Chondrocyte deformation and local tissue strain in articular cartilage: A confocal microscopy study

It is well accepted that mechanical forces can modulate the metabolic activity of chondrocytes, although the specific mechanisms of mechanical signal transduction in articular cartilage are still unknown. One proposed pathway through which chondrocytes may perceive changes in their mechanical environment is directly through cellular deformation. An important step toward understanding the role of chondrocyte deformation in signal transduction is to determine the changes in the shape and volume of chondrocytes during applied compression of the tissue. Recently, a technique was developed for quantitative morphometry of viable chondrocytes within the extracellular matrix using three-dimensional confocal scanning laser microscopy. In the present study, this method was used to quantify changes in chondrocyte morphology and local tissue deformation in the surface, middle, and deep zones in explants of canine articular cartilage subjected to physiological levels of matrix deformation. The results indicated that at 15% surface-to-surface equilibrium strain in the tissue, a similar magnitude of local tissue strain occurs in the middle and deep zones. In the surface zone, local strains of 19% were observed, indicating that the compressive stiffness of the surface zone is significantly less than that of the middle and deep zones. With this degree of tissue deformation, significant decreases in cellular height of 26, 19, and 20% and in cell volume of 22, 16, and 17% were observed in the surface, middle, and deep zones, respectively. The deformation of chondrocytes in the surface zone was anisotropic, with significant lateral expansion occurring in the direction perpendicular to the local split-line pattern. When compression was removed, there was complete recovery of cellular morphology in all cases. These observations support the hypothesis that deformation of chondrocytes or a change in their volume may occur during in vivo joint loading and may have a role in the mechanical signal transduction pathway of articular cartilage.     

Intra-articular injections and articular cartilage metabolism. An experimental study in rabbits.

We studied the effects of repeated intra-articular injections of sterile 140 mM NaCl solution on articular cartilage in adult rabbits. After 20 injections into the knee joints over a period of 4 weeks, chondrocyte glucosaminoglycan synthesis was evenly reduced in all cartilage layers, accompanied by a significant proteoglycan depletion of the matrix which was most marked in the superficial half of the cartilage. These and other changes only partially reversed during a further 4-week period after the injections had been stopped. Our data underline the need for a clear-cut indication for intra-articular injections. The microtrauma caused by injection, in conjunction with the introduction of a carrier solution into the joint, may, at least when repeated at short intervals, lead to measurable damage to the articular cartilage.     

Effects of various irrigating solutions on articular cartilage. An experimental study in rabbits

The effect of six different solutions (normal saline, ringer's lactate, chlorhexidine, povidone iodine, ceftriaxone, chloramphenicol) on articular cartilage was investigated in an in vivo rabbit model study. The right knees were aspirated and injected with one of these solutions for five days and, three days later, the patellae of the rabbits were excised and investigated histologically. Left knees were used as controls. There was no difference between the groups and the controls with respect to structure, cell density, and nuclei-to-lacunae ratio. These results suggest that, these solutions have no noxious effects on articular cartilage when used as irrigating fluids in orthopaedic practice.     

Effects of immobilization on the articular cartilage in young rabbits. A quantitative light microscopic stereological study

Eight young New Zealand White rabbits were immobilized by splinting the hind limb to extension for eight weeks. Eight mobile animals served as controls. The articular cartilage of the lateral tibial condyle of control and of both splinted and contralateral knee joints were studied using a stereological method, which allowed the authors to derive, from quantitative histologic measurements, three-dimensional parameters defining the tissue structure. Qualitatively, osteoarthrotic changes occurred in both the splinted and the contralateral knee joints. Quantitatively, osteoarthrotic changes produced an increase in cell density but a decrease in cell size in the superficial zone of articular cartilage, and an increase in cell size in the deep zone. Combined qualitative and quantitative histologic characteristics were also analyzed. Different types of osteoarthrosis were observed in the splinted and the contralateral knees.     

Chondrocyte necrosis and apoptosis in impact damaged articular cartilage.

