Biomechanical, structural, and biochemical indices of degenerative and osteoarthritic deterioration of adult human articular cartilage of the femoral condyle.

OBJECTIVE: To compare the tensile biomechanical properties of age-matched adult human knee articular cartilage exhibiting distinct stages of degenerative or osteoarthritic deterioration and to determine the relationships between tensile properties and biochemical and structural properties hypothesized to underlie functional biomechanical deterioration. METHODS: Age-matched articular cartilage samples, obtained from the lateral and medial femoral condyles (LFC and MFC), exhibited (1) minimal fibrillation, characteristic of normal aging (NLA), (2) overt fibrillation associated with degeneration (DGN), or (3) overt fibrillation associated with osteoarthritis (OA). DGN samples were from knees that exhibited degeneration but not osteophytes while OA samples were from fragments removed during total knee arthroplasty. Cartilage samples were analyzed for tensile properties, cell and matrix composition, and histopathological structure. RESULTS: Differences in tensile, compositional and surface structural properties were indicative of distinct stages of cartilage degeneration, early (OA) advanced (DGN) and late (OA) with early degenerative changes in NLA samples being more advanced in the MFC than the LFC, including higher surface fibrillation, lower intrinsic fluorescence, and lower mechanical integrity. The transition from early to advanced degeneration involved a diminution in mechanical function, surface integrity, and intrinsic fluorescence. The transition from advanced to late degeneration involved an increase in cartilage water content, an increase in degraded collagen, and loss of collagen. CONCLUSIONS: These results provide evidence of coordinated mechanical dysfunction, collagen network remodeling, and surface fibrillation. Even in the cartilage of knees exhibiting overt fibrillation but not extensive erosions characteristic of clinical osteoarthritis, most features of advanced cartilage degeneration were present.

Development of a novel spontaneous metastasis model of human osteosarcoma transplanted orthotopically into bone of athymic mice.

There is a pressing need for in vivo models in which potential antitumor agents can be tested for their ability to inhibit the growth and metastatic spread of human sarcomas. A recent advance in this regard has been the development of a v-Ki-ras-oncogene-transformed human osteosarcoma cell line (KRIB) that efficiently colonizes the lungs of athymic nude mice when cells (1 x 10(5)) are administered by i.v. injection. In the present study, we have utilized this cell line to develop a spontaneous metastasis model in which a small number of tumor cells are injected into the tibial bones of athymic mice. When as few as 1000 KRIB cells are orthotopically implanted into the tibial bones of nude mice, bone tumors, which are radiographically and histologically similar to primary human osteosarcoma, develop within 4 weeks. Furthermore, as in the human disease, cells from these primary tumors subsequently seed the animals' lungs, resulting in reproducible and quantifiable pulmonary metastasis evident both upon gross inspection of the lungs and histologically 6 weeks after tumor inoculation. Surgical amputation of the tumor inoculation site up to 2 weeks after tumor injection prevents pulmonary metastasis, indicating that substantial local (tibial) growth and invasion of the primary tumor for at least 2 weeks is required for subsequent metastasis. Implantation of s.c. 5000 KRIB cells fails to produce local or metastatic tumors. We anticipate that this model will prove to be a powerful tool with which to study the mechanisms of human osteosarcoma growth and pulmonary metastasis, and to assess the efficacy of promising therapeutic agents.

The tetrabasic KKKK(147-150) motif determines intracrine regulatory effects of PthrP 1-173 on chondrocyte PPi metabolism and matrix synthesis.

