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On the conditional equivalence of chemical loading and mechanical loading on articular cartilage.

Osmotic pressure loading of articular cartilage has been customarily invoked to be equivalent to mechanical loading. In the literature, this equivalence is defined by the amount of water squeezed from the tissue, i.e. if the amount of water content lost by these two modes of loading are the same, it has been generally regarded that the two loadings are equivalent. This assumption has never been proven. Using the water content lost concept, in this paper, we derived the exact conditions under which an osmotic pressure loading of cartilage can be considered to be equivalent to a mechanical loading. However, the mechanical loading condition satisfying this equivalency criterion, i.e. an isotropic loading delivered via a porous permeable rigid platen uniformly applied all around the specimen, is not practically achievable. Moreover, even if this were achieved experimentally, the interstitial fluid pressure caused by the two loading conditions are not the same. This result has important ramifications for interpretation of experimental data from mechanical stimulations of cartilage explant studies.

Algorithms↗

Scaffolds for articular cartilage repair.

Tissue engineering of articular cartilage seeks to restore the damaged joint surface, inducing repair of host tissues by delivering repair cells, genes, or polypeptide stimulatory factors to the site of injury. A plethora of devices and materials are being examined for their potential to deliver these agents to wound sites, and to act as scaffolds for ingrowth of new tissue. This review will discuss various promising scaffolds for cartilage tissue engineering applications.

Animals↗

Postoperative magnetic resonance imaging of articular cartilage repair.

The treatment of articular cartilage injuries remains a challenge because cartilage has a limited capacity for spontaneous repair. Over the past decade, a number of surgical interventions have been developed to produce a durable repair. This article reviews the available treatment options, including their indications, technique, and clinical results, and illustrates the MR imaging appearance of normal repair sites and postoperative complications.

Bone Nails↗

A microstructural model for the anisotropic drained stiffness of articular cartilage.

A constitutive model for articular cartilage is developed to study directional load sharing within the soft biological tissue. Cartilage is idealized as a composite structure whose static mechanical response is dominated by distortion of a sparse fibrous network and by changes in fixed charge density. These histological features of living cartilage are represented in a microstructural analog of the tissue, linking the directionality of mechanical stiffness to the orientation of microstructure. The discretized 'model tissue' is used to define a stiffness tensor relating drained stress and strain over a regime of large deformation. The primary goal of this work was to develop a methodology permitting more complete treatment of anisotropy in the stiffness of cartilage. The results demonstrate that simple oriented microscopic behaviors can combine to produce complicated larger scale response. For the illustrative example of a homogeneous specimen subjected to confined compression, the model predicts a nonlinear anisotropic drained response, with inherent uncertainty at cellular size scales.

Biomechanical Phenomena↗

Focal lacunar resorption in the articular cartilage of femoral heads.

Human articular cartilage taken from 92 femoral heads at autopsy was examined macroscopically and microscopically. Fifty-two showed no changes except for occasional slight degeneration in the non-pressure areas; these changes were visible only microscopically. In the remaining 40 heads, different degrees of osteoarthrosis were seen; half the heads also showed focal lacunar resorptive lesions in the cartilage. The origin of this focal cartilage resorption is discussed and its possible association with necrosis, pannus formation and enzymatic synovial activities. We conclude that there is no evidence of a direct relationship between focal cartilage resorption and osteoarthrosis.

Adult↗

Phosphorylation of proteoglycans in human articular cartilage.

A study of human articular cartilage indicated that componenet proteoglycans can be phosphorylated. Phosphorylation, also found in a specimen of human epiphysial cartilage, occurred when [gamma-32P]-ATP or 32Pi was included in the in vitro incubation medium. Treatment of the phosphorylated proteoglycans with chondroitinase and chondrosulfatases effectively removed the chondroitin sulfate without dephosphorylating the remaining molecule. Since phosphorylation could be effected in a totally chemically defined medium, it appears that the necessary enzyme systems for this reaction are contained entirely within chondrocytes.

Adult↗

Age-related changes in the synthesis of matrix macromolecules by bovine articular cartilage.

