Rate Of Proteoglycan Synthesis Research Articles

Very Rapid Clearance after a Joint Bleed Knee Can Not Prevent Adverse Effects on Cartilage and Synovial Tissue: A Canine in Vivo Study.

Purpose Joint bleeds lead to joint destruction. In vitro exposure of human and canine cartilage to blood results in long lasting severe adverse changes in cartilage. An in vivo joint haemorrhage in the canine knee joint demonstrates similar adverse effects although less outspoken and long-lasting. We investigated the clearance rate of blood from canine knee joints as a possible explanation for this discrepancy.Methods Blood was injected into the knee joint of Beagle dogs, either 48h, 24h or 15m before termination. The amount of red and white blood cells present in the joint cavity was determined. Chondrocyte activity and cartilage matrix integrity as well as cartilage destructive activity of synovial tissue were determined biochemically. Additionally, synovial tissue was analyzed by use of histochemistry.Results Fifteen minutes after the injection of autologous blood, the red blood cell count was 5,7*1012/L, comparable to the amount present in whole blood, and gradually decreased (1,6*1012/L at 24 hours) to 0,2*1012/L within 48 hours (less than 5%). The amount of white blood cells increased in the first 24 hours, and was still increased after 48 hours, although less than after 24 hours. The proteoglycan synthesis rate and -release were adversely affected already within 24 hours (−22% and +24% respectively), and these effects were more severe 48 hours post-injection (−34% and +53% resp.). Synovial tissue culture supernatants demonstrate cartilage destructive properties as expressed by an increased release, a decreased synthesis rate, and decreased content of cartilage proteoglycans; increasing with time after the experimental haemorrhage (+207%/+247%; −58%/−62%; −8%/−28% respectively, for 24/48 hours). Evaluation of the synovial tissue revealed at 15 minutes post-injection countless numbers of intact RBC that were almost completely disappared after 48 hours, withonly limited recruitment of macrophages and iron deposition.Conclusions Blood is cleared very rapidly from the canine knee joint, but in that short time span already has adverse effects on both cartilage and synovial tissue. This rapid clearance can play a role in the discrepancy between long-term in vitro and in vivo effects of blood-induced joint damage since more than 10% v/v blood for 48 hours is needed induced to long-term adverse effects in vitro. Irrespectively, blood has devastating effects on articular cartilage very rapidly, and in this respect it is important to prevent (traumatic) joint haemorrhages and if they occur, to treat them properly.

Intra-individual comparison of human ankle and knee chondrocytes in vitro: relevance for talar cartilage repair

As compared to knee chondrocytes (KC), talar chondrocytes (TC) have superior synthetic activity and increased resistance to catabolic stimuli. We investigated whether these properties are maintained after TC are isolated and expanded in vitro. Human TC and KC from 10 cadavers were expanded in monolayer and then cultured in pellets for 3 and 14 days or in hyaluronan meshes (Hyaff-11) for 14 and 28 days. Resulting tissues were assessed biochemically, histologically, biomechanically and by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The proteoglycan and collagen synthesis rates in the pellets were also measured following exposure to Interleukin-1 beta (IL-1 beta). After 14 days of pellet culture, TC and KC expressed similar levels of type I collagen (CI) and type II collagen (CII) mRNA and the resulting tissues contained comparable amounts of glycosaminoglycans (GAG) and displayed similar staining intensities for CII. Also proteoglycan and collagen synthesis were similar in TC and KC pellets, and dropped to a comparable extent in response to IL-1 beta. Following 14 days of culture in Hyaff-11, TC and KC generated tissues with similar amounts of GAG and CI and CII. After 28 days, KC deposited significantly larger fractions of GAG and CII than TC, although the trend was not reflected in the measured biomechanical properties. After isolation from their original matrices and culture expansion, TC and KC displayed similar biosynthetic activities, even in the presence of catabolic stimuli. These in vitro data suggest a possible equivalence of TC and KC as autologous cell sources for the repair of talar cartilage lesions.

Degenerated and healthy cartilage are equally vulnerable to blood-induced damage

Background:Joint bleeds have a direct adverse effect on joint cartilage, leading to joint deterioration and, ultimately, to disability.Objective:To examine the hypothesis that because degenerated cartilage has a limited repair capacity,...

