Biomedical subjects

R L Sah

Publications and source records attributed to R L Sah.

At least 37 records · Page 2Linked to original sources

PubMed2001

Depth-dependent compressive properties of normal aged human femoral head articular cartilage: relationship to fixed charge density.

OBJECTIVES: Determine the depth-varying confined and osmotic compression moduli of normal human articular cartilage from the femoral head, and test whether these moduli are dependent on fixed charge density. METHODS AND RESULTS: Using an automated instrument to allow epifluorescence microscopy analysis during confined compression testing on cartilage samples, the equilibrium confined compression modulus (H(A 0)) was found to vary markedly with depth (z=0-1500 microm) from the articular surface. H(A 0) increased from 1.16+/-0.20 MPa in the superficial (0-125 microm) layer to 7.75+/-1.45 MPa in the deepest (1250-1500 microm) layer tested, and was fit by the expression, H(A 0)(z) [MPa]=1.44 exp(0.0012.z [microm]). Also, in successive slices of cartilage extending from the articular surface to the middle-deep regions, the bulk modulus (K(0)) and fixed charge density (FCD) increased, consistent with previous findings. While H(A 0), K(0), and FCD each varied with depth from the articular surface, the dependence of H(A 0) and K(0) on depth did not appear to be completely related to variations in FCD. CONCLUSIONS: The confined compression modulus of normal aged human femoral head articular cartilage increases markedly with depth from the articular surface, a trend similar to that observed for articular cartilage from other joints in animals but with an absolute amplitude that is several-fold higher. The compressive properties were not simply related to FCD at different depths from the articular surface, suggesting that other as yet undefined factors also contribute to compressive properties.

Aged↗

PubMed2001

Growth responses of cartilage to static and dynamic compression.

During skeletal development, growth, and maturation, gradual changes in the material properties and physical dimensions of cartilage occur under the influence of mechanical loading. The objective of the current study was to compare glycosaminoglyean biosynthesis and cell proliferation in fetal, calf, and adult bovine cartilage explants, isolated from defined depths from the articular surface, in response to controlled compressive loads. Mechanical testing confirmed that for all cartilage samples subjected to load, there was a marked time-averaged (static) compression, whereas the addition of dynamic load at a frequency of 0.01 Hz induced dynamic strain with amplitude and phase shift characteristics typical of stimuli that previously were found to be associated with stimulation of glycosaminoglycan synthesis. In metabolic studies, the application of static loading (84 kPa) for 24 hours inhibited glycosaminoglycan and deoxyribonucleic acid synthesis in all cultured cartilage samples. The superposition of dynamic loading (200 kPa, 0.01 Hz) induced a 20% stimulation of glycosaminoglycan biosynthesis in calf cartilage from the middle-deep zones over statically-loaded samples and an additional approximate 50% suppression of deoxyribonucleic acid synthesis in fetal and calf cartilage from the articular surface. These results indicate that synthesis of glycosaminoglycan and deoxyribonucleic acid, two distinct indices of cartilage growth, are regulated independently by mechanical loading and that cartilage responds differently to static and dynamic loading at different stages of maturation.

Age Factors↗

PubMed2001

Matrices for cartilage repair.

Techniques for repairing focal articular cartilage defects are evolving from methods that induce a local stimulation of fibrocartilaginous repair to methods that will lead to a hyaline articular cartilage repair. Mosaicplasty and autologous chondrocyte implantation are examples of the latter. A tissue engineered hyaline cartilage implant that could be used off the self would minimize the morbidity of these techniques. However, there are significant questions that still need to be resolved before such tissue-engineered implants will be practical. Principally among these is the question of what is the ideal matrix for such an implant, particularly from the standpoint of the best material and architecture. Second, what is the ideal cell source to use with these implants. A third major unknown is what is the most ideal way to use growth factors to enhance the repair. As these issues are resolved, the prospects of a tissue engineered cartilage replacement will advance from theory to practice.

Biocompatible Materials↗

PubMed2000

Mechanical compression modulates proliferation of transplanted chondrocytes.