Calf and mature cow articular cartilage was labeled in vitro with [35S]SO4 and [3H]glycine and kinetics of incorporation of both isotopes by cartilage fragments was determined by scintillation spectroscopy. The cartilage fragments were then extracted in sequence with 4M GuHCl (Guanidium chloride) and pepsin. The pepsin digest was adjusted to 1.3 M NaCl and pepsin-solubilized collagen salted out. The 4M GuHCl extract, collagen and pepsin-resistent residue were then freeze-dried. The 4M GuHCl extract was further fractionated by DEAE (Diethylaminoethyl) 52 ion exchange chromatography to obtain protein and PG (Proteoglycan) fractions. The protein fraction was also characterised by SDS-PAGE and PG fraction by Sepharose C1-2B chromatography under associative conditions in the presence and absence of an exogenous HA (Hyaluronic acid). The GAG (Glycosaminoglycan) side chains of the PG samples were analysed by Sephadex G-200 column chromatography and their composition determined by paper chromatography after chondroitinase ABC digestion. Linear incorporation of both isotopes was observed from 1 to 18 hours of incubation and roughly equal amounts of [35S]SO4 counts were found on per cell bases in both cartilages although less [3H]glycine was incorporated by cow chondrocytes. It was also found that calf chondrocytes synthesize much greater proportion of the collagen whereas the cow cells synthesize PGs of smaller hydrodynamic sizes, bearing shorter GAG side chains that are enriched in KS (Keratan sulfate) and Ch-6S (Chondroitin-6 sulfate isomer). A failure of cow 35S-PGs monomers to interact with an exogenous HA in the presence of other extracted components was also demonstrated. The relevance of these findings for the mechanism of cartilage damage in aging and osteoarthritis is discussed.

Aging↗

[Mosaicplasty in articular cartilage injuries of the knee].

Articular cartilage has little ability to regenerate. Cartilage lesions usually persist, may provoke pain, swelling, locking and disability, and may predispose for development of osteoarthritis. Long-term results after traditional surgical techniques are unsatisfactory. In recent years, auto-transplantation of chondrocytes and osteochondral cylinder grafts aimed at reconstituting the chondral surface has been introduced. By the mosaicplasty technique, osteochondral grafts are transplanted from areas of little weight bearing at the outer limits of the trochlea to the focal lesion in the same knee. Since March 1998, 35 consecutive mosaicplasties in 33 patients (median age 35 years) have been performed at our hospital. The median area of the lesions was 2.8 sq. cm and the lesions were located at the femoral condyles, trochlea or patella. Six to twelve months post-operatively we found significant improvement of the Lysholm kneescore, from 40 (SD 14) pre-operatively to 84 (SD 14) (p < 0.001), and on the Cincinnati kneescore (pain, swelling, giving way, catching and as total evaluation). Short-term results are good regarding symptoms and knee function. The method is some-what complicated, especially when performed arthroscopically. There are few complications. Mosaicplasty may be considered in cartilage lesions of moderate size (1 to 5 sq. cm) on the femoral condyles, trochlea and patella.

Adolescent↗

[Relationship between exposure time and cryopreservation of articular cartilage].

OBJECTIVE: To search an optimal method for improving viability of cryopreserved articular cartilage. METHODS: Articular cartilage which was sampled from the rabbits were randomly divided into 5 groups. Fresh cartilage was group I, other groups were frozen. Before frozen, other cartilage was exposured in 10% DMSO at 4 degrees C for 30 minutes(group II), 1 hour(group III), 2 hours (group IV), 4 hours(group V), then were stored in liquid nitrogen for 1 week. Viabilities of the chondrocytes were detected by Typan-blue staining, electron transmission microscope, and determination of incorporation 3H-TdR after the temperature returned to normal. RESULTS: 1. The cells were injuried at different extent after the cartilage was frozen. In group I, survival rate of cells was 96% and incorporation of 3H-TdR was (4,953.13 +/- 583.27)%, statistic difference was significant between group I and other groups(P < 0.01). The microstructure of group I was normal while other groups all had damage of the organella, 2. Structures and functions of cells in group IV were best among frozen groups. Organella were less damaged than group II, III, V, survival rate of cells was 56% and incorporation of 3H-TdR was (1,139.88 +/- 146.39)%, statistic difference was significant between group IV and group II, III, V(P < 0.01). CONCLUSION: If cartilage are exposured in 10% DMSO at 4 degrees C for 2 hours before frozen, optimal cryopreservation can be achieved.