An injectable cross-linked scaffold for nucleus pulposus regeneration

Incorporation of scaffolds has long been recognized as a critical element in most tissue engineering strategies. However with regard to intervertebral disc tissue engineering, the use of a scaffold containing the principal extracellular matrix components of native disc tissue (i.e. collagen type II, aggrecan and hyaluronan) has not been investigated. In this study the behavior of bovine nucleus pulposus cells that were seeded within non-cross-linked and enzymatically cross-linked, atelocollagen type II based scaffolds containing varying concentrations of aggrecan and hyaluronan was investigated. Cross-linking atelocollagen type II based scaffolds did not cause any negative effects on cell viability or cell proliferation over the 7-day culture period. The cross-linked scaffolds retained the highest proteoglycan synthesis rate and the lowest elution of sulfated glycosaminoglycan into the surrounding medium. From confined compression testing and volume reduction measurements, it was seen that the cross-linked scaffolds provided a more stable structure for the cells compared to the non-cross-linked scaffolds. The results of this study indicate that the enzymatically cross-linked, composite collagen–hyaluronan scaffold shows the most potential for developing an injectable cell-seeded scaffold for nucleus pulposus treatment in degenerated intervertebral discs.

Proteoglycan Synthesis in Porcine Nasal Cartilage Grafts Following Nd:YAG (λ= 1.32 μm) Laser-Mediated Reshaping

Mechanically deformed morphologic cartilage grafts undergo temperature-dependent stress relaxation during sustained laser irradiation resulting in stable shape changes. In this study, porcine nasal septal cartilage specimens were evaluated for viability by measuring the incorporation of Na235SO4 into proteoglycan (PTG) macromolecules in whole tissue culture following laser-mediated reshaping. Synthesis rates of PTG were determined by scintillation counting lyophilized specimens and normalizing these values by total protein content. Positive controls were established by inducing chondrocyte apoptosis using prolonged exposure to nitric oxide (NO). In chondrocytes, apoptosis induced using NO resulted in significantly lower PTG synthesis rates compared to untreated native specimens. Cartilage specimens were irradiated with light emitted from a Nd:YAG laser (25 W/cm2, λ= 1.32 μm) while recording simultaneously radiometric surface temperature, internal stress and back-scattered light intensity from a probe laser. Each specimen received one, two or three sequential laser exposures. The duration of each exposure was determined from real-time measurements of characteristic changes in back-scattered light intensity that correlate with accelerated stress relaxation. A 5 min time interval between each laser exposures allowed the cartilage specimen to return to thermal equilibrium. Average PTG synthesis rates decreased with successive laser exposures, though these were always higher than baseline rates established for NO-treated tissues, suggesting that laser-mediated cartilage reshaping acutely does not eliminate the entire population of viable chondrocytes. The reduction in PTG synthesis is correlated with the time–temperature-dependent heating profile created during laser irradiation, supporting our hypothesis that careful monitoring of laser dosimetry is required to ensure chondrocyte viability.

Characterization of an in vitro intervertebral disc organ culture system

Intervertebral disc organ culture has the capacity to control mechanical and chemical boundary conditions while keeping the tissue largely intact, and allowing interventions that would be impossible or unethical on animal studies. Recent studies on ex vivo organ culture has mostly involved small animals, or been limited to development and validation studies. In this study, bovine caudal discs were used. The large animal model design ensures that sufficient tissue is available for measurement of multiple dependent variables on the same disc, and a similar aspect ratio, diffusion distance, composition and rate of proteoglycan synthesis to human lumbar discs. The first goal of this study was to refine a set of dependent variables capable of characterizing the response of the intervertebral disc to culturing and to develop a technique to measure cell viability in all three regions of the disc. The second goal was to use these variables to compare static and diurnal loading as a method of maintaining intervertebral disc structure, composition, and cell metabolism similar to the in vivo state. Static (0.2 MPa) and diurnal loading (0.1 and 0.3 MPa alternating at 12 h intervals) were applied and intervertebral discs were examined after 4 or 8 days with dependent variables including changes in geometry (disc height and diameter), composition (tissue water content, tissue proteoglycan content and proteoglycan content lost to the culture media), cell viability and metabolism (proteoglycan synthesis). Results indicate that there was a decrease in disc height and water content after culture regardless of culture duration or loading condition. Cell viability significantly decreased with culture duration in the inner annulus and nucleus; however, a significant reduction in cell viability for the diurnal versus static loading condition was only observed after 8 days in the nucleus region. No significant differences were seen in viability of the outer annulus region with time, or in any loading groups. We conclude that our system is capable of keeping bovine caudal discs alive for at least 8 days without significant changes in GAG content, or cell metabolism, and that static loading was slightly better able to maintain cell viability than diurnal loading. This system offers promise for the future studies on large intervertebral discs requiring measurements of multiple mechanical and biological dependent variables on the same tissue.