The presence of an appropriate number of reparative cells in an articular cartilage defect is probably necessary for consistent and successful repair. Following the transplantation of chondrocytes into a defect, cell proliferation may modulate local defect cellularity. Transplanted cells can be compressed during cartilage repair as a result of joint-loading or press-fitting a graft into a cartilage defect. The objective of this study was to characterize the proliferative response of chondrocytes after attachment to cartilage and application of static compressive stress between cartilaginous surfaces in an ex vivo model. The chondrocytes were isolated from adult bovine cartilage, cultured in high-density monolayer, resuspended, and then transplanted onto the surface of devitalized cartilage at a density of 250,000 cells/cm2. The total DNA content of transplanted cell layers increased steadily to a plateau by 5 days and represented a 4-fold increase in cell number during incubation in medium including serum and ascorbate. Over the culture period, the level of DNA synthesis ([3H]thymidine incorporation), on a per cell basis, decreased steadily (88% between days 0 and 6). The application of 24 hours of static compressive stress (0.06-0.4 MPa) to the adherent cells at 1 and 4 days after transplantation inhibited overall DNA synthesis by 70-approximately 87% compared with unloaded controls. After release from load, cell proliferation generally remained at low levels. The marked proliferation of chondrocytes when attached to cartilage without applied load and the inhibition of this proliferation by relatively low-amplitude static compressive stress may be relevant to the occasional overgrowth of tissue in some chondrocyte transplantation procedures. The dosimetry of these effects suggests that the in vivo mechanical environment may have a marked effect on proliferation of transplanted chondrocytes.

Animals↗

PubMed2000

Biomechanical regulation of matrix metalloproteinase-9 in cultured chondrocytes.

Abnormal mechanical loading of joints may induce degeneration of articular cartilage. Shear stress is one mode of mechanical loading that may regulate chondrocyte metabolism. We investigated the mechanism by which shear stress induces the gene encoding matrix metalloproteinase-9, a mediator of the progressive degradation of articular cartilage in osteoarthritis. In vitro experiments using passaged rabbit chondrocytes in monolayer culture subjected to a shear stress of 16 dyn/cm2 (1.6 Pa) in a flow channel showed increased expression of the matrix metalloproteinase-9 gene. The induction of matrix metalloproteinase-9 appeared to depend on a region in the 5' promoter of the gene that contains a 12-0-tetradecanoylphorbol 13-acetate-responsive element. Transfection experiments using a construct containing a luciferase reporter driven by a 12-0-tetradecanoylphorbol 13-acetate-responsive element indicated that shear stress activated a 12-0-tetradecanoylphorbol 13-acetate-responsive element-mediated transcription in chondrocytes. Similar experiments showed that shear stress induced a matrix metalloproteinase-9 promoter construct (matrix metalloproteinase-9-luciferase). Shear stress activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38. Transfection of matrix metalloproteinase-9-luciferase together with the dominant negative mutant of c-Jun NH2-terminal kinase, but not with that of extracellular signal-regulated kinase or p38, attenuated the shear-induced matrix metalloproteinase-9 promoter activity. In addition, transfection of constructs encoding dominant negative mutants of Ras, Rac, and Cdc42 attenuated the induction of c-Jun transcriptional activity by shear stress. Thus. shear stimulation of chondrocytes stimulates Ras, Rac, and Cdc42, which subsequently activate c-Jun NH2-terminal kinase to induce a 12-0-tetradecanoylphorbol 13-acetate-responsive element-mediated expression of matrix metalloproteinase-9.

Animals↗

PubMed2000

Response of chickens to infection with Newcastle disease virus isolated from a guinea fowl.

An isolate of Newcastle disease virus obtained from a guinea fowl was characterized as a viscerotropic velogenic strain based upon pathogenicity index studies. Following inoculation of the viral isolate oronasally into 3-week-old chickens, clinical signs appeared after an incubation period of 4-5 days and included dullness, depression, dyspnoea, diarrhoea and leg paralysis. The virus caused a mortality of 56% with haemorrhages at the tip of the glands of the proventriculus and caecal tonsil. Histopathological changes were prominent in the lymphoid organs, being characterized by depletion, degeneration and necrosis of the lymphoid tissues. The brain was the first organ affected, with changes being noticed 3 days after infection. Isolation of virus from various organs was more frequent from 5 to 10 days after infection, but the virus persisted in some of the organs until 21 days after infection. In spite of the high mortality, a good immune response was elicited by the isolate, as was evident from the antibody titre.

