To characterize age-related changes in the matrix of human intervertebral disc (IVD) specimens, human specimens from the third to the eighth decade of life were collected and analyzed for collagen ...and proteoglycan (PG) composition.
To identify age-related changes in the concentration of matrix macromolecules (collagen and PGs, including the small leucine-rich PGs biglycan, decorin, fibromodulin, and lumican) in human anulus fibrosus (AF) and nucleus pulposus (NP).
IVD degeneration is associated with changes in the concentration and fragmentation of matrix molecules. Deciphering age-related matrix alterations may help us to better understand the regulatory mechanisms underlying IVD degeneration.
Forty-six whole IVDs were obtained from the thoracolumbar spines (T11-L5) of humans aged between 32 and 80 years. All specimens were classified as Thompson grade 1 or 2 according to MRI criteria. Specimens were separated into (i) outer-and (ii) inner AF, and (iii) NP. DNA, collagen, and PG contents were measured using chemical assays, whereas small nonaggregating PG levels were analyzed by comparative Western blotting.
Total PG and collagen contents in both the AF and NP consistently decreased with aging. The concentrations of small nonaggregating PGs varied. In the outer anulus, decorin levels decreased, whereas biglycan and fibromodulin levels increased with age. In the inner anulus and nucleus, biglycan demonstrated a significant increase with aging. These changes differed in most cases from those previously reported for degenerating disc tissues.
Collagen and PGs appeared to undergo specific age-related changes in the human IVD. Although the total contents of these 2 families of molecules decreased during aging, individual species of small nonaggregating PGs showed species-specific age-related changes. Interestingly, the level of biglycan rose and remained elevated in all 3 compartments of the disc with aging. The functional significance of these alterations is yet to be determined.
Most attempts to tissue-engineer cartilage have involved seeding of cultured cells into a biological or synthetic scaffold. We have developed a novel two-step culture approach that makes possible the ...in vitro formation of cartilaginous-like tissue by mature adult bovine chondrocytes without the aid of a synthetic matrix. The first step consists of culturing chondrocytes under conditions that maintain their rounded shape and their molecular phenotype as assessed by type II collagen and aggrecan production. This step was accomplished by culturing the isolated chondrocytes in alginate beads until the cells have reestablished a proteoglycan-rich cell-associated matrix (CM). The second step consists of culturing the cells with their CM, after recovery from the beads, on a tissue culture insert with a porous membrane. In this study, young adult bovine articular chondrocytes were cultured in alginate beads in the presence of 10% or 20% fetal bovine serum (FBS). After 7 days of culture, the alginate beads were dissolved by incubating the beads for 20 min in sodium citrate buffer, a calcium chelator. Following a brief centrifugation, the cells with their CM were recovered, resuspended in medium containing 10% or 20% FBS and seeded onto a tissue culture insert. After 1 week of culture on the insert, the individual cells with their CM progressively became incorporated into a mass of cartilaginous tissue. Culture with 20% FBS resulted in the best formation of tissues. These tissues, easily recovered from the insert, were then subjected to biochemical and histological analyses. The biochemical results showed that the chondrocytes remain phenotypically stable in the tissues. The
de novo tissue has a relatively high ratio of PG/collagen. Histological examination of the tissue revealed it contained a cartilage-like matrix strongly stained with toluidine blue. This scaffold-free system appears ideal to study, in vitro, the development of transplantable cartilaginous tissue.
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.
One approach to repairing articular defects is to regenerate cartilage by recapitulating the changes that occur during fetal and postnatal growth into adulthood, and to thereby restore functional ...biomechanical properties, especially those of the normally strong superficial region. The objectives of this study were (1) to characterize and compare tensile biomechanical properties of the superficial region of articular cartilage of the patellofemoral groove (PFG) and femoral condyle (FC) from bovine animals over a range of growth stages (third-trimester fetal, 1–3 week-old calf, and adult), and (2) to determine if these properties were correlated with collagen network components. With growth from the fetus to the adult, the equilibrium and dynamic tensile moduli and strength of cartilage samples increased by an average of 391–1060%, while the strain at the failure decreased by 43%. The collagen concentration (per wet weight) increased by 98%, and the pyridinoline cross-link concentration increased by 730%, while the glycosaminoglycan concentration remained unchanged or decreased slightly. Some growth-associated changes were location-specific, with tensile moduli and strength attaining higher values in the PFG than the FC. The growth-associated variation in tensile moduli and strength were associated strongly with variation in the contents of collagen and pyridinoline cross-link, but not sulfated glycosaminoglycan. The marked changes in the tensile properties and collagen network components of articular cartilage with growth suggest that such parameters may be used to evaluate the degrees to which regenerated cartilage recapitulates normal development and growth.
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.
An in vitro biologic study of the effects of adenovirus expressing bone morphogenetic proteins (BMPs) and adenovirus expressing Sox9 on extracellular matrix metabolism by bovine nucleus pulposus ...cells.
