Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel

Full-thickness articular cartilage defects are a major clinical problem; however, presently there is no treatment available to regeneratively repair these lesions. The current therapeutic approach is to drill the base of the defect to expose the subchondral bone with its cells and growth factors. This usually results in a repair tissue of fibrocartilage that functions poorly in the loaded joint environment. The use of phenotypically appropriate chondrocytes embedded in a collagen gel delivery vehicle may provide a method that could be used to repair full-thickness articular cartilage defects with functionally satisfactory hyaline cartilage. Allograft articular chondrocytes embedded in a type I collagen gel were transplanted into large (6 x 3 x 3 mm), full-thickness articular cartilage defects in condylar and patellar weight-bearing surfaces to develop clinically applicable methods to repair articular cartilage defects. Chondrocytes were isolated from the articular cartilage of 4-week-old New Zealand rabbits and embedded in type I collagen gels. This composite was transplanted into a full-thickness defect on the medial femoral condyle and patellar groove of adolescent host rabbits. The repair cartilage was assessed histologically by a semiquantitative scoring system and biomechanically with a microindentation technique of specimens 4-48 weeks after chondrocyte transplantation. Defects in both locations were repaired with histologically apparent hyaline cartilage observed from as early as 4 weeks until 48 weeks after transplantation. The repair cartilage in the medial femoral condyle was more irregular than in the patellar groove, but in all other respects was similar. The grafted tissue did not remodel and differentiate into the morphological zones seen in normal articular cartilage. No tidemark or subchondral bony plate formed even 48 weeks after transplantation. Biomechanically, the repaired cartilage demonstrated indentation values similar to normal articular cartilage 12 weeks after transplantation and remained the same 48 weeks after transplantation. By contrast, the control (i.e., empty) defects healed with tissue that exhibited very poor metachromatic staining and exhibited very high indentation values. Incomplete bonding of the repair tissue to the normal cartilage was seen, and the surface was significantly irregular with major discontinuities. These observations provide the basis for considering the use of allograft articular chondrocytes to repair articular cartilage defects in the weight-bearing regions of the knee.     

Rabbit articular cartilage defects treated with autologous cultured chondrocytes

Adult New Zealand rabbits were used to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously and were 3 mm in diameter, extending down to the calcified zone. Healing of the defects was assessed by gross examination, light microscope, and histological-histochemical scoring at 8, 12, and 52 weeks. Chondrocyte transplantation significantly increased the amount of newly formed repair tissue compared to the found in control knees in which the lesion was solely covered by a periosteal flap. In another experiment, carbon fiber pads seeded with chondrocytes were used as scaffolds, and repair significantly increased at both 12 and 52 weeks compared to knees in which scaffolds without chondrocytes were implanted. The histologic quality scores of the repair tissue were significantly better in all knees in which defects were treated with chondrocytes compared to knees treated with periosteum alone and better at 52 weeks compared to knees in which defects were treated with carbon scaffolds seeded with chondrocytes. The repair tissue, however, tended to incomplete the bonding to adjacent cartilage. This study shows that isolated autologous articular chondrocytes that have been expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesions. A gradual maturation of the hyalinelike repair with a more pronounced columnarization was noted as late as 1 year after surgery.     

Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation

alpha 1-Adrenergic receptor-mediated responses are overwhelming in adult rat hepatocytes. Inversely, beta-responses are predominant over alpha 1-responses in the hepatocytes that have been cultured at a low cell density (10(4) cells/cm2) for 24 h. The insulin-EGF-induced DNA synthesis in the beta-response-dominant hepatocytes was doubled by beta-agonists or cAMP-generating agents added far behind (16-20 h) the addition of insulin/EGF; i.e., immediately before the entry into the S-phase of the cell cycle. Agonists of alpha 1-adrenergic or other Ca2+, mobilising receptors added to the alpha 1-response-dominant hepatocytes increased DNA synthesis only if they were added within 1-2 h after the addition of insulin/EGF, at the early stage of G1-phase. Agonists of "non-dominant" receptors were rather antagonistic to agonists of "dominant" receptors. Thus, agonists of alpha 1-adrenergic (and other Ca2+ mobilising) receptors and agonists of beta-adrenergic (and other cAMP-generating) receptors acted as comitogens in their own particular manners in the presence of growth factors in hepatocytes in which the respective receptor functions were dominant.     