Animals↗

PubMed2000

Novel method for the quantitative assessment of cell migration: a study on the motility of rabbit anterior cruciate (ACL) and medial collateral ligament (MCL) cells.

A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.

Animals↗

PubMed2000

Adhesive force of chondrocytes to cartilage. Effects of chondroitinase ABC.

Chondrocyte transplantation is a clinical procedure for cartilage repair. Transplanted cells may have difficulty attaching to the surface of chondral lesions because of the anti-adhesive properties of the proteoglycan rich matrix. This study used micromanipulation methods to determine if pretreatment of cartilage with chondroitinase ABC affects chondrocyte adhesion to cartilage and if chondrocytes adhere preferentially to the superficial, middle, or deep layers of cartilage. Bovine chondrocytes were transplanted in vitro on articular cartilage sections cut perpendicular to the articular surface. At various times between 15 and 75 minutes after seeding, a micropipette micromanipulation system was used to measure the adhesion force of individual chondrocytes to cartilage. The chondrocyte adhesion force increased with chondroitinase ABC treatment and seeding time but generally was similar for the different regions of articular cartilage (superficial, middle, deep layer) to which the cells were attached. For normal cartilage, the adhesion force increased from 1.29 +/- 0.24 mdyne after 15 to 30 minutes seeding to 5.29 +/- 0.25 mdyne after 60 to 75 minutes. Treatment with chondroitinase ABC at certain concentrations and durations (1.0 U/mL for 5 minutes or 0.5 or 1 U/mL for 15 minutes) led to an increase in adhesion force, whereas relatively low concentration or treatment time (0.25 U/mL for 15 minutes or 0.5 U/mL for 5 minutes) had little or no detectable effect. The increase in adhesion attributable to chondroitinase ABC treatment appeared most marked (+144% to +292%) for short (15 to 30 minutes) seeding durations but was still significant (+46%) for the longest seeding period (60 to 75 minutes) studied after the 1 U/mL for 15 minute treatment condition. These results provide direct biomechanical evidence that enzymatic treatment of a cartilage surface can enhance chondrocyte adhesion.

Analysis of Variance↗

PubMed2000

Cartilage repair with autogenic perichondrium cell and polylactic acid grafts.

The repair of articular cartilage injuries remains a challenge, with many of the current therapeutic strategies based on the grafting or recruitment of chondrogenic tissues or cells. This 1-year study compared the repair of a 3.7-mm diameter by 3-mm deep osteochondral defect in the medial femoral condyle of 24 New Zealand White rabbits; the defect was obtained using an autogenic perichondrium cell polylactic acid composite graft with a contralateral control in which the osteochondral defect remained empty. To elucidate the effect of host immune responses on the repair process after perichondrium cell transplantation, the results of the autogenic perichondrium cell polylactic acid graft group were compared with those obtained in the authors' previous 1-year study of allogenic perichondrium cell polylactic acid composite grafts implanted in a similar model. One year after surgery, the repair site underwent gross inspection and histologic, histomorphometric, biochemical, and biomechanical analyses. The autogenic perichondrium cell polylactic acid graft group (92%) and the control group in which the osteochondral defect remained empty (88%) resulted in a high percentage of grossly acceptable repairs. The autogenic grafts appeared to augment the intrinsic healing capacity of the animals (as compared with the animals in the No Implant Group). The autogenic perichondrium cell polylactic and grafts improved the histologic appearance and percentage of Type II collagen of the cartilaginous repair tissue. Compared with allogenic grafts, the autogenic grafts had better reconstitution of the subchondral bone. However, the results of this experimental model suggest a suboptimal concentration of glycosaminoglycans in the neocartilage matrix, a depressed surface of the repair tissue, a histologic appearance that was not equivalent to that of normal articular cartilage, and reduced biomechanical properties for the repair tissue. The future application of growth factors to this model may yield a treatment that can be applied in the clinical arena.