Animals↗

Autologous chondrocytes implantation: indications and results in the treatment of articular cartilage lesions of the knee.

Articular cartilage lesions of the knee constitute a frequent event and one that is difficult to treat. In time, different surgical methods have been used with results that are often contradictory. The authors report the indications and results of different methods used in the treatment of cartilage lesions: in particular, the experience initiated in 1997 with autologous chondrocytes implantation is discussed. Between 1997 and 2000, a total of 40 patients were submitted to autologous chondrocytes implantation injected in suspension under a periosteal flap. This method includes wide exposure of the joint lesion by arthrotomy. Between 1999 and 2001, a total of 30 patients were submitted to autologous chondrocytes implantation using a tridimensional matrix. The availability of biomaterial simplified the implant method and made arthroscopy possible. All of the patients were submitted to serial clinical follow-ups. MRI was also conducted for a second arthroscopic look. The results obtained in both series after a mean follow-up of 4 years (range 2 to 6 years) are good, and confirm the effectiveness of the method that allows for complete morphological and structural repair of lesions of the joint cartilage.

Adolescent↗

Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage.

Proteoglycan aggregates from bovine articular cartilage have been visualized by electron microscopy of mixed proteoglycan-cytochrome c monolayers. The proteoglycan aggregates consist of proteoglycan subunits arising laterally at fairly regular intervals (20 to 30 nm) from the opposite sides of an elongated filamentous structure. The filamentous backbone in individual aggregates varies in length from 400 to 4000 nm. The individual proteoglycan subunits in the aggregate vary in length from 100 to 400 nm. However, there is no difference in the average size of the proteoglycan subunits associated with the largest or smallest aggregates. The sizes of the individual aggregates are determined mainly by the lengths of their filamentous backbones. The stoichiometry of binding of subunits to filament, calculated from the data reported here, is close to that for the binding of subunits to hyaluronic acid reported by others.

Animals↗

Effect of nalidixic acid, pipemidic acid and cinoxacin on chondrocyte metabolism in explants of articular cartilage.

Explants of immature bovine articular cartilage were exposed to nalidixic acid, pipemidic acid and cinoxacin at one and ten times the human therapeutic plasma level for 7 days. Only nalidixic acid had significant effects on the chondrocyte metabolism. 20 micrograms/ml nalidixic acid caused an increase of 35S-sulfate incorporation into glycosaminoglycans at day 7. Two hundred micrograms/ml nalidixic acid inhibited the incorporation of 3H-thymidine into DNA. The incorporation of 35S-sulfate into glycosaminoglycans was decreased at day 0, while at day 7 the incorporation had returned to the control value. Pipemidic acid and cinoxacin had no significant effects on either the 3H-thymidine or the 35S-sulfate incorporation.

Animals↗

Two latent metalloproteases of human articular cartilage that digest proteoglycan.

Human articular cartilage contains very low levels of metalloprotease activity; the activity in 1 g of cartilage is approximately equivalent to the activity of 1 microgram of trypsin. Development of a sensitive assay, based on the digestion of radioactive proteoglycan, has made it possible to study protease activity in 1-2-g specimens of cartilage. Cartilage was extracted with Tris buffer in the cold and with Tris buffer containing 10 mM CaCl2 at 60 degrees C. The extracts were passed through Sepharose 6B; two major and two minor metalloprotease activities were detected. A neutral metalloprotease activity, pH optimum 7.4, was found as a latent form of Mr = 56,000. It could be activated with aminophenylmercuric acetate or trypsin with a resultant decrease of Mr to 40,000. An acid metalloprotease, pH optimum 5.3, also occurred as a latent form of Mr = 50,000. Activation converted this to Mr = 35,000. Removal of calcium ions by dialysis reduced the activity of the neutral enzyme by 80-85% and of the acid enzyme by 100%. Both activities were restored by 10 mM Ca2+. Both enzymes were completely inhibited by 1 mM o-phenanthroline in the presence of excess calcium. This inhibition was overcome by 1 mM Zn2+ and, to a lesser extent, by Co2+. These proteases may be important in the metabolism of the cartilage matrix and in its destruction in osteoarthritis.

