilage of human subjects in groups more likely to have cartilage degeneration. This series of integrated bench to bedside studies demonstrates translational feasibility to use OCT for clinical studies on whether human cartilage degeneration can be diagnosed early enough for intervention that may delay or prevent the onset of osteoarthritis.

p38 MAPK inhibition in nucleus pulposus cells: a potential target for treating intervertebral disc degeneration.

STUDY DESIGN: Human nucleus pulposus cells were cultured in alginate beads and activated with IL-1 beta or TNF-alpha, with and without inhibition of p38 mitogen activated protein kinase (p38 MAPK) activity. Cell production of factors modulating the anabolic/catabolic balance of the disc was determined. OBJECTIVE: To determine the role of signaling through p38 MAPK in nucleus pulposus cell's response to inflammatory cytokines and whether it might be a valid target for the development of molecular therapies for disc degeneration. SUMMARY OF BACKGROUND DATA: Multiple factors contribute to intervertebral disc degeneration (IDD), and development of effective therapies depends on understanding the underlying cellular pathophysiology. Interleukin-1 beta and tumor necrosis factor-alpha are implicated in the development of IDD, and p38 MAPK is part of cytokine and mechanical stress signal pathways in other cells. These studies determine whether inhibiting p38 MAPK can decrease factors that negatively affect the metabolic balance and viability of nucleus pulposus cells. MATERIALS AND METHODS: Degenerated intervertebral disc tissue was obtained from patients undergoing elective surgical procedures. Nucleus pulposus cells in alginate bead culture were exposed to IL-1 or TNF-alpha, with or without p38 MAPK inhibition, and conditioned media analyzed for accumulation of nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) through 10 days. RESULTS: Inhibition of p38 MAPK decreased PGE2 in conditioned medium of control, unstimulated cells while not affecting TIMP-1 accumulation. Blocking cytokine activation of p38 MAPK reduced IL-1 and TNF-alpha induced PGE2 and IL-6 accumulation. p38 MAPK inhibition increased the ratio of TIMP-1 to MMP-3 in conditioned medium of cells activated by IL-1 or TNF-alpha. CONCLUSION: Inhibition of p38 MAPK in cytokine-activated disc cells blunts production of factors associated with inflammation, pain, and disc matrix catabolism. The data support further analysis of these effects on the anabolic/catabolic balance of nucleus pulposus cells and suggest that molecular techniques blocking this signal could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

p38 MAPK and COX2 inhibition modulate human chondrocyte response to TGF-beta.

These studies compare actions of p38 MAPK inhibition and COX2 inhibition to modulate human arthritic chondrocyte responses to TGF-beta and FCS under basal and IL-1 activated conditions. Chondrocytes isolated from arthritic human femoral condyle cartilage obtained at total knee replacement were grown to 80% confluence. Proteoglycan synthesis and proliferation were measured with and without IL-1 activation in the presence and absence of growth factors and with and without inhibition of p38 MAPK or COX2 activity. Experiments to evaluate TIMP-1 production under these conditions were done using cartilage organ cultures. Neither p38 MAPK inhibitors nor COX2 inhibition affected basal proliferation. However both inhibitors enhanced the proliferative response to TGF-beta and FCS in IL-1 activated chondrocytes. TGF-beta stimulated proteoglycan synthesis was decreased by p38 MAPK inhibition, however COX2 inhibition restored the response to TGF-beta in IL-1 activated cells. In contrast, COX2 inhibition did not modulate TIMP-1 production while p38 MAPK inhibitors potentiated TGF-beta stimulated production of TIMP-1 in IL-1 activated cartilage. p38 MAPK inhibition and COX2 inhibition have unique and similar abilities to counteract some of the effects of IL-1 on human chondrocyte/cartilage metabolism. Both will partially restore the proliferative response to growth factors. p38 MAPK inhibition blunts TGF-beta stimulation of proteoglycan synthesis, but increases TIMP-1 synthesis. COX2 inhibition can restore the proteoglycan synthetic response to TGF-beta, but has no effect on cartilage TIMP-1 production. Use of these inhibitors to minimize cartilage damage in arthritic and mechanically stressed joints should reflect these characteristics.

Mini-pig fresh osteochondral allografts deteriorate after 1 week of cold storage.

As a well-defined animal transplantation model, the mini-pig potentially is well-suited for large animal studies of fresh osteochondral allograft transplantation. This study was done to determine the histologic characteristics and function of proteoglycan synthesis of mini-pig articular cartilage after refrigeration in basal media for as much as 6 weeks. Osteochondral sections of 10 mini-pig knees were refrigerated in various media at 4 degrees C for 1 to 42 days after slaughter. Four hundred twenty samples were evaluated by 35S uptake and 260 samples by histologic evaluations. Proteoglycan synthesis declined by 7 days to 21% of the level measured on Day 1 and was undetectable at 42 days. Histologic evaluation revealed progressive degeneration. Mankin scores rose from 3.69 +/- 0.27 on Day 1 to 6.40 +/- 0.18 on Day 7, and logarithmically increased to 10.83 +/- 0.07 on Day 42. These results indicate that the metabolic characteristics of porcine articular cartilage were not retained after refrigeration in basal media for 7 days. Optimum cold storage of porcine osteochondral allografts for cartilage transplantation research may be less than 7 days. Because osteochondral grafts for clinical use currently are stored for greater than 7 days, similar studies of the viability of human articular cartilage are needed.