Increase of decorin content in articular cartilage following running

The effect of long distance running exercise (40 km/day for 15 weeks, five days a week) on the decorin content of articular cartilage from the knee joint was studied in female beagle dogs. Samples from load bearing sites on the lateral plateau of the tibia (TL), and pooled material from two minimum load bearing sites on the posterior section of lateral (FLP) and medial (FMP) femoral condyles were analyzed. The running exercise protocol did not lead to significant changes in the overall glycosaminoglycan content of the cartilage. However, the amount of decorin significantly increased in the TL samples, and also in the FMP pool. These results support earlier in vitro observations that decorin synthesis is stimulated by loading, independent of the synthesis of aggrecan.     

Regulation of cartilage oligomeric matrix protein synthesis in human synovial cells and articular chondrocytes

OBJECT: The relationship between glial fibrillary acidic protein (GFAP) expression and glial tumor cell behavior has not been well defined. The goal of this study was to examine this relationship further. METHODS: To investigate the relationship between GFAP expression and glial tumor cell behavior, the authors isolated clones from the human glioblastoma cell line, U-373MG, according to their level of GFAP expression. Immunochemical analysis demonstrated that one clone had consistently low GFAP expression (approximately 93% of cells were GFAP negative), whereas a second clone had consistently high GFAP expression (approximately 80% of the cells were GFAP positive). The structure, population doubling time, saturation density, anchorage-independent growth, migratory rate, and invasive potential of these two clones were determined in relation to their level of GFAP expression. Morphologically, both clones were composed of ameboid as well as stellate components. Although the population doubling times of the two clones were equally rapid, the clone with low GFAP expression demonstrated a slightly higher saturation density compared with the clone with high GFAP expression. In an anchorage-independent environment (soft agar), a greater difference in growth characteristics was noted between the two clones: the high-expression clone formed more colonies and these colonies were compact, well defined, and spherical, whereas the low-expression clone formed predominantly smaller, two-dimensional colonies with vague boundaries and isolated cells or groups of cells at the periphery. In contrast to these minor differences between the clones, the low-expression clone showed a markedly increased migratory rate and invasive potential compared with the high-expression clone. Therefore, the clone with reduced GFAP expression appeared more aggressive, demonstrating decreased contact inhibition, increased migratory rate, and increased invasive potential. CONCLUSIONS: These results suggest a direct correlation between GFAP expression and some measures of aggressive tumor growth and transformation properties.     

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

Mutant mice that lack either protein zero (P0) or connexin 32 (Cx32) were generated previously to investigate the function of these myelin proteins in peripheral nerves and to assess the value of these mice as animal models for hereditary human peripheral neuropathies. Mice that are completely devoid of P0 expression (P0(+/0)) show a complex phenotype that is characterized by hypomyelination, compromised myelin compaction, and degeneration of myelin and axons early in life. In contrast, young mouse mutants that have retained one wild-type allele of the P0 gene (P0(+/0)) reveal morphologically normal myelin but start to develop signs of demyelination and remyelination at 4 months of age. A similar late-onset myelin deficiency was observed in Cx32-deficient mice (Cx32(0/0)). We have now generated mice deficient for Cx32 and P0. In animals that lack both proteins (Cx32(0/0)/P0(0/0), the phenotype is morphologically identical to mice that solely lack P0. Animals that lack Cx32 and carry one functional P0 allele (Cx32(0/0/P0(+/0)) revealed demyelination and remyelination as evidenced by thin myelin and Schwann cell onion bulb formation already at the age of 4 weeks, a time point when no pathology was observed in the single mutants. These morphological deficits were also more prominent in 4-month-old Cx32(0/0)/P0(+/0)animals compared to the single mutants. Our data support the view that Cx32 and P0 are crucial molecules for the maintenance of myelin. Furthermore, the function of Cx32 in the peripheral nervous system appears to be largely dispensable when myelin compaction is impaired.     