Expression of PTHrP is a major regulator of growth cartilage development and also becomes robust in osteoarthritic cartilage. We further defined how PTHrP 1-173, which we observed to be the preferentially expressed PTHrP isoform in normal and osteoarthritic cartilage, functions in chondrocytes. We transfected both immortalized human juvenile costal chondrocytes (TC28 cells) and rabbit articular chondrocytes with wild-type PTHrP 1-173 and mutants of putative PTHrP 1-173 endoproteolytic processing sites. Wild-type PTHrP 1-173 inhibited collagen synthesis and decreased extracellular PPi (which critically regulates hydroxyapatite deposition) by 50-80% in both chondrocytic cell types. In contrast, PTHrP 1-173 mutated at the PTHrP 147-150 motif KKKK (but not the other site-directed mutants) and increased both extracellular PPi and collagen synthesis by >50%. Synthetic PTHrP 140-173 mutated at amino acids 147-150 and also increased extracellular PPi, and wild-type 140-173 decreased extracellular PPi in permeabilized cells. The 147-nuclear localization of PTHrP. We conclude that the tetrabasic 147-150 motif functions to determine how PTHrP 1-173 regulates collagen synthesis and levels of extracellular PPi by an intracrine mechanism in chondrocytes, and it may prove useful as a therapeutic target for regulation of mineralization.

Magnetic resonance imaging in assessing cartilage changes in experimental osteoarthrosis of the knee.

Cartilage degeneration in osteoarthrosis (OA) of the knee generally is believed to precede osseous abnormalities. Because cartilage abnormalities are not readily detected by routine radiography, we investigated the role of magnetic resonance imaging (MRI) in assessing cartilage damage in a goat model for OA. Four goats had the anterior cruciate ligament of one knee severed surgically to create instability and accelerate OA. Two goats each were killed at 4 and 6 weeks, respectively, after walking on the unstable knees. MRI of the knees was performed with Hybrid fat suppression sequences. The images were correlated with gross anatomic sections and histologic analysis of the knees. On gross examination, the unstable knees showed rapid development of thinning, surface irregularity, and focal defects of the cartilage. These findings correlated well with abnormalities detected on the MRIs. In addition, areas of decreased signal intensity in cartilage correlated with histologic evidence of degenerative changes in the cartilage substance, including fragmentation, fibrillary and eosinophilic changes, and chondrocyte proliferation, indicating attempted cartilage repair. Precise correlation of pathologic and MRI data, however, was lacking, related in part to inability to match perfectly the level and orientation of the gross section with that on the MRI examination.

In situ assessment of cell viability within biodegradable polylactic acid polymer matrices.

Efforts to expand treatment options for articular cartilage repair have increasingly focussed on the implantation of cell polymer constructs. Primary cells cultured from perichondrium, a chondrogenic tissue, were found to survive in vitro within a biodegradable porous polylactic acid matrix. The novel application of an in situ fluorescent double-stain protocol to cell polymer constructs was supported by increased 3H-thymidine uptake and the ability of cell seeded polylactic acid to form first passage explant cultures. This in situ viability staining technique allowed for rapid determination of cell viability and, in conjunction with confocal microscopy, assessment of cellular distribution within a biodegradable scaffold. Advantages of using this method over histological and electron microscopic analysis include in situ observation, absence of distortion in scaffold architecture due to polymer dissolution and disruption during processing, and obtaining a viability assessment within 30 min. Potential applications of this protocol as a screening tool for laboratory engineered tissues and in the evaluation of cellular injury in natural tissues are discussed.

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.

Differential metabolic responses of periarticular ligaments and tendon to joint immobilization.

Parameters of collagen metabolic behavior were analyzed in the periarticular connective tissues, i.e., medial collateral ligament (MCL), anterior cruciate ligament (ACL), and patellar tendon (PT), of control and immobilized rabbit knees. Two periods of immobilization were studied: 9 and 12 wk. Collagen turnover and collagen cross-links were quantitatively assessed in the three tissues. The results showed that after 9 wk both synthesis and degradation were significantly increased in the MCL and ACL, whereas the PT showed lesser effects. After 12 wk all three tissues experienced significant losses of collagen mass, which resulted in tissue atrophy. The concentrations of the reducible collagen cross-links dihydroxylysinonorleucine and hydroxylysinonorleucine in the immobilized MCL and ACL were greater than their respective controls, indicating an increase in collagen synthesis, whereas concentrations of the nonreducible cross-link hydroxypyridinoline were observed to be decreased in these tissues. Of the reducible cross-links in the PT, only hydroxylysinonorleucine was found to be increased over control, whereas hydroxypyridinoline was slightly less concentrated. These results taken together have demonstrated that the ligamentous tissues are more susceptible to the effects of stress deprivation secondary to joint immobilization than the PT, and, in particular, the ACL of the three tissues studied appears to be most vulnerable.