Chondrocyte response to growth factors is modulated by p38 mitogen-activated protein kinase inhibition.

Inhibitors of p38 mitogen-activated protein kinase (MAPK) diminish inflammatory arthritis in experimental animals. This may be effected by diminishing the production of inflammatory mediators, but this kinase is also part of the IL-1 signal pathway in articular chondrocytes. We determined the effect of p38 MAPK inhibition on proliferative and synthetic responses of lapine chondrocytes, cartilage, and synovial fibroblasts under basal and IL-1-activated conditions.Basal and growth factor-stimulated proliferation and proteoglycan synthesis were determined in primary cultures of rabbit articular chondrocytes, first-passage synovial fibroblasts, and cartilage organ cultures. Studies were performed with or without p38 MAPK inhibitors, in IL-1-activated and control cultures. Media nitric oxide and prostaglandin E2 were assayed.p38 MAPK inhibitors blunt chondrocyte and cartilage proteoglycan synthesis in response to transforming growth factor beta; responses to insulin-like growth factor 1 (IGF-1) and fetal calf serum (FCS) are unaffected. p38 MAPK inhibitors significantly reverse inhibition of cartilage organ culture proteoglycan synthesis by IL-1. p38 MAPK inhibition potentiated basal, IGF-1-stimulated and FCS-stimulated chondrocyte proliferation, and reversed IL-1 inhibition of IGF-1-stimulated and FCS-stimulated DNA synthesis. Decreases in nitric oxide but not prostaglandin E2 synthesis in IL-1-activated chondrocytes treated with p38 MAPK inhibitors are partly responsible for this restoration of response. Synovial fibroblast proliferation is minimally affected by p38 MAPK inhibition.p38 MAPK activity modulates chondrocyte proliferation under basal and IL-1-activated conditions. Inhibition of p38 MAPK enhances the ability of growth factors to overcome the inhibitory actions of IL-1 on proliferation, and thus could facilitate restoration and repair of diseased and damaged cartilage.

Recovery of articular cartilage metabolism following thermal stress is facilitated by IGF-1 and JNK inhibitor.

BACKGROUND: The safety of intra-articular use of thermal probes is related to whether chondrocytes can tolerate exposure to high temperatures and whether cytoprotective agents may improve chondrocyte survival after thermal injury. PURPOSE: This study was conducted to characterize the metabolic responses of articular cartilage after short-term exposure to temperatures between 50 degrees C and 60 degrees C with and without addition of insulin-like growth factor 1 (IGF-1) and c-Jun N-terminal kinase (JNK) inhibitor. METHODS: Human articular cartilage from osteoarthritic knees was subjected to defined thermal stress. RESULTS: Although significant reduction of proteoglycan synthesis was observed after 5 seconds of exposure to 55 degrees C and 60 degrees C and after 10- to 30-second exposures to 53 degrees C, recovery of metabolic activity levels was observed after 7 days. CONCLUSION: Addition of IGF-1 and JNK inhibitor Sp600125 enabled the cartilage to maintain significantly higher levels of proteoglycan synthesis immediately after thermal stress. IGF-1 also enhanced recovery of metabolic activity after 7 days. CLINICAL SIGNIFICANCE: Results from this study indicate that there may be time and temperature parameters within which thermal chondroplasty can be safely performed. The data additionally suggest that inadvertent chondrocyte injury may be minimized through potential addition of substances like IGF-1 or JNK inhibitor.

Recovery of chondrocyte metabolic activity after thermal exposure.

BACKGROUND: The relationship between temperature elevation and thermal exposure time during thermal chondroplasty has implications for cell viability and subsequent articular cartilage function. PURPOSE: To characterize cartilage metabolic changes after exposure to thermal stress and to determine whether changes seen acutely are reversible. STUDY DESIGN: Controlled laboratory study. METHODS: Human cartilage was exposed to a 45 degrees, 50 degrees, or 55 degrees C bath for up to 3 minutes. Untreated control specimens were analyzed with each group. Viability and metabolic capability of treated and untreated specimens were evaluated immediately or 1 week after thermal stress by using methylthiotetrazole conversion, (3)H-serine incorporation into protein, and (35)S-sulfate incorporation into newly synthesized proteoglycan. RESULTS: Nonarthritic and arthritic articular cartilage metabolic activity declined with increasing thermal exposure. Articular cartilage displayed a recovery from thermal stress after exposure to the 50 degrees C but not the 55 degrees C bath. Arthritic cartilage displayed increased sensitivity with higher temperatures. CONCLUSIONS: Understanding of the increased sensitivity to thermal stress of arthritic articular cartilage may be helpful in thermally based treatments. CLINICAL RELEVANCE: Further correlation with the temperatures attained during thermal chondroplasty will be necessary to confirm the clinical relevance of these in vitro observations to the use of radiofrequency energy devices to treat partial-thickness chondral lesions.