Animals↗

PubMed1999

Effect of seeding duration on the strength of chondrocyte adhesion to articular cartilage.

Chondrocyte adhesion to cartilage may play an important role in the repair of articular defects by maintaining cells in positions where their biosynthetic products can contribute to the repair process. The objective of this in vitro study was to determine the effect of the duration of seeding time on the ability of chondrocytes to resist detachment from cartilage when subjected to mechanical perturbation (fluid-induced shear stress). Suspensions of adult bovine articular chondrocytes were prepared from primary, high-density monolayer cultures and infused into a parallel-plate shear-flow chamber where they settled onto 50-microm-thick sections of bovine articular cartilage at a density of approximately 20,000 cells/cm2. The chondrocytes were seeded and allowed to attach to the cartilage surface for specific durations (5-40 minutes) in medium including 10% serum at 22 degrees C, after which the cells were exposed to fluid flow-induced shear stresses (6-90 Pa). The fraction of detached cells at each shear stress was calculated from microscopic images. Shear stress was applied for 1 minute because this length of time was sufficient to induce steady-state cell detachment. Increasing the duration of cell seeding led to a more firm attachment of chondrocytes to cartilage. After 9 minutes of seeding, 50% cell detachment was induced by gravitational force alone. After 40 minutes of seeding, 50% detachment required 26 Pa of shear stress. Extrapolation of the data to account for the effect of repeated applications of cell suspensions to an individual cartilage substrate indicated that for a freshly prepared cartilage section, 50% detachment was induced by gravity after 25 minutes of seeding and by 2.3 Pa of shear stress after 40 minutes of seeding. The increase in resistance to shear stress-induced cell detachment with increasing seeding duration suggests that it may be beneficial to allow chondrocytes to stabilize in the absence of applied load for some time after chondrocyte transplantation for cartilage repair in vivo.

Animals↗

PubMed1999

Integrative cartilage repair: inhibition by beta-aminopropionitrile.

The effects of beta-aminopropionitrile, a known inhibitor of lysyl oxidase, on the extractability of newly synthesized collagen and integrative cartilage repair were determined in explant cultures of adult bovine articular cartilage. Dose-escalation studies indicated that treatment of cartilage explants for 6 days with beta-aminopropionitrile caused a dose-dependent inhibition of proteoglycan synthesis ([35S]sulfate incorporation) with a 50% inhibition at 2.2 mM. However, 0.25 mM beta-aminopropionitrile had no detectable effect on proteoglycan synthesis and was thus used for subsequent experiments. Treatment of cartilage with beta-aminopropionitrile for 14 days increased the extractability of newly synthesized collagen with 4 M guanidine-HCl while having little effect on proteoglycan synthesis, proteoglycan deposition, collagen synthesis (formation of [3H]hydroxyproline after labeling with [3H]proline), collagen deposition, or cartilage cellularity (DNA content). In untreated cultures, the percentage of radiolabeled collagen ([3H]hydroxyproline) that was extractable after 1 day of radiolabeling, 6 days of radiolabeling, or 6 days of label and 6 days of chase decreased from 81 to 25 and 9%, respectively. In beta-aminopropionitrile-treated cultures, the extractability was relatively higher (96, 62, and 47%, respectively). Treatment with beta-aminopropionitrile after radiolabeling with [14C]lysine also significantly inhibited the formation of the reducible crosslink [14C]dihydroxylysinonorleucine without affecting the overall deposition in cartilage of [14C]lysine and [14C]hydroxylysine. In functional repair studies, treatment with beta-aminopropionitrile caused an almost complete inhibition of integration between pairs of cartilage explants maintained in apposition for 2 weeks. These results indicate that beta-aminopropionitrile blocks the formation of collagen crosslinks in cartilage explants and suggest that such crosslinks are critical to integrative cartilage repair.

Aminopropionitrile↗

PubMed1999

Biomechanics of integrative cartilage repair.