Exposure of human cartilage tissue to low concentrations of blood for a short period of time leads to prolonged cartilage damage: An in vitro study

Joint bleeding, or hemarthrosis, leads in time to severe joint damage. This study was carried out to test the in vitro thresholds of exposure time and concentration that lead to irreversible joint damage, to add to the discussion on the usefulness of aspiration of the joint after a hemorrhage. Explants of healthy human articular cartilage tissue were cultured in the presence or absence of 50% (volume/volume) blood for 1, 2, 3, or 4 days or in the presence of 0%, 5%, 10%, 20%, 30%, or 50% (v/v) blood for 4 days, followed by a 12-day period of recovery after withdrawal of blood. The effect of blood exposure on cartilage was determined by measuring the rate of proteoglycan synthesis as well as the release and content of cartilage matrix proteoglycans and the activity of matrix metalloproteinases. Exposure of cartilage to 50% (v/v) blood led to adverse changes that were largely independent of the exposure time. The adverse effects persisted after an initial exposure of up to or exceeding 2 days. Exposure of cartilage to increasing concentrations of blood for 4 days led to concentration-dependent adverse changes. These effects persisted when the concentration equaled or exceeded 10% (v/v) blood. Moreover, after 2 days of exposure to a blood load of 10% (v/v), the adverse effects on cartilage were not reversible. A 2-day exposure of cartilage in vitro to 10% (v/v) blood leads to prolonged impairment of joint cartilage. This suggests that aspiration of blood from the joint within 2 days after hemarthrosis should be considered to prevent blood-induced joint damage in the long term.

Incremental retinal-defocus theory of myopia development—Schematic analysis and computer simulation

Previous theories of myopia development involved subtle and complex processes such as the sensing and analyzing of chromatic aberration, spherical aberration, spatial gradient of blur, or spatial frequency content of the retinal image, but they have not been able to explain satisfactorily the diverse experimental results reported in the literature. On the other hand, our newly proposed incremental retinal-defocus theory (IRDT) has been able to explain all of these results. This theory is based on a relatively simple and direct mechanism for the regulation of ocular growth. It states that a time-averaged decrease in retinal-image defocus area decreases the rate of release of retinal neuromodulators, which decreases the rate of retinal proteoglycan synthesis with an associated decrease in scleral structural integrity. This increases the rate of scleral growth, and in turn the eye's axial length, which leads to myopia. Our schematic analysis has provided a clear explanation for the eye's ability to grow in the appropriate direction under a wide range of experimental conditions. In addition, the theory has been able to explain how repeated cycles of nearwork-induced transient myopia leads to repeated periods of decreased retinal-image defocus, whose cumulative effect over an extended period of time results in an increase in axial growth that leads to permanent myopia. Thus, this unifying theory forms the basis for understanding the underlying retinal and scleral mechanisms of myopia development.

Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts

In order to understand the effect of mechanical strain on scleral extracellular matrix remodeling, human scleral fibroblasts were subjected to equibiaxial stretch in vitro and the expression of proteoglycans, metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were evaluated. Isolated human scleral fibroblasts were seeded onto flexible bottom culture plates, and subjected to a cyclic stretch regimen of 15% equibiaxial stretch for 45 s followed by 15 s of rest for 6–48 h in the presence of 35SO 4. Newly synthesized proteoglycans were measured in the medium by CPC precipitation of radiolabelled glycosaminoglycans. MMP-2 activity and expression levels were measured in the medium by, Western blot, gel zymography and real-time PCR. Steady state levels of TIMP-2 mRNA and membrane-type MMP, MT1-MMP (MMP-14) mRNA were measured in the cell layer using real-time PCR. The predominant gelatinolytic enzyme secreted by scleral fibroblasts was the pro-enzyme form of MMP-2 ( ProMMP-2). Mechanical stretch resulted in a significant increase of ProMMP-2 after 12 and 48 h (+76.28%, p < 0.05; +19.56%, p < 0.01, respectively). Mechanical stretch significantly increased the production of the active form of MMP-2 ( ActiveMMP-2) after 48 h (+59.72%, p < 0.05) and decreased levels of TIMP-2 mRNA (−22%, p < 0.05). The rate of scleral proteoglycan synthesis and the steady state levels of MMP-2 and MMP-14 mRNA were not significantly affected by mechanical stretch. These results suggest that mechanical strain stimulates the activation of MMP-2 by scleral fibroblasts, possibly through increased levels of ProMMP-2 and reduced levels of TIMP-2. Increased levels of ActiveMMP-2 in the sclera would be expected to contribute to scleral extracellular matrix degradation, scleral thinning and possible ocular ectasia.

Impact of Expansion and Redifferentiation Conditions on Chondrogenic Capacity of Cultured Chondrocytes

Cartilage regeneration based on isolated and culture-expanded chondrocytes is studied in a variety of in vitro models, but with varying morphological quality of tissue synthesized. The goal of the present study was to investigate the extent of the influence of expansion and redifferentiation conditions on final tissue morphology by comparing 2 expansion and redifferentiation methods. Chondrocytes from 9 human donors were expanded in medium without growth factor supplementation (basic expansion condition [BEC]) or in medium with basic fibroblast growth factor (bFGF) supplementation (growth factor supplemented expansion condition [GFSEC]). After expansion, cells were either redifferentiated in pellet culture or seeded on collagen type II-coated filters. Post-expansion mRNA levels of collagen type I and II and Sox-5, -6, and 9, measured by semiquantitative real-time polymerase chain reaction (PCR), suggested that expansion in GFSEC results in increased dedifferentiation compared to BEC. However, after 28 days of redifferentiation culture, morphology of tissue synthesized by GFSEC-expanded chondrocytes scored significantly higher on the Bern scale compared to BEC (6.4 +/- 0.3 points vs. 4.5 +/- 0.3 points in pellet culture and 6.0 +/- 0.4 points vs. 4.5 +/- 0.3 points on collagen-coated filters; p < 0.05). Expansion in GFSEC compared to BEC increased proteoglycan (PG) synthesis rate at day 9 (4.0-fold in pellet culture and 1.9-fold on collagen-coated filters; p < 0.01), PG release (6.7-fold in pellet culture and 3.2-fold on collagen-coated filters; p < 0.001), and final PG content at day 28 (1.6-fold in pellet culture and 1.5-fold on collagen-coated filters; p < 0.05). Redifferentiation on collagen-coated filters compared to pellet culture increased PG synthesis rate at day 9 (5.2-fold in BEC-expanded chondrocytes and 2.6-fold in GFSEC-expanded chondrocytes; p < 0.01), PG release (4.2-fold in BEC-expanded chondrocytes and 3.1-fold in GFSECexpanded chondrocytes; p < 0.01), and final PG content (1.3-fold in BEC-expanded chondrocytes and 1.9- fold in GFSEC-expanded chondrocytes; p < 0.01). Moreover, as visualized via electron microscopy, chondrocytes and organization of extracellular matrix cultured on filters was more similar to those found for hyaline cartilage. In conclusion, chondrocyte expansion in GFSEC and redifferentiation on collagen-coated filters resulted in most optimal chondrogenesis.

Comparison of cellular response in bovine intervertebral disc cells and articular chondrocytes: effects .of lipopolysaccharide on proteoglycan metabolism