Calcium↗

The cellular origin of cartilage-like tissue after periosteal transplantation of full-thickness articular cartilage defects: an experimental study using transgenic rats expressing green fluorescent protein.

BACKGROUND: Periosteal transplantation is commonly used for the treatment of articular cartilage defects. However, the cellular origin of the regenerated tissue after periosteal transplantation has not been well defined. The objective of this study was to investigate the cellular origin of the regenerated tissue after periosteal transplantation. METHOD: Free periosteum was harvested from the tibia of 10-week-old adolescent enhanced green fluorescent protein (GFP-) expressing transgenic Sprague Dawley (SD) rats and was transplanted to full-thickness articular cartilage defects of the patellar groove in normal 10-week-old adolescent SD rats. The periosteum was sutured to the defect with the cambium layer facing the joint cavity. 8 SD rats were killed at 4 weeks and 8 SD rats were killed at 8 weeks after surgery. The repaired tissue was assessed histologically and histochemically. GFP-positive cells derived from the donor periosteum could easily be detected in the repaired tissue by use of a fluorescent microscope. RESULTS: At both 4 and 8 weeks after transplantation, the entire area of the defects had been repaired, with the regenerated tissue being well stained histologically with safranin-O. Most cells in the whole area of the regenerated tissue were GFP-positive, indicating that very few of the cells were GFP-negative cells originating from the recipient rats. INTERPRETATION: This experiment demonstrates that most cells in regenerated tissue after periosteal transplantation using adolescent animals do not originate from recipient cells but from the periosteal cells of the donor.

Animals↗

Collagen network primarily controls Poisson's ratio of bovine articular cartilage in compression.

The equilibrium Young's modulus of articular cartilage is known to be primarily determined by proteoglycans (PGs). However, the relation between the Poisson's ratio and the composition and structure of articular cartilage is more unclear. In this study, we determined Young's modulus and Poisson's ratio of bovine articular cartilage in unconfined compression. Subsequently, the same samples, taken from bovine knee (femoral, patellar and tibial cartilage) and shoulder (humeral cartilage) joints, were processed for quantitative microscopic analysis of PGs, collagen content, and collagen architecture. The Young's modulus, Poisson's ratio, PG content (estimated with optical density measurements), collagen content, and birefringence showed significant topographical variation (p < 0.05) among the test sites. Experimentally the Young's modulus was strongly determined by the tissue PG content (r = 0.86, p < 0.05). Poisson's ratio revealed a significant negative linear correlation (r = -0.59, p < 0.05) with the collagen content, as assessed by the Fourier transform infrared imaging. Finite element analyses, conducted using a fibril reinforced biphasic model, indicated that the mechanical properties of the collagen network strongly affected the Poisson's ratio. We conclude that Poisson's ratio of articular cartilage is primarily controlled by the content and organization of the collagen network.

Animals↗

Apoptosis and p53 expression in chondrocytes relate to degeneration in articular cartilage of immobilized knee joints.