Collagenase activity in anterior cruciate ligament: protective role of the synovial sheath.

To evaluate the protective role of the synovial sheath of the anterior cruciate ligament (ACL), we have developed a synovectomy model that exposes the ACL substance to the intra-articular environment with and without hemarthrosis. Histology and the level of collagenase activity were studied to assess intrinsic ligament alterations. The treatment groups studied were as follows: ACLs of sham-operated knees receiving arthrotomy only, ACLs of knees receiving arthrotomy and acute hemarthrosis, ACLs of knees that underwent synovectomy, and ACLs of knees that underwent both synovectomy and acute hemarthrosis. All animals were killed 10 days postoperatively for gross, histological, and biochemical assessment. Histologically at 10 days ACLs experiencing synovectomy and ACLs having synovectomy plus hemarthrosis revealed marked hypocellular areas. Biochemical results indicate that synovectomy is the treatment mainly responsible for the observed increase in ACL collagenase activity. Hemarthrosis alone clearly had no effect, although hemarthrosis coupled with synovectomy appeared to further increase the amount of active collagenase present in the ACLs. This study indicates that, with exposure of the ACL substance to the synovial fluid or with hemarthrosis after synovectomy, there is an increase in the degradative activity of the ACL. The protective role of the synovial sheath suggests that the synovial sheath injury associated with acute ACL rupture may allow for exposure of the ligament substance to the degradative effects of the synovial environment and associated hemarthrosis.

Medical collateral ligament healing subsequent to different treatment regimens.

The response of transected canine medical collateral ligaments (MCL) to clinical treatment regimens was investigated. These regimens included no surgical repair with no immobilization and surgical repair with various periods of immobilization. The biomechanical, biochemical, and histological properties of the healing MCL were examined 6 and 12 wk postoperatively. At 6 wk, all healing MCLs had increased cellularity with decreased levels of total collagen and increased amounts of reducible Schiff base cross-links and type III collagen. Biomechanically, the varus-valgus (V-V) knee laxity was significantly increased, and no group achieved normal structural or mechanical properties. At 12 wk the histological appearance of the MCL became more normal but still had increased cellularity. Biochemically, the total collagen levels in experimental MCLs were not statistically different from the controls, but these MCLs still had high amounts of type III collagen and an even higher number of reducible cross-links. From knees in which the MCL was not treated, the V-V knee laxity and the ultimate loads of the femur-MCL-tibia complex achieved normal values. However, the stress-strain properties for these MCLs and those treated with repair and immobilization did not completely recover.

Vascular assessment of the periarticular ligaments of the rabbit knee.

While the rabbit is being extensively utilized in animal models for orthopaedic research, the vascular anatomy of the knee ligaments has not been thoroughly described in this species. This study demonstrates the blood supply to the infrapatellar fat pad, the cruciate ligaments, the medial collateral ligament (MCL), and the menisci, such that the effects of manipulating these tissues may be properly interpreted. Vascular injection with India ink and iodinated i.v. contrast dye was performed in 11 New Zealand white rabbits, and routine histology done on six. The large vessel anatomy is similar to that described for humans and dogs, with a descending geniculate artery, medial and lateral superior and inferior geniculate arteries, a posterior geniculate artery, and a recurrent anterior tibial artery. The microvascular anatomy is also similar in that the infrapatellar fat pad and synovial membrane are highly vascular, the menisci are vascularized from their periphery (being avascular centrally), and the medial collateral ligament is relatively well vascularized. A difference from dogs and humans is present in the anterior cruciate ligament (ACL), which is poorly vascularized, with a single artery on its anterior aspect. High magnification histologic evaluation reveals numerous capillaries in the substance of the MCL, while the ACL is nearly devoid of such vessels. The interspecies variation in vascular anatomy is a variable that must be taken into consideration in any surgical or traumatic animal model investigation of knee pathology.