Rehabilitation following surgical procedures to address articular cartilage lesions in the knee

Knowledge of the structure and function of articular cartilage is important when considering rehabilitation following surgical procedures for articular cartilage lesions of the knee. Articular cartilage is avascular and derives its nutrition primarily from synovial fluid, resulting in a limited potential for regeneration. Basic science evidence has demonstrated that compressive loading may have a positive impact on articular cartilage healing; however, excessive shear loading may be detrimental. Rehabilitation following surgical procedures for articular cartilage lesions should include controlled range of motion exercises. Exercises to enhance muscle function must be done in a manner which minimizes shear loading of the joint surfaces in the area of the lesion. A period of protected weight bearing is often necessary and should be followed by progressive loading of the joint. This article will: 1) provide a brief review of the structure and function of articular cartilage lesions as it relates to rehabilitation; 2) describe common surgical procedures to address articular cartilage lesions; and 3) provide guidelines for rehabilitation following surgical management of articular cartilage lesions.     

Differential expression of aggrecanase and matrix metalloproteinase activity in chondrocytes isolated from bovine and porcine articular cartilage

The release of aggrecan catabolites from cartilage is an early event in the pathogenesis of degenerative joint diseases. The enzymes involved in this process are unknown, controversial, and the subject of intense investigation. In this paper we have utilized a recombinant substrate containing the interglobular domain (IGD) of aggrecan to study specifically aggrecanase versus matrix metalloproteinase (MMP) catabolism in this domain of aggrecan. Our studies have shown that (i) there are species differences in the expression of latent versus active MMP activity on the aggrecan IGD; (ii) interleukin-1alpha exposure induces both aggrecanase and MMP activities, whereas retinoic acid induces only aggrecanase activity and inhibits the MMP activity on the aggrecan IGD; (iii) activators of latent MMP activity (p-aminophenylmercuric acetate and trypsin) significantly reduce aggrecanase activity; (iv) the time course of the appearance of aggrecanase versus the MMP catabolism of aggrecan IGD differs; (v) aggrecanase is a protease with metalloprotease characteristics; however (vi) the physiological (tissue) inhibitors of MMPs show weak inhibition (TIMP-1) or no inhibition (TIMP-2) of aggrecanase activity. Collectively, these studies show that aggrecanase and MMP catabolism of the aggrecan IGD are independent and uncoupled.     

Treatment of articular cartilage defects in athletes: an analysis of functional outcome and lesion appearance

This article characterizes chondral injuries and reviews the results of microfracture treatment in high-level competitive and recreational athletes. Thirty-eight high-level and 140 recreational athletes completed functional questionnaires preoperatively and yearly postoperatively, recording symptoms, function, and activity level. Second-look arthroscopy tapes were available in 26 high-level and 54 recreational athletes. The mean follow-up for the high-level athletes was 3.7 +/- 1.4 years. Chondral defects averaged 223 +/- 180 mm2. Lesion size and follow-up were not significantly different in the recreational group. Functional questionnaire responses demonstrated significant improvements from the time of microfracture to final follow-up. Improvement in function and symptoms was similar for the competitive and recreational athletes.     