Cartilage repair is required in a number of orthopaedic conditions and rheumatic diseases. From a macroscopic viewpoint, the complete repair of an articular cartilage defect requires integration of opposing cartilage surfaces or the integration of repair tissue with the surrounding host cartilage. However, integrative cartilage repair does not occur readily or predictably in vivo. Consideration of the 'integrative cartilage repair process', at least in the relatively early stages, as the formation of a adhesive suggests several biomechanical approaches for characterizing the properties of the repair tissue. Both strength of materials and fracture mechanics approaches for characterizing adhesives have recently been applied to the study of integrative cartilage repair. Experimental configurations, such as the single-lap adhesive test, have been adapted to determine the strength of the biological repair that occurs between sections of bovine cartilage during explant culture, as well as the strength of adhesive materials that are applied to opposing cartilage surfaces. A variety of fracture mechanics test procedures, such as the (modified) single edge notch, 'T' peel, dynamic shear, and trouser tear tests, have been used to assess Mode I, II, and III fracture toughness values of normal articular cartilage and, in some cases, cartilaginous tissue undergoing integrative repair. The relationships between adhesive biomechanical properties and underlying cellular and molecular processes during integrative cartilage repair remain to be elucidated. The determination of such relationships may allow the design of tissue engineering procedures to stimulate integrative cartilage repair.

Animals↗

PubMed1998

Effect of static compression on proteoglycan biosynthesis by chondrocytes transplanted to articular cartilage in vitro.

Transplantation of chondrocytes by injection or within carrier matrices has shown promise for augmenting the repair of articular cartilage defects. In vivo, transplanted chondrocytes are exposed to mechanical forces. This in vitro study examined the effect of a step application of compressive load to chondrocytes after the cells had been seeded onto a cartilage surface. Bovine chondrocytes were transplanted onto bovine cartilage disks, allowed to attach for 1 hour or 4 days, and subjected to compression through overlying cartilage disks in a confined compression configuration. Before use, the disks were lyophilized to lyse the endogenous chondrocytes and thereby allow assessment of the metabolic activity of the transplanted cells. During a 16-hour application of compressive stress of 0.24-0.72 MPa, proteoglycan synthesis, assessed as [35S]sulfate incorporation into macromolecules, was inhibited by approximately 68% after the 1-hour attachment and by approximately 45% after the 4-day attachment. Cell retention after the application of load was assessed by use of [3H]thymidine-tagged chondrocytes and quantitation of the displacement of radioactivity. After the 1-hour seeding period, loading induced a dose-dependent dislodgment of [3H]radioactivity (as much as 35%) from the tissue bilayer. In contrast, after the 4-day seeding period, there was no detectable effect of loading on chondrocyte dislodgment with an 8-12% release of radioactivity. The inhibitory effect of a 16-hour compression of 0.48 MPa applied after the 4-day seeding period was studied further. This protocol did not appear to have an irreversible effect on chondrocyte metabolism; at 2 days after the release of load, proteoglycan synthesis by the loaded cells was stimulated by 41% compared with transplanted cells that were not subjected to loading. These results suggest that the application of static compressive stress to chondrocytes at a cartilage surface may affect biosynthesis by these cells and thus subsequent integrative cartilage repair. Such an effect may have implications for optimization of the tightness of the press fit of a cell-laden cartilaginous construct into an articular defect.

Animals↗

PubMed1998

Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation.