Cartilage repair with autogenic perichondrium cell/polylactic acid grafts: a two-year study in rabbits.

The repair of articular cartilage injuries remains a challenge. In this 2-year study, osteochondral defects in the femoral condyles of five rabbits were repaired with an autogenic perichondrium cell/polylactic acid graft and compared with a contralateral control in which the defect remained empty. The rabbits in the group with the grafts had a higher percentage of biologically acceptable repairs (100%) than did those in the control group (80%). According to the histologic and histomorphometric analyses, the grafts augmented the intrinsic healing. Nonetheless, the results for the grafts were tarnished by a depressed repair surface and a histologic appearance not equivalent to that of normal cartilage. The application of growth factors to this model may yield a clinical treatment.

Synergistic effect of growth factors on cell outgrowth from explants of rabbit anterior cruciate and medial collateral ligaments.

Cellular migration and proliferation are integral aspects of wound healing. An in vitro assay for cellular migration and proliferation using explants of rabbit anterior cruciate and medial collateral ligaments was developed previously. This study presents the effects of serum-free culture medium supplemented with basic fibroblast growth factor, bovine insulin, transforming growth factor-beta 1, and platelet-derived growth factor-B, added either individually or in combination, on cell outgrowth in explants of rabbit anterior cruciate and medial collateral ligaments. Outgrowth was assessed at 3 and 6 days by counting the number of cells surrounding the tissue explants. For explants of both ligaments, cell outgrowth was dependent on the presence of 10% fetal bovine serum or the combination of growth factors. Little outgrowth occurred in explants of either ligament in the presence of basic fibroblast growth factor, transforming growth factor-beta 1, or bovine insulin. Platelet-derived growth factor-B at concentrations of 20 and 100 ng/ml seemed to increase cell outgrowth from medial collateral ligament explants at 6 days. The outgrowth from the explants of both ligaments was much greater in the presence of all four growth factors than the sum of the outgrowth with the individual factors. This synergistic effect was as much as 10-fold and 20-fold for the anterior cruciate ligament explants at days 3 and 6, respectively, but no more than 3-fold for the medial collateral ligament explants at these times. Medial collateral ligament explants exhibited greater cell outgrowth than anterior cruciate ligament explants in 10% serum and in the presence of the four growth factors.

Tendons and ligaments: a morphological and biochemical comparison.

The purpose of this study was to compare selected rabbit tendons and ligaments morphologically and biochemically. Five representative structures from each of six age- and sex-matched rabbits were compared. Biochemical analyses included total collagen, reducible collagen cross-links, quantitative collagen typing, DNA, and glycosaminoglycans. Histological and chemical differences were demonstrated between the tendons and the ligaments. Smaller differences were also found between the individual ligaments (collateral and cruciate) and between the two tendons (patellar and Achilles) that were examined. These findings suggest that ligaments are more metabolically active than tendons, having more plump cellular nuclei, higher DNA content, larger amounts of reducible cross-links, and the presence of more type III collagen, as compared with tendons. They also contain slightly less total collagen than tendons and more glycosaminoglycans. We conclude that the tendons and ligaments studied have unique histological and biochemical characteristics, despite their gross similarities. Relatively increased metabolic activity in ligaments, implied by our findings, may be species specific, age related (transient), or may truly represent a structural expression of functional need for more rapid adaptation. Further investigation of other similarities or differences between particular ligaments (or tendons) is indicated, and attention is directed toward the importance of such variables in development of models for tendon and ligament studies.