To compare the effects of recombinant adenoviral vectors expressing various BMPs (2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15) and Sox9 on extracellular matrix accumulation by bovine nucleus pulposus cells.
Nucleus pulposus matrix production may be promoted by transducing the cells with genes that permit the sustained expression of growth factors. The choice of the particular factors or BMPs to be studied for these applications has been largely based on the commercial availability of such products. To our knowledge, this study is the first effort to evaluate systematically the relative effectiveness of the various members of the BMP family in promoting intervertebral disc matrix repair.
Adult bovine nucleus pulposus cells cultured in monolayer were transduced with adenoviruses expressing human BMP-2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15, and adenovirus expressing Sox9. Proteoglycan and collagen accumulation, and cell proliferation were measured 6 days after viral transduction. As a positive control, cells were cultured without any exogenous gene in the presence of recombinant human (rh)BMP-7.
Nucleus pulposus cells transduced with adenoviruses expressing BMP-2, 3, 4, 5, 7, 8, 10, 13, 15, and Sox9 accumulated more proteoglycans than nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein (control). It is noteworthy that nucleus pulposus cells transduced with adenoviruses expressing BMP-2 and 7 resulted in essentially as great a stimulation of proteoglycan accumulation as nucleus pulposus cells maintained in the presence of rhBMP-7 (adenoviruses expressing BMP-2: 104% increase; adenoviruses expressing BMP-7: 162% increase; and rhBMP-7: 120% increase). Nucleus pulposus cells transduced with BMP-2, 4, 5, 7, 8, 10, 14, 15, and Sox9 accumulated significantly more collagen compared to nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein; adenoviruses expressing BMP-4 and 14 were the most effective (552% and 661% increase, respectively). Nucleus pulposus cells also proliferated, as measured by deoxyribonucleic acid content, when transduced with adenoviruses expressing BMP-2 and 8.
To our knowledge, for the first time, we have shown the relative effectiveness of 12 different BMPs and Sox9 in stimulating proteoglycan and collagen production by nucleus pulposus cells. Adenoviruses expressing BMP-2 and 7 were the most effective in stimulating proteoglycan accumulation, while adenoviruses expressing BMP-4 and 14 were the most effective in stimulating collagen accumulation. To our knowledge, this study is the first to compare the relative effectiveness of various BMPs and Sox9 on extracellular matrix accumulation by nucleus pulposus cells, and could help to develop more efficacious approaches to the treatment of degenerating intervertebral discs.
Articular chondrocytes embedded in alginate gel produce de novo a matrix rich in collagens and proteoglycans. A major advantage of this culture system is that the cells can be recovered by chelating ...the calcium, which otherwise maintains the alginate in its gel state. Chondrocytes thus released are surrounded by tightly bound cell-associated matrix, which seems to correspond to the pericellular and territorial matrices identified in cartilage by electron microscopy. The cells and their associated matrix can be easily separated by mild centrifugation from more soluble matrix components derived principally from the 'interterritorial' matrix. This new cell culture system thus makes it possible to study the assembly and turnover of molecules present in two distinct matrix pools. Importantly, a significant proportion of the aggrecan molecules in each of these two pools can be extracted using a non-denaturing solvent, thereby making possible studies of the metabolism and turnover of native proteoglycan aggregates. We show in this report that chondrocytes isolated from the full depth of adult bovine articular cartilage and maintained for 8 months in alginate gel are still metabolically active and continue to synthesize cartilage-specific type II collagen and aggrecan. The cells did not synthesize large amounts of type I collagen or of the small nonaggregating proteoglycans as usually occurs when chondrocytes lose their phenotypic stability. After this extended period of time in culture, the cells were present as two populations exhibiting differences in size, shape and amount of extracellular matrix surrounding them. The first population was found only near the surface of the bead: these cells were flattened and surrounded by a matrix sparse in proteoglycans and collagen fibrils. The second population was found throughout the remaining depth of the bead: the cells were more round and almost always surrounded by a basket-like meshwork consisting of densely packed fibrils running tangential to the surface.
Rabbit knee articular chondrocytes overexpressing human growth factors were injected into cultured intervertebral disc explants. Survival of the injected cells and accumulation of extracellular ...matrix were assessed.
To define the utility of cell-based gene delivery approach for repair of the intervertebral disc.
Back pain associated with symptomatic disc degeneration is a common clinical condition. Growth factors stimulate disc cell metabolism, but the ideal method for in vivo delivery has not been established. Cells as a vehicle for delivering growth factors to the disc offer potential advantages, including prolonged production of the growth factor within the disc and vital cells to participate in the repair process.