We examined the ability of local anesthetics to correct altered inactivation properties of rat skeletal muscle Na+ channels containing the equine hyperkalemic periodic paralysis (eqHPP) mutation when expressed in Xenopus oocytes. Increased time constants of current decay in eqHPP channels compared with wild-type channels were restored by 1 mM benzocaine but were not altered by lidocaine or mexiletine. Inactivation curves, which were determined by measuring the dependence of the relative peak current amplitude after depolarization to -10 mV on conditioning prepulse voltages, could be shifted in eqHPP channels back toward that observed for wild-type (WT) channels using selected concentrations of benzocaine, lidocaine, and mexiletine. Recovery from inactivation at -80 mV (50-ms conditioning pulse) in eqHPP channels followed a monoexponential time course and was markedly accelerated compared with wild-type channels (tauWT = 10.8 +/- 0.9 ms; taueqHPP = 2.9 +/- 0.4 ms). Benzocaine slowed the time course of recovery (taueqHPP,ben = 9.6 +/- 0.4 ms at 1 mM) in a concentration-dependent manner. In contrast, the recovery from inactivation with lidocaine and mexiletine had a fast component (taufast,lid = 3.2 +/- 0.2 ms; taufast,mex = 3.1 +/- 0.2 ms), which was identical to the recovery in eqHPP channels without drug, and a slow component (tauslow,lid = 1,688 +/- 180 ms; tauslow,mex = 2,323 +/- 328 ms). The time constant of the slow component of the recovery from inactivation was independent of the drug concentration, whereas the fraction of current recovering slowly depended on drug concentrations and conditioning pulse durations. Our results show that local anesthetics are generally incapable of fully restoring normal WT behavior in inactivation-deficient eqHPP channels.

Anesthetics, Local↗

PubMed1997

Physical properties of rabbit articular cartilage after transection of the anterior cruciate ligament.

The effect of unilateral transection of the anterior cruciate ligament on the confined compression and swelling properties of the distal femoral articular cartilage of skeletally mature rabbits at 9 weeks after surgery was determined. Gross morphological grading of the transected and contralateral control distal femora stained with India ink confirmed that cartilage degeneration had been induced by ligament transection. Osteochondral cores, 1.8 mm in diameter, were harvested from the medial femoral condyles. The modulus, permeability, and electrokinetic (streaming potential) coefficient of the articular cartilage of the osteochondral cores were assessed by confined compression creep experiments. The properties (mean +/- SD) of control cartilage were: confined compression modulus, 0.75 +/- 0.28 MPa; hydraulic permeability, 0.63 +/- 0.28 x 10(-15) m2/Pa*sec; and electrokinetic coefficient, 0.16 +/- 0.31 x 10(-9) V/Pa. In transected knees, the modulus was reduced by 18% (p = 0.04), while the permeability and electrokinetic coefficient were not detectably altered. The change in modulus was accompanied by a trend (p = 0.07) toward a decrease (-11%) in the glycosaminoglycan density within the tissue, a significant increase (p < 0.001) in the water content of the cartilage after equilibration in 1 x phosphate buffered saline from 70.3 +/- 4.1% in control knees to 75.2 +/- 4.0% in transected knees, and little further swelling after tissue equilibration in hypotonic saline. The compressive modulus of the cartilage from both control and transected knees was positively correlated with the density of tissue glycosaminoglycan. The alterations in the physical properties of the articular cartilage after transection of the anterior cruciate ligament in the rabbit show trends similar to those observed in human and other animal models of osteoarthritis and provide further support for the use of this model in the study of cartilage degeneration.

Animals↗

PubMed1997

Depth-dependent confined compression modulus of full-thickness bovine articular cartilage.

The objective of this study was to determine the equilibrium confined compression modulus of bovine articular cartilage as it varies with depth from the articular surface. Osteochondral samples were compressed by 8, 16, 24, and 32% of the cartilage thickness and allowed to equilibrate. Intratissue displacement within the cartilage was measured with use of fluorescently labeled chondrocyte nuclei as intrinsic, fiducial markers. Axial strain was then calculated in nine sequential 125 microns thick cartilage layers comprising the superficial 1,125 microns and in a 250 microns thick layer of cartilage adjacent to the cartilage-bone interface. Adjacent osteochondral cores were also tested in confined compression to determine the equilibrium stresses required to achieve the same levels of compression. Stress-strain data for each layer of each sample were fit to a finite deformation stress-strain relation to determine the equilibrium confined compression modulus in each tissue layer. The compressive modulus increased significantly with depth from the articular surface and ranged from 0.079 +/- 0.039 MPa in the superficial layer to 1.14 +/- 0.44 MPa in the ninth layer. The deepest layer 250 microns thick, had a modulus of 2.10 +/- 2.69 MPa. These moduli were markedly different from the apparent "homogeneous" modulus for full-thickness cartilage (0.38 +/- 0.12 MPa) and ranged from 21 to 560% of that value. The relatively low moduli and the compression-induced stiffening of the superficial layers suggest that these layers greatly affect the biomechanical behavior of cartilage, such as during confined compression testing. The delineation of the depth-dependent modulus provides a basis for detailed study of the relationship between the composition, structure, and function of cartilage in such processes as aging, repair, and degeneration.