Chondrocyte-seeded hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat

The aim of this study was to evaluate the potential for restoration of a large cartilage defect in the goat knee with hydroxyapatite (HA) loaded with chondrocytes. Isolated chondrocytes were suspended in fibrin glue, seeded on top of the HA, and then the composite graft was implanted in the defect. After transplantation, cell behaviour, newly synthesised matrix and the HA-glue interface were assessed histologically after 2, 4, 12, 26 and 52 weeks. Special attention was paid to the incorporation process of HA in the subchondral bone and interactions between this biomaterial and the fibrin-glue-chondrocyte suspension. Chondrocytes in the glue proved to survive the transplantation procedure and produced new metachromatically stained matrix two weeks after implantation. The glue-cell suspension had penetrated the superficial porous structure of the HA. Four weeks after surgery, islands of hyaline-like cartilage were observed at the HA-glue interface. A layer of fibrous tissue was formed surrounding the HA graft, resulting in a relatively instable fixation of the HA in the defect. This instability of the graft in the defect, possibly together with early weight bearing, resulted in a gradual loss of the newly formed hyaline cartilage-like repair tissue. Progressive resorption of the HA occurred without any sign of active bone remodelling from the host site. One year after surgery part of the defect which extended down to the cancellous bone had been predominantly restored with newly formed lamellar bone. Only small HA remnants were still present at the bottom of the original defect. Resurfacing of the joint had occurred with fibrocartilaginous repair tissue. The absence of adequate fixation capacity of the HA near the joint space resulted in a relative instability of the graft with progressive resorption. Therefore, HA is not a suitable biomaterial to facilitate the repair of large articular cartilage defects.     

Regeneration of defects in articular cartilage with callus and cortical bone grafts. An experimental study

Luteal-phase estrogen and progesterone concentrations were measured every other day and used to monitor the corpus luteum activity. The patterns of estrogen and progesterone concentrations were compared relative to the day of endogenous human chorionic gonadotropin (hCG) detection (defined as the day of implantation). The relationship between estrogen and progesterone and hCG concentrations was studied in 71 viable pregnancies, 12 clinical abortions, five preclinical abortions and 84 non-pregnant cycles after IVF/ET. Although all patients received luteal-phase progesterone support (25-50 mg/ml), low late luteal-phase progesterone concentrations of < 30 ng/ml from day + 11 to day + 15 were found in 64 patients (17% of viable pregnancies, 33.3% of clinical abortions, 60% of preclinical abortions and 53.6% of non-pregnant cycles) day + 1 was the day of retrieval). Implantation always occurred before or on day + 13 and 86% of pregnant cycles implanted on day + 8 to day + 11. Viable pregnancies had significantly higher mean progesterone concentrations on day + 3 to day + 7 (pre-implantation) and on day + 9 to day + 15 (postimplantation) than those of non-pregnant cycles or abortions. On the day of implantation, the mean +/- standard of deviation of estrogen (pg/ml) and progesterone (ng/ml) levels for viable pregnancies, clinical abortion and preclinical abortions were 314 +/- 210, 40.5 +/- 25; 226.7 +/- 246, 48.7 +/- 31; and 39.6 +/- 24.5, 28.6 +/- 24.5, respectively. On the same day, 73.2% of viable pregnancies, 41.7% of clinical abortions, and 20% preclinical abortions had a progesterone concentration > 30 ng/ml; 73.2% of viable pregnancies, 41.7% of clinical abortions and 20% of preclinical abortions had an estrogen concentration > 100 pg/ml. Although not precluding implantation completely, late luteal-phase hormonal deficiencies may impair endometrial growth and might ultimately lead to failure or abnormal implantation. A viable pregnancy requires not only a functional corpus luteum in the early luteal phase to develop a receptive endometrium, but also a responsive corpus luteum in the late luteal phase to support pregnancy. The time of implantation is critical. Implantation that occurs before the demise of the corpus luteum will facilitate a normal pregnancy.     

Growth profiles and articular cartilage characterization in a goat model of Legg-Calve-Perthes disease