Early histologic, metabolic, and vascular assessment of anterior cruciate ligament autografts.

A rabbit model for anterior cruciate ligament (ACL) reconstruction using autogenous patellar tendon was utilized to study the early events of autograft cellular dynamics. Biochemical, autoradiographic, histological, and vascular injection techniques demonstrated that the native autograft cell population rapidly necroses. This repopulation occurs without a vascular contribution; cells entering the autograft are reliant upon synovial fluid nutrition.

PT-12, a putative ras-activated proliferation-dependent gene, is expressed in patellar tendon and not in anterior cruciate ligament.

We describe a gene (PT-12) that is expressed in the patellar tendon and not in the anterior cruciate ligament. We used a recently developed polymerase chain reaction-based subtractive cDNA analysis to discover genes that are overexpressed in the patellar tendon but not expressed in the anterior cruciate ligament; the long-term objective was to find genes that are central to the self-repair of the patellar tendon, in contrast with the inability of the anterior cruciate ligament to launch a repair response following injury. PT-12 is a homologue of human S2 or mouse LLRep3 ribosomal genes, which are known to be overexpressed in highly proliferating cells. This study opens a new vista to the development of techniques and reagents to study the differences between two periarticular tissues (i.e., the patellar tendon and anterior cruciate ligament) that differ primarily in their ability to self-repair.

Origin of replacement cells for the anterior cruciate ligament autograft.

A rabbit model for anterior cruciate ligament (ACL) reconstruction using autogenous avascular patellar tendon (PT) was utilized to study the early events of graft incorporation. Histological observations demonstrated that autografts were centrally acellular with a peripheral rim of cells at 2 weeks, a central focal proliferation of cells at 3 weeks, and a cellular homogeneous distribution by 4-weeks postoperation. Graft necrosis followed by cellular proliferation suggested that a different population of cells other than the native PT fibroblasts may be inhabiting the graft. The extrinsic contribution of cells was studied by selective destruction of native PT cells with liquid nitrogen immersion prior to reconstruction of the ACL. The intrinsic contribution of cells was evaluated by sequestration of the PT graft in a semipermeable membrane before it was used to reconstruct the ACL. Histological analysis of tissue that was liquid N2 treated, used as an autograft, and then harvested 3-weeks postoperation revealed fibroblastic incorporation of the graft. In contrast, no cells were observed in semipermeable membrane sequestered autografts. These data suggest that autogenous ACL autografts of PT origin are repopulated by cells of external origin. In vitro control studies that were carried out in parallel demonstrated that PT fibroblasts could survive in tissue culture, but not in the synovial environment of the ACL. This suggests that fibroblasts from different sources have different, tissue-specific nutritional requirements.

Acute hemarthrosis: a histological, biochemical, and biomechanical correlation of early effects on the anterior cruciate ligament in a rabbit model.

The early histological, biochemical, and biomechanical characteristics of the anterior cruciate ligament (ACL) were determined in a rabbit model of acute hemarthrosis. The ACLs of 19 rabbits were given seven consecutive daily knee injections of 2 ml of fresh autologous blood, and then compared to contralateral ACLs from control knees injected with 2 ml of lactated Ringer's solution daily for 7 days. The rabbits were then sacrificed. Synovial proliferation with iron deposition within synoviocytes was observed; however, the architecture of the ACL was maintained. Additionally, the total collagen content, collagenase activity, and biomechanical properties of the ACL were unaltered.

Hyaluronan suppressed nitric oxide production in the meniscus and synovium of rabbit osteoarthritis model.