Lipopolysaccharide (LPS) induces matrix degradation and markedly stimulates the production of several cytokines, i.e., interleukin-1beta, -6, and -10, by disc cells and chondrocytes. We performed a series of experiments to compare cellular responses of cells from the bovine intervertebral disc (nucleus pulposus and annulus fibrosus) and from bovine articular cartilage to LPS. Alginate beads containing cells isolated from bovine intervertebral discs and articular cartilage were cultured with or without LPS in the presence of 10% fetal bovine serum. The DNA content and the rate of proteoglycan synthesis and degradation were determined. In articular chondrocytes, LPS strongly suppressed cell proliferation and proteoglycan synthesis in a dose-dependent manner and stimulated proteoglycan degradation. Compared with articular chondrocytes, nucleus pulposus cells responded in a similar, although less pronounced manner. However, treatment of annulus fibrosus cells with LPS showed no significant effects on proteoglycan synthesis or degradation. A slight, but statistically significant, inhibition of cell proliferation was observed at high concentrations of LPS in annulus fibrosus cells. Thus, LPS suppressed proteoglycan synthesis and stimulated proteoglycan degradation by articular chondrocytes and nucleus pulposus cells. The effects of LPS on annulus fibrosus cells were minor compared with those on the other two cell types. The dissimilar effects of LPS on the various cell types suggest metabolic differences between these cells and may further indicate a divergence in pathways of LPS signaling and a differential sensitivity to exogenous stimuli such as LPS.

Altered in vitro chondrogenic properties of chondrocytes harvested from unaffected cartilage in osteoarthritic joints

In vitro models of chondrogenesis often depart from chondrocytes harvested from less-affected areas of osteoarthritic joints. However, there are indications that these chondrocytes are phenotypically different from chondrocytes from healthy joints and thus might differ in their capacity to generate hyaline cartilage. The goal of this study was to compare the chondrogenic capacity of chondrocytes from healthy and OA joints. Chondrocytes isolated from nine healthy and nine OA knee joints were expanded in monolayer for two passages. Chondrocytes from passages 1 and 2 were analyzed for expression of (de)differentiation and hypertrophy markers and were seeded at passage 2 on collagen-coated filters for redifferentiation culture to study cartilage matrix formation. The collagen II/I mRNA ratio, reflecting differentiation, decreased from passage 1 to 2 in both chondrocytes from OA joints and chondrocytes from healthy joints (P<0.05), without a significant difference between the two donor types. At passage 1, levels of the cartilage transcription factors Sox-5, Sox-6 and Sox-9 appeared to be higher in chondrocytes from OA joints (n.s.), but this was not seen at passage 2. However, a clear difference was observed in collagen type X expression, which was high in chondrocytes from OA joints at both passages, while undetectable in chondrocytes from healthy joints (P<0.01). Tissue generated by chondrocytes from healthy joints redifferentiated for 28 days, showed a significantly better morphology, as assessed by histological scoring (P<0.01) and higher proteoglycan content (P<0.05), compared to chondrocytes from OA joints. Matrix turnover parameters, i.e., proteoglycan synthesis and degradation rate, were not significantly affected by donor tissue origin. These results suggest that clear differences between chondrocytes from healthy and OA joints exist and that these are not completely abolished during the process of de- and redifferentiation. Therefore, in vitro cartilage regeneration models, which use chondrocytes from OA joints, should be interpreted with care.

Impairment of chondrocyte biosynthetic activity by exposure to 3-tesla high-field magnetic resonance imaging is temporary

The influence of magnetic resonance imaging (MRI) devices at high field strengths on living tissues is unknown. We investigated the effects of a 3-tesla electromagnetic field (EMF) on the biosynthetic activity of bovine articular cartilage. Bovine articular cartilage was obtained from juvenile and adult animals. Whole joints or cartilage explants were subjected to a pulsed 3-tesla EMF; controls were left unexposed. Synthesis of sulfated glycosaminoglycans (sGAGs) was measured by using [35S]sulfate incorporation; mRNA encoding the cartilage markers aggrecan and type II collagen, as well as IL-1β, were analyzed by RT–PCR. Furthermore, effects of the 3-tesla EMF were determined over the course of time directly after exposure (day 0) and at days 3 and 6. In addition, the influence of a 1.5-tesla EMF on cartilage sGAG synthesis was evaluated. Chondrocyte cell death was assessed by staining with Annexin V and TdT-mediated dUTP nick end labelling (TUNEL). Exposure to the EMF resulted in a significant decrease in cartilage macromolecule synthesis. Gene expression of both aggrecan and IL-1β, but not of collagen type II, was reduced in comparison with controls. Staining with Annexin V and TUNEL revealed no evidence of cell death. Interestingly, chondrocytes regained their biosynthetic activity within 3 days after exposure, as shown by proteoglycan synthesis rate and mRNA expression levels. Cartilage samples exposed to a 1.5-tesla EMF remained unaffected. Although MRI devices with a field strength of more than 1.5 T provide a better signal-to-noise ratio and thereby higher spatial resolution, their high field strength impairs the biosynthetic activity of articular chondrocytes in vitro. Although this decrease in biosynthetic activity seems to be transient, articular cartilage exposed to high-energy EMF may become vulnerable to damage.