Animals↗

PubMed1997

Chondrocyte transplantation to articular cartilage explants in vitro.

The transplantation of chondrocytes has shown promise for augmenting the repair of defects in articular cartilage. This in vitro study examined the efficiency of the transplantation of bovine chondrocytes onto articular cartilage disks and the ability of the transplanted chondrocytes to subsequently synthesize and deposit proteoglycan. The radiolabeling of chondrocyte cultures with [3H]thymidine, followed by 4 days of chase incubation, resulted in the incorporation of 98% of the radiolabel into DNA (as assessed by susceptibility to DNase). At the end of the culture period, the [3H]DNA was stable, with a half-life of radioactivity loss into the medium of 73 days. With use of radiolabeled chondrocytes for quantitation, the efficiency of transplantation onto a cartilage substrate was 93 +/- 4% for seeding densities of as much as 650,000 cells per cm2 and a seeding duration of 1 hour. These findings were confirmed both by tracking cells stained with 5-chlormethylfluorescein diacetate and by quantitating DNA. During the 16 hours after seeding onto a cartilage substrate (in which the endogenous cells had been lysed by lyophilization), the transplanted cells synthesized sulfated proteoglycan in direct proportion to the number of cells seeded. Most (83%) of the newly synthesized proteoglycan was released into the medium rather than retained within the layer of transplanted cells and the recipient cartilage substrate. Comparative studies with lyophilized-rehydrated or live cartilage as the recipient substrate indicated a similar efficiency of chondrocyte seeding and proteoglycan synthesis by the seeded chondrocytes. The transplanted cells retained the chondrocyte phenotype, as judged by a high proportion of the [35S]macromolecules being in the form of aggrecan that was capable of aggregating with hyaluronan and link protein, as well as by immunostaining within and around the transplanted cells for type-II, but not type-I, collagen. These results indicate that the number of chondrocytes transplanted onto a cut cartilage surface greatly affects the level of matrix synthesis; this in turn may affect repair.

Animals↗

PubMed1997

Quantitation and localization of cartilage degeneration following the induction of osteoarthritis in the rabbit knee.

OBJECTIVE: To develop and apply a new video imaging technique to quantify and localize Indian ink staining of cartilage of the rabbit femorotibial joint after the induction of osteoarthritis by unilateral transection of the anterior cruciate ligament (ACLT). METHODS: Nine weeks after surgery, femora and tibiae from 11 ACLT and contralateral control knees were harvested and positioned to obtain calibrated gray-scale images of the ink-painted articular cartilage surfaces that are opposed with the knee in 90 degrees flexion. Images were processed so that areas of normal cartilage gave a relatively high reflectance score, whereas ink-stained fibrillated cartilage and exposed bone gave low scores. RESULTS: Comparison of the medial and lateral femoral condyles and tibial plateaus (MFC, LFC, MTP, LTP) of control and ACLT knees showed that the area of the MTP not covered by the meniscus had a significantly lower reflectance score (P < 0.001) than other areas. ACLT led to an 11% decrease (P < 0.001) in the overall reflectance score. The reflectance score decreased as a traditional morphological grading of degeneration increased. ACLT-induced degeneration had a predilection for the posteromedial aspects of the joint, and to a lesser extent, the anterolateral aspects. In the tibial plateaus, ACLT caused significant degeneration in the covered, but not the uncovered, areas. Image scores of opposing cartilage surfaces (i.e., MFC vs MTP and LFC vs LTP) were significantly (R = 0.56-0.70, P < 0.001) correlated in ACLT and control knees. DISCUSSION: Identification and characterization of cartilage areas prone to degeneration may be particularly useful for further analysis of biochemical and biomechanical mechanisms in osteoarthritis, as well as the efficacy of therapeutic interventions.

Animals↗