New Zealand white rabbit articular chondrocytes transduced with adenovirus expressing human bone morphogenetic protein-7 and green fluorescence protein (GFP) (AdhBMP-7), human bone morphogenetic protein-10 and GFP (AdBMP-10), or GFP alone (AdGFP, as a control) were injected into whole disc explants. Discs were maintained in culture for 1 to 2 months. At the conclusion of the culture periods, cell survival was assessed by fluorescence microscopy and extracellular matrix accumulation was assessed with biochemical methods.
Chondrocytes achieved long-term survival in the cultured disc explants. The discs treated with chondrocytes/BMP-7 demonstrated a 50% increase in proteoglycan content within the nucleus pulposus compared to control (chondrocytes/GFP), while discs injected with chondrocytes/BMP-10 failed to show a significant increase in proteoglycan accumulation.
Our study demonstrates the ability of transduced articular chondrocytes to survive and promote proteoglycan accumulation when transplanted into the intervertebral disc. These data support the potential of a cell-based gene therapy approach for disc repair. Further studies using this approach in animal models are indicated as a step towards achieving disc repair in humans.
Summary Objective To determine the effects of exercise and weight loss interventions on serum levels of four biomarkers and to examine if changes in biomarker levels correlate with clinical outcome ...measures in obese and overweight adults with knee osteoarthritis (OA). Methods Serum was obtained at baseline, 6 and 18 months from 193 participants in Arthritis, Diet and Activity Promotion Trial. This was a single-blind 18-month trial with subjects randomized to four groups: healthy-lifestyle (HL), diet (D), exercise (E) and diet plus exercise (D + E). Serum levels of cartilage oligomeric matrix protein (COMP), hyaluronan (HA), antigenic keratan sulfate (AgKS), and transforming growth factor-β1 (TGF-β1) were measured by enzyme linked immunosorbent assay. Results At baseline there were no significant differences in biomarker levels between intervention groups. When results for all the intervention groups were combined, the levels of HA were found to be negatively correlated with medial joint space width and positively correlated with Kellgren–Lawrence scores (K–L scores) while TGF-β1 levels negatively correlated with K–L scores. When biomarker levels measured at 6 and 18 months were adjusted for baseline values, age, gender, and body mass index, weak but significant differences between intervention groups were present for mean levels of COMP and TGF-β1. Furthermore, AgKS levels averaged over all groups tended to decrease over time. There were no significant associations of baseline biomarkers and the follow-up outcomes. Weak associations were noted between change in the biomarkers at 18 months and change in outcome measures that included change in weight with AgKS and COMP and change in Western Ontario and McMaster Universities Osteoarthritis Index pain with AgKS. Conclusion Overall, the E and D interventions did not show a consistent effect on levels of potential OA biomarkers. The four biomarkers showed differences in correlations with outcome measures suggesting that they may measure different aspects of disease activity in OA. The strongest correlations were between serum HA and radiographic measures of OA at baseline.
Biologic study on the effects of coculture of bovine articular chondrocytes transduced ex vivo with genes expressing bone morphogenetic proteins (BMPs) on nucleus pulposus (NP) cells.
To evaluate the ...effects of bovine articular chondrocytes transduced with adenoviruses expressing various BMPs on proteoglycan and collagen production, and cellular proliferation of NP cells in vitro.
Matrix synthesis by intervertebral disc cells is promoted by exposing the cells to growth factors or delivering genes that permit sustained expression of growth factors. We propose a novel therapeutic approach involving delivery of autologous chondrocytes, transduced ex vivo with bioactive proteins, to provide both the cells and proteins required to stimulate disc healing.
Adult bovine articular chondrocytes were transduced with adenoviruses (Ads) expressing either BMP-2, 4, 5, 7, 10, or 13 and plated as monolayers. Bovine NP cells encapsulated in alginate beads were cocultured, floating in the medium. Proteoglycan and collagen accumulation, and NP cell proliferation were measured after 6 days of coculture. As a positive control, beads were cocultured with articular chondrocytes in the presence of rhBMP-7.
NP cells cocultered with articular chondrocytes transduced with BMPs-2, 4, 7, and 10 accumulated significantly (P < 0.05) more proteoglycan than when cocultured with chondrocytes transduced with AdGFP (control) AdBMP-2: 23.6%; AdBMP-4: 27.0%; AdBMP-7: 129.1%; AdBMP-10: 102.1% increases respectively. Collagen accumulation was significantly (P < 0.05) increased by NP cells cocultured with articular chondrocytes transduced with BMPs-2, 4, 5, and 7. AdBMP-2: 104.6%; AdBMP-4: 40.6%; AdBMP-5: 58.6%; AdBMP-7: 55.5% increases respectively. NP cells proliferated when cocultured with articular chondrocytes transduced with AdBMP-2 and -7.
Bovine NP cells are stimulated to produce proteoglycans and collagen when exposed to chondrocytes transduced with genes for various BMPs. If applied to the treatment of disc degeneration, this strategy could provide the disc with not only metabolically active chondrocytes but also promote matrix replenishment by stimulating native NP cells.
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