The influence of mechanical compression on the induction of osteoarthritis-related biomarkers in articular cartilage explants

Macromolecules of the articular cartilage extracellular matrix released into synovial fluid, blood, or urine can serve as potentially useful biomarkers of the severity of osteoarthritis (OA). Biomechanical factors play an important role in OA pathogenesis, yet their influence on biomarker production is not well understood. The goal of this study was to examine the hypothesis that dynamic mechanical stress influences the release of these biomarkers from articular cartilage. Explants of porcine cartilage were subjected to dynamic compression at 0.5 Hz for 24h at stresses ranging from 0.006 to 0.1 MPa. The concentrations of cartilage oligomeric matrix protein (COMP), keratan sulfate (KS measured as the 5 D 4 epitope), total sulfated glycosaminoglycan (S-GAG), and the KS (keratanase-digestible) and chondroitin sulfate (CS) (chondroitinase-digestible) fractions of S-GAG were measured. Radiolabel incorporation was used to determine the rates of proteoglycan and protein synthesis. The magnitudes of mechanical stress applied in this study induced nominal tissue strains of 4-23%, consistent with a range of physiological to hyperphysiologic strains measured in situ. COMP release increased in proportion to the magnitude of dynamic mechanical stress, while KS, CS and total S-GAG release increased in a bimodal pattern with increasing stress. Protein and proteoglycan synthesis were significantly decreased at the highest level of stress. Mechanical stress differentially regulates the turnover of distinct pools of cartilage macromolecules. These findings indicate that mechanical factors, independent of exogenous cytokines or other stimulatory factors, can influence the production and release of OA-related biomarkers from articular cartilage.

Low-Dose Interleukin-1 Partially Counteracts Osteogenic Protein-1???Induced Proteoglycan Synthesis by Adult Bovine Intervertebral Disk Cells

Low back pain associated with degenerative disk disease is a common clinical problem that has enormous socioeconomic impact in today's aging population. As an alternative to the surgical removal of the diseased intervertebral disk, the direct application of a purified growth factor into the intervertebral disk may provide physiatrists a valuable tool to halt or slow down disk degeneration. Our goal here is to determine if low levels of interleukin-1 (IL-1), a proinflammatory cytokine present in the degenerating disk, could interfere with the potentially beneficial effects of growth factors on proteoglycan synthesis. New knowledge gained from this study will prove useful in the development of new treatment modalities that take advantage of the stimulatory effects of growth factors such as osteogenic protein-1 (OP-1). This was an in vitro study of proteoglycan accumulation and synthesis by cells from the nucleus pulposus, inner annulus fibrosus, and outer annulus fibrosus in the bovine intervertebral disk. In cells cultured with serum and no additional exogenous growth factor, treatment with low-dose IL-1 does not result in a significant decrease in proteoglycan synthesis. However, in the case of cells stimulated with OP-1, treatment with IL-1 resulted in a statistically significant decrease in sulfur-35-proteoglycan synthesis by cells derived from all three zones of the bovine intervertebral disk (nucleus pulposus, 60.3% [P < 0.0001]; inner annulus fibrosus, 18.4% [P = 0.0330]; outer annulus fibrosus, 12.3% [P = 0.0255]). Proteoglycan accumulation over the 12-day culture period also decreased significantly (nucleus pulposus, 26.8% [P < 0.0001]; inner annulus fibrosus, 15.8% [P = 0.0276]; outer annulus fibrosus, 16.8% [P = 0.0102]). It is worth noting that cells cultured in the presence of both OP-1 and IL-1 synthesized proteoglycan at a faster rate than cells cultured in the presence of IL-1 alone (nucleus pulposus, 58.5% [P < 0.0001]; inner annulus fibrosus, 39.7% [P = 0.0055]; outer annulus fibrosus, 45.1% [P = 0.0164]). Likewise, cells treated with OP-1 and IL-1 accumulated more proteoglycan in their newly formed matrix than cells cultured in the presence of IL-1 alone (nucleus pulposus, 65.3% [P < 0.0001]; inner annulus fibrosus, 60.5% [P = 0.0034]; outer annulus fibrosus, 19.4% (P = 0.0840]). Intervertebral disk cells that are stimulated by the growth factor OP-1, to increase the rate at which they produce a proteoglycan-rich matrix become more susceptible to the inhibitory effects of the proinflammatory cytokine IL-1 on the rate of proteoglycan synthesis and accumulation in the matrix over time. This notwithstanding, IL-1 at the low dose used did not totally obliterate the stimulatory effects of OP-1 on matrix formation. Consequently, this growth factor may remain partially effective in stimulating disk repair in vivo even when proinflammatory cytokines such as IL-1 are present.