Numerous animal species, including the goat, have been evaluated as potential models for human Legg-Calve-Perthes disease (LCPD). These models disrupt the vasculature of the femoral head, causing it to collapse, and therefore do not mimic all the clinical patterns of the human disease. Baseline data regarding the weight and femoral length in the growing goat are not available. This study characterized the goat's normal growth for comparison with that of humans. The growth aberrations in the proximal femur created by surgically ablating the capital physis were described and compared with the aberrations observed in human LCPD cases. Age, weight, and femoral length (test and control) data were obtained for goats approximately 1 to 14 months of age. At 4 months of age, a craniolateral surgical approach was used to expose the cranial lateral capital physis so that it could be cauterized. Postoperative radiographs were evaluated by graphic analysis to assess the resultant changes in the morphology of the proximal femur. The articular cartilage of the femoral head and acetabulum was evaluated mechanically, using indentation testing, to determine the apparent modulus of elasticity, and histopathologically regarding its thickness and proteoglycan content. The proximal femurs of goats and humans exhibit similar morphology and growth patterns. There was a positive correlation between age, weight, and femoral lengths in the goat. The surgical procedure was effective in ablating the capital femoral physis as indicated by shorter femoral lengths and fragmented, flattened, and mushroomed femoral heads. The histopathological data revealed that the articular cartilage was significantly thicker in the operated hip joints at the ventrocaudal and cranial acetabula and the dorsal and ventral femoral heads. The test cartilage exhibited significantly less positive staining for proteoglycans in the dorsocaudal and the cranial acetabula as well as the ventral femoral head. The apparent modulus of elasticity, of the test cartilage was significantly lower than the control value at the dorsocaudal acetabulum. These data show that the surgical procedure produced morphological changes that mimic those in human LCPD. The increased thickness of the articular cartilage of the LCPD femoral head may account for the articular degeneration observed in older patients with LCPD, as increased cartilage thickness is associated with decreased tissue quality.     

Gene expression of matrix metalloproteinases 1, 3, and 9 by chondrocytes in osteoarthritic human knee articular cartilage is zone and grade specific

OBJECTIVES: Matrix metalloproteinases (MMPs) are thought to be major mediators of cartilage destruction. Osteoarthritis (OA) is characterised by cartilage degradation. This study explores gene expression of three MMPs in articular chondrocytes during the histological development of the cartilage lesion of OA. METHODS: Biopsy specimens of human normal and OA cartilage, classified into four grades on the basis of histology, were probed for MMPs 1, 3, and 9 using 35S-labelled cDNA probes. The signal was measured at four different depths (zones) using an automated image analyser and compared with signal from sections probed with lambda DNA. Rheumatoid synovium was used as a positive control for MMP gene expression. RESULTS: Rheumatoid tissue contained mRNA for all three MMPs. Expression in chondrocytes varied with the depth of the chondrocyte in the cartilage and the histomorphological extent of the OA changes. There was no detectable mRNA signal for these three MMPs in normal cartilage. In general, in OA, MMP-1 gene expression was greatest in the superficial cartilage in established disease. By contrast mRNAs for MMP-3 and 9 were expressed deeper in the cartilage, MMP-9 early in disease and MMP-3 with a biphasic pattern in early and late stage disease, most pronounced in the latter. This was a consequence of differential expression in single cells and chondrocyte clusters in late disease. CONCLUSION: The data indicate that expression of genes for MMPs 1, 3, and 9 is differentially regulated in human articular chondrocytes and, in individual cells, is related to the depth of the chondrocyte below the cartilage surface and the nature and extent of the cartilage lesion.     

Effect of cultured autologous chondrocytes on repair of chondral defects in a canine model