OBJECTIVE: We have reported that articular cartilage showed early stage degeneration at 7 and 14 days after immobilization, moderate degeneration at 28 days, and severe degeneration at 42 days in rabbits. To test whether apoptosis occurs in association with p53 expression in chondrocytes during the process of articular cartilage degeneration, we investigated the degree of cartilage degeneration, the frequency of apoptotic cells, and the levels of p53 mRNA in rabbits and mice after knee immobilization. METHODS: Right knees of male Japanese white rabbits were immobilized in full extension with fiberglass casts for up to 42 days. Similarly, right knees of male p53 wild-type [p53 (+/+)] and p53 null [p53 (-/-)] mice were immobilized in full extension with bandage tape for up to 84 days. Apoptotic cells were confirmed by TUNEL staining on the sections of knee joints. Total RNA of articular chondrocytes obtained from Day 0 or immobilized knees was analyzed semiquantitatively by RT-PCR using specific primers for p53. RESULTS: Articular cartilage degenerated after immobilization of p53 (+/+) mouse knees, but not after immobilization of p53 (-/-) knees. Apoptotic cells were observed in articular cartilage in the femur and tibia of rabbits and p53 (+/+) mice after immobilization. However, only a few apoptotic cells were observed at the same sites in p53 (-/-) mice. In RT-PCR analysis, the levels of p53 mRNA obtained from immobilized groups were significantly higher than those of Day 0 groups in rabbit and p53 (+/+) mouse knees. CONCLUSION: Apoptosis and p53 expression in chondrocytes relate to degeneration in articular cartilage of immobilized knee joints.

Animals↗

The effect of maturation and aging on the structure and content of link proteins in rabbit articular cartilage.

We have examined extracts of articular cartilage from rabbits aged 3-100 weeks for evidence of age-related changes in the structure and content of link protein (LP) in this tissue, with the following findings: (a) Two major molecular weight forms of LP were seen on SDS-PAGE (41 and 48 kDa) and the proportion of these changed markedly with age. The 48 kDa species was predominant in young animals (representing about 78% of the total LP at 5 weeks) whereas the 41 kDa species increased in amount with age (representing 35% of the total LP at 100 weeks). A minor form of about 43 kDa, representing less than 20% of the total, was present only during the growth phase. A small amount of fragmented link protein (less than 5% of the total) of about 25-30 kDa was present in samples from mature and aged rabbits only. (b) The quantitation of LP in guanidinium: HCl extracts of cartilage, by radioimmunoassay with monoclonal antibody 8-A-4, was markedly influenced by the conditions of preparation and pretreatment of samples. Assays of dialyzed guanidine extracts following treatment at 80 degrees C for 15 min in 0.025% (w/v) SDS indicated that immature and mature cartilage contains about 50 and 180 micrograms of LP/g of tissue, respectively. On the other hand, assays following treatment at 100 degrees C for 20 min in 0.1% (w/v) SDS suggested that rabbit cartilage contains about 300 micrograms of LP/g of tissue at all ages; finally, assay of CsCl purified proteoglycan samples under these conditions indicated a content of about 500 micrograms of LP/g at all ages. (c) Calculations based on the analysis of proteoglycan preparations for aggregating monomer and link protein suggest that a LP:aggregating monomer molar ratio of about 0.9 is maintained in the articular cartilage throughout maturation and aging in the rabbit.

Aging↗

Anisotropic hydraulic permeability in compressed articular cartilage.

The extent to which articular cartilage hydraulic permeability is anisotropic is largely unknown, despite its importance for understanding mechanisms of joint lubrication, load bearing, transport phenomena, and mechanotransduction. We developed and applied new techniques for the direct measurement of hydraulic permeability within statically compressed adult bovine cartilage explant disks, dissected such that disk axes were perpendicular to the articular surface. Applied pressure gradients were kept small to minimize flow-induced matrix compaction, and fluid outflows were measured by observation of a meniscus in a glass capillary under a microscope. Explant disk geometry under radially unconfined axial compression was measured by direct microscopic observation. Pressure, flow, and geometry data were input to a finite element model where hydraulic permeabilities in the disk axial and radial directions were determined. At less than 10% static compression, near free-swelling conditions, hydraulic permeability was nearly isotropic, with values corresponding to those of previous studies. With increasing static compression, hydraulic permeability decreased, but the radially directed permeability decreased more dramatically than the axially directed permeability such that strong anisotropy (a 10-fold difference between axial and radial directions) in the hydraulic permeability tensor was evident for static compression of 20-40%. Results correspond well with predictions of a previous microstructurally-based model for effects of tissue mechanical deformations on glycosaminoglycan architecture and cartilage hydraulic permeability. Findings inform understanding of structure-function relationships in cartilage matrix, and suggest several biomechanical roles for compression-induced anisotropic hydraulic permeability in articular cartilage.

Animals↗