Differential Effects of Fibronectin Fragment on Proteoglycan Metabolism by Intervertebral Disc Cells: A Comparison With Articular Chondrocytes

This in vitro study used the alginate bead culture system to probe for differences in the effects of fibronectin fragment on cell proliferation and proteoglycan metabolism by different populations of intervertebral disc cells and articular chondrocytes. To compare the effects of fibronectin fragment on cell proliferation, and proteoglycan synthesis and degradation by cells from the nucleus pulposus, the anulus fibrosus, and articular cartilage. In articular cartilage, the administration of fibronectin fragment stimulates cartilage degeneration. Fibronectin fragment levels were increased in human intervertebral discs with increased disc degeneration. Fibronectin fragment injected into the central region of the rabbit intervertebral disc induced a progressive degeneration of that disc. Bovine tails and metacarpophalangeal joints from 14- to 18-month-old animals were used. Alginate beads containing cells isolated from intervertebral discs and articular cartilage were cultured with (1-100 nmol/L) or without (control) fibronectin fragment in the presence of 10% fetal bovine serum. In these cultures, deoxyribonucleic acid and proteoglycan contents, as well as the rate of proteoglycan synthesis were determined. Proteoglycan degradation was measured in cultures with or without 10 nmol/L fibronectin fragment. In articular chondrocytes, fibronectin fragment strongly suppressed proteoglycan synthesis and stimulated proteoglycan degradation; the total proteoglycan content was diminished in a dose-dependent manner. Compared to articular chondrocytes, nucleus pulposus cells responded to fibronectin fragments in a similar, although less pronounced manner. On the other hand, anulus fibrosus cells treated with fibronectin fragment did not show any significant effects on proteoglycan degradation. A slight but statistically significant up-regulation of proteoglycan synthesis was observed at 10 nmol/L fibronectin fragment in outer anulus fibrosus cells. However, total proteoglycan content was decreased significantly at high concentrations of fibronectin fragment. Fibronectin fragment has different effects on cell proliferation, proteoglycan synthesis, degradation, and accumulation by articular chondrocytes and intervertebral disc cells. The different effects of fibronectin fragment in those different cell types suggest metabolic differences between these cells, and may further suggest differences in pathways of fibronectin fragment signaling as well as a differential need of these cells to be involved in tissue remodeling in which both anabolic and catabolic pathways might be altered.

Effect of Extracellular Matrix Protein on the Rate of Proteoglycan Synthesis in Rabbit Intervertebral Disc Cells

The extracellular matrix (ECM) is very important for fundamental cellular processes. However, the effects of ECM proteins on intervertebral disc (IVD) cell proliferation and metabolism have not been clarified. To verify the effects of ECM proteins on DNA and proteoglycan (PG) synthesis of IVD cells, PG synthesis rate was measured in IVD cells cultured in monolayer with or without ECM protein. Nucleus pulposus (NP) cells and anulus fibrosus (AF) cells isolated from adolescent rabbits were cultured in monolayer with or without ECM protein and at different concentrations of ECM protein for 4-6 days. [S]Sulfate incorporation into PG in the cell-associated matrix (CM) formed around cells and the further-removed matrix (FRM) in labeling medium was measured and standardized to DNA content. NP cells in type I or type II collagen-coated plates significantly increased the rate of PG synthesis in both the CM and the FRM compared with those in uncoated plates and in fibronectin-coated plates; however, AF cells with ECM proteins did not increase the rate significantly. The rate of PG synthesis of nucleus cells was contra-dose dependent on both type I and type II collagen.