Articular cartilage has a limited capacity for repair. In recent clinical and animal experiments, investigators have attempted to elicit the repair of defects of articular cartilage by injecting cultured autologous chondrocytes under a periosteal flap (a layer of periosteum). The objective of the present study was to determine the effect of cultured autologous chondrocytes on healing in an adult canine model with use of histomorphometric methods to assess the degree of repair. A total of forty-four four-millimeter-diameter circular defects were created down to the zone of calcified cartilage in the articular cartilage of the trochlear groove of the distal part of the femur in fourteen dogs. The morphology and characteristics of the original defects were defined in an additional six freshly created defects in three other dogs. Some residual noncalcified articular cartilage, occupying approximately 2 per cent of the total cross-sectional area of the defect, was sometimes left in the defect. The procedure sometimes damaged the calcified cartilage, resulting in occasional microfractures or larger fractures, thinning of the zone of calcified cartilage, or, rarely, small localized penetrations into subchondral bone. The forty-four defects were divided into three treatment groups. In one group, cultured autologous chondrocytes were implanted under a periosteal flap. In the second group, the defect was covered with a periosteal flap but no autologous chondrocytes were implanted. In the third group (the control group), the defects were left empty. The defects were analyzed after twelve or eighteen months of healing. Histomorphometric measurements were made of the percentage of the total area of the defect that became filled with repair tissue, the types of tissue that filled the defect, and the integration of the repair tissue with the adjacent cartilage at the sides of the defects and with the calcified cartilage at the base of the defect. In histological sections made through the center of the defects in the three groups, the area of the defect that filled with new repair tissue ranged from a mean total value of 36 to 76 per cent, with 10 to 23 per cent of the total area consisting of hyaline cartilage. Integration of the repair tissue with the adjacent cartilage at the edges of the defect ranged from 16 to 32 per cent in the three groups. Bonding between the repair tissue and the calcified cartilage at the base of the defect ranged from 41 to 89 per cent. With the numbers available, we could detect no significant difference among the three groups with regard to any of the parameters used to assess the quality of the repair. In the two groups in which a periosteal flap was sutured to the articular cartilage surrounding the defect, the articular cartilage showed degenerative changes that appeared to be related to that suturing.     

Variations in thickness of articular cartilage in the human sacroiliac joint

Differences in articular cartilage thickness in the sacroiliac joint were investigated in different regions of the sacral and the iliac articular surfaces in the embalmed cadavers of five males and six females. The mean thickness of the sacral articular cartilage was greater than that of the iliac articular cartilage (P < 0.001) and the sacral articular cartilage of the female was thicker than that of the male (P < 0.02). Differences between thicknesses of the iliac articular cartilage in the male and female and in different regions of the sacral and iliac articular cartilages were found to be not significant.     

Expression of members of a novel membrane linked metalloproteinase family (ADAM) in human articular chondrocytes

The I1-imidazoline receptor is expressed in the rostral ventrolateral medulla (RVLM) where it mediates vasodepression, and in PC12 pheochromocytoma cells where it elicits generation of diacylglycerol independent of phosphatidylinositol turnover or activation of phospholipase D. We hypothesized that the I1-imidazoline receptor couples to a phosphatidylcholine-selective phospholipase C (PC-PLC). The I1-agonist moxonidine elicited diacyglyceride accumulation and release of [3H]phosphocholine from PC12 cells prelabeled with [3H]choline. The PC-PLC inhibitor D609 abolished both responses. Microinjection of D609 into the RVLM of hypertensive rats blocked the vasodepressor response to intravenous moxonidine. These data implicate PC-PLC in cellular and organismic responses to I1-receptor stimulation.     

Biomechanical topography of human articular cartilage in the first metatarsophalangeal joint

The objective of this study was to provide a map of cartilage biomechanical properties, thickness, and histomorphometric characteristics in the human, cadaveric first metatarsophangeal joint, to determine if normal articular cartilage was predisposed topographically to biomechanical mismatches in articulating surfaces. Cartilage intrinsic material properties and thickness were obtained from seven pairs of human, freshly frozen, cadaveric, metatarsophalangeal joints using an automated creep indentation apparatus under conditions of biphasic creep. Eight sites were tested: four on the metatarsal head, two on the proximal phalanx base, and one on each sesamoid bone to obtain the aggregate modulus, Poisson's ratio, permeability, shear modulus, and thickness. Cartilage in the lateral phalanx site of the left metatarsal head had the largest aggregate modulus (1.34 MPa), whereas the softest tissue was found in the right medial sesamoid (0.63 MPa). The medial phalanx region of the right joint was the most permeable (4.56 x 10(-15) meter4/Newton-second), whereas the medial sesamoid articulation of the metatarsal head of the left joint was the least permeable (1.26 x 10(-15) meter4/Newton-second). Material properties and thickness are indicative of the tissue's functional environment. The lack of mismatches in cartilage biomechanical properties of the articulating surfaces found in this study may be supportive of clinical observations that early degenerative changes, in the absence of traumatic events, do not occur at the selected test sites in the human first metatarsophalangeal joint.