Nitric oxide (NO) plays an important role in cartilage degeneration, and NO donors induce meniscus degeneration and synovium inflammation. This study evaluated the effect of intraarticular injections of hyaluronan (HA) on NO production in meniscus and synovium using an experimental osteoarthritis (OA) model. Thirty-six New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT), and were divided into three groups. Four weeks after ACLT, the HA group started to receive intraarticular HA injections once a week for 5 weeks; the vehicle group started to receive the carrier of HA; and the no injection group, no treatment. All ACLT knees were harvested at the 9th week. Meniscus and synovium sections were examined by immunohistochemistry for nitrotyrosine. The pieces of these two tissues were cultured for 24 h. Culture supernatants were analyzed for nitrite concentration. The amount of NO produced by the meniscus was much larger than that produced by the synovium. NO productions in the meniscus and synovium of the HA group were significantly lower than those of the other groups. The results suggest that the inhibition of NO production in meniscus and synovium might be a part of the mechanism of the therapeutic effect of HA on OA.

Microstructural properties of the distal growth plate of the rabbit radius and ulna: biomechanical, biochemical, and morphological studies.

The purposes of this study were to define the tensile properties of each zone of the rabbit growth plate and to correlate them with the microarchitecture and biochemical composition of the zones. The epiphysis-growth plate-metaphysis complex was obtained from the radius and ulna of 20 8-week-old rabbits. Four dye markers were placed on the growth plate. The complex was loaded to failure with a tensile testing machine, and the strain behavior was recorded simultaneously with a microscope, a charge-coupled device camera, and a video dimension-analyzer system. The collagenous fiber architecture of each zone was examined with a microscope, and the collagen content of each zone was also determined. The tangent modulus of the resting zone was 75% stiffer than that of the other two zones. The highest values for strain at failure and energy absorbed to failure were observed in the hypertrophic zone, and the total collagen content was highest in the proliferating zone. The collagen fibers were more randomly aligned in the resting zone than in the other two zones. The diversity observed in the microarchitecture of the rabbit growth plate correlates with the zone-dependent differences in its mechanical properties.

Characterization of the intrinsic properties of the anterior cruciate and medial collateral ligament cells: an in vitro cell culture study.

The poor healing abilities of the anterior cruciate ligament (ACL) in contrast to those of the medial collateral ligament (MCL) are well known. Different intrinsic properties of the constituent cells of these ligaments have been proposed to be one of the factors in the differential repair mechanisms. To examine this hypothesis, we have established primary cell lines of ACL and MCL from the tissue explants of approximately similar dimensions and have studied their behavior in vitro. The outgrowth of cells from ACL explants was slower than from MCL explants, as shown by the size of the surrounding clusters of cells. Both ACL and MCL cultures exhibited typical fibroblastic morphology. No significant differences were observed in either attachment or growth of cells from the attached explants derived from various segments of ACL and MCL. Growth curves of ACL and MCL cultures at both passage numbers 2 and 6 showed a slower rate of proliferation of ACL cells than MCL cells (p less than 0.005). DNA synthesis measured in terms of [3H]thymidine incorporation (CPM/10(3) cells) of both log phase (ACL = 607.5 +/- 5.4 vs. MCL = 1356.4 +/- 11.3) and confluent (ACL = 83.0 +/- 3.6 vs. MCL = 189.8 +/- 5.4) cultures, supports the conclusion that differential proliferation rates of these cells exist in culture. FITC-phalloidin staining (for actin) of later passage cultures (P3-P5) showed a spread-out appearance of ACL cells and an elongated appearance of MCL cells. Relatively more stress fibers were seen within ACL cells. SDS-PAGE and Western blot analysis of cellular proteins revealed higher actin (43 kDa) content in ACL cells than in MCL cells. In vitro wound closure assay was performed by creating a uniform wound of 0.6 mm width in the confluent layer of ACL and MCL cultures. By 48 h postwounding, cell-free zones created in ACL cultures were occupied partially by single cells in a nonconfluent fashion. In contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. Results presented in this report demonstrate a lower proliferation and migration potential of ACL cells in comparison with MCL cells. These differences in intrinsic properties of ACL and MCL cells that were observed in vitro might contribute to the differential healing potentials of these ligaments in vivo.