The effects of cyclic mechanical strain and tumor necrosis factor alpha on the response of cells of the meniscus

Cells of the knee meniscus respond to changes in their biochemical and biomechanical environments with alterations in the biosynthesis of matrix constituents and inflammatory mediators. Tumor necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine that is involved in the pathogenesis of both osteoarthritis and rheumatoid arthritis, but its influence on meniscal physiology or mechanobiology is not fully understood. The objectives of this study were to examine the hypothesis that cyclic mechanical strain of meniscal cells modulates the biosynthesis of matrix macromolecules and pro-inflammatory mediators, and to determine if this response is altered by TNF-alpha. Cells were isolated from the inner two-thirds of porcine medial menisci and subjected to biaxial tensile strain of 5-15% at a frequency of 0.5Hz. The synthesis of proteoglycan, protein, nitric oxide (NO), and prostaglandin E(2) were determined. Cyclic tensile strain increased the production of nitric oxide through the upregulation of nitric oxide synthase 2 (NOS2) and also increased synthesis rates of prostaglandin E(2), proteoglycan, and total protein in a manner that depended on strain magnitude. TNF-alpha increased the production of NO and total protein, but inhibited proteoglycan synthesis rates. TNF-alpha prevented the mechanical stimulation of proteoglycan synthesis, and this effect was not dependent on NOS2. These findings indicate that pro-inflammatory cytokines can modulate the responses of meniscal cells to mechanical signals, suggesting that both biomechanical and inflammatory factors could contribute to the progression of joint disease as a consequence of altered loading of the meniscus.

Growth factor osteogenic protein-1: differing effects on cells from three distinct zones in the bovine intervertebral disc.

The objective of this study was to determine if cells isolated from three distinct zones in the bovine intervertebral disc (IVD) differ in their response to growth factor osteogenic protein-1 (OP-1) because of inherent biological differences. The new knowledge gained will help determine if treatment of degenerative disc disease with purified growth factors is effective and will provide guidance in terms of the injection technique and frequency of treatments required. This was an in vitro study measuring the effects of OP-1 on proteoglycan accumulation and synthesis by cells from the nucleus pulposus, inner-annulus fibrosus (inner-AF), and outer-annulus fibrosus (outer-AF) in the bovine IVD. Growth factor OP-1, at 100 ng/ml, stimulated proteoglycan accumulation and resulted in a statistically significant increase in the proteoglycan content of cells derived from three zones of the bovine IVD: 97% in the nucleus pulposus, 40% in the inner-AF, and 75% in the outer-AF. To elucidate the mechanism of enhanced proteoglycan accumulation in response to OP-1, we studied the rate of proteoglycan synthesis and cell proliferation. OP-1 stimulation resulted in a statistically significant increase in the DNA content in cultures containing cells from all three zones of the IVDs: 79% in the nucleus pulposus, 100% in the inner-AF, and 73% in the outer-AF. After dividing by DNA content, OP-1 resulted in a statistically significant increase in the rate of proteoglycan synthesis in the nucleus pulposus (78%) and outer-AF (17%) cells, but the increase in inner-AF cells (23%) did not achieve statistical significance. OP-1 stimulates proteoglycan accumulation by bovine IVD cells isolated from all three zones of the bovine IVDs. Cells from all three zones proliferated significantly. Individual cells derived from nucleus pulposus and outer-AF, but not those from the inner-AF, synthesized proteoglycans at a significantly faster rate with OP-1 stimulation.

Effects of the Degradation Rate of Collagen Matrices on Articular Chondrocyte Proliferation and Biosynthesis in Vitro

The objective of this study was to evaluate effects of the degradation rate of type II collagen scaffolds on the proliferation and biosynthetic activity of adult canine chondrocytes in vitro. The lower number of cells in more rapidly degrading scaffolds appeared to be related to the loss of scaffold material with dissolution. After 14 days in culture, protein and proteoglycan synthesis rates per cell for rapidly degrading scaffolds were comparable to rates for nondegraded matrices. This result suggests that decoupling of the degradation and formation phases of tissue remodeling may occur under certain circumstances.