A decrease in chondrocyte numbers is one characteristic of osteoarthritic cartilage. This decrease may be the result of apoptosis or other forms of cell death induced by mechanical damage. Furthermore, cell death may contribute to the structural and metabolic changes found in osteoarthritic cartilage. Therefore, we investigated cell viability and the mode of cell death in cartilage subjected to an increasing severity of impact loads expected to cause compositional damage and osteoarthritic-like metabolic alterations. Canine cartilage explants were subjected to cyclic indentation impacts of 5 megapascals at 0.3 Hz for 0, 2, 20, and 120 min and then kept in culture for 2, 4, 48, and 144 h. Cell death was assessed by the TUNEL assay and by uptake of propidium iodide. Viable cells were detected by the ability to metabolize fluorescein diacetate. Nuclear morphology and ultrastructure of the cell were examined using Hoechst 33342 fluorescent staining and transmission electron microscopy (TEM). As controls for necrosis and apoptosis, cartilage was, respectively, frozen and thawed or incubated with mitomycin-C, an apoptosis inducer. In cartilage that had been loaded for 2 h, 32% of the chondrocytes in the loaded core took up propidium iodide within 2 h after loading. Most of these were in the middle to superficial zones and reflected leaky cell membranes usually characteristic of necrosis. Less than 1% of these chondrocytes were positive in the TUNEL assay after 4 h. After additional culture for 2 days, however, the proportion of chondrocytes which were positive in the TUNEL assay reached 73%. A dose dependent response to duration of loading was detected with the TUNEL assay at this time. The TUNEL assay was not specific for apoptosis since 92% of chondrocytes in freeze/thawed cartilage were TUNEL positive. However, some cells with apoptotic bodies and chromatin condensation characteristic of apoptosis were found in the transition zone between necrotic and normal chondrocytes, but not in the superficial and upper zones, in impact damaged cartilage. We concluded that in this study, necrosis occurred first, followed by apoptosis.     

Long-lasting tolerance of articular cartilage after experimental intraoperative radiation in rabbits.

In experimental intraoperative irradiation, 18 adult rabbits received a single, 50-Gy dose of x-radiation at a unilateral knee joint, and subsequent changes in the articular cartilage were examined over a 15-month period by histology, scanning electron microscopy, and autoradiography. Although the subchondral bone showed histologically typical findings of osteonecrosis three to nine months postirradiation, the articular cartilage revealed no obvious degenerative changes during the entire study period. Scanning electron microscopy revealed normal collagen architecture in the irradiated cartilage for as long as 15 months postirradiation. Autoradiography demonstrated active RNA synthesis by the irradiated chondrocytes during the same period. These results indicate that articular cartilage tissue tolerates intraoperative radiotherapy without sustaining serious degenerative changes, unless possible collapse or contracture disturbs its biomechanical integrity. The survival of articular cartilage can be advantageous for this type of limb-salvage surgery in the treatment of malignant bone tumors around a synovial joint.     

The influence of weight-bearing exercise on articular cartilage of meniscectomized joints. An experimental study in sheep

Unilateral medial meniscectomy was undertaken in 29 purebred adult merino sheep. Arthrotomy (without meniscectomy) was conducted in 11 animals, and six were used as nonoperated controls. All animals were followed with either three- or six-month active or passive postoperative management. Active animals traversed a total of 360 km after three months and 1040 km after six months. The passive group was housed in pens allowing limited weight-bearing exercise. After death, morphologic changes were recorded and cartilages from the medial and lateral joint regions were examined for water, collagen, and proteoglycan (as hexuronate) content. Proteoglycan aggregation and extractability under nondissociative (0.4 molar GuHCl) and dissociative (4.0 molar GuHCl) conditions were also determined. The collagen content of the medial cartilage of the passively maintained meniscectomized animals was reduced, relative to external controls. Although proteoglycan content was elevated in medial cartilage of the passive group three months postoperatively, these levels returned to the control range after six months. However, low-salt (0.4 molar GuHCl) extractability of proteoglycans still remained high. All cartilages of control and meniscectomized joints showed an elevation of proteoglycan content in the active groups three months postoperatively. The cartilages in the medial region of meniscectomized animals showed the largest increase, but these levels declined after six months. Proteoglycan aggregation and water content were still elevated relative to controls six months postoperatively. The collagen levels in the three-month or six-month actively maintained meniscectomized group were not distinguishable from control values. Morphologically, joints of the passively and actively maintained animals showed focal surface fibrillation and erosions. However, in the active group, osteophytes were common and well developed six months postoperatively. These studies indicate that, while weight-bearing exercise after meniscectomy appears to be beneficial to the quality of the cartilaginous matrix, it is also accompanied by osteophytosis and cartilage hyperplasia.     

Chondrocyte mitosis in the articular cartilage of femoral heads with various diseases.

Autoradiographic studies using thymidine-3H reveal the mitosis of chondrocytes in degenerated joints, i.e. joints having secondary osteoarthritis or aseptic necrosis of the femoral head. The findings obtained provide additional support for the recent investigations regarding chondrocyte mitosis in primary osteoarthritic cartilage. Histologic and histochemical examinations suggest that a loss of glycosaminoglycans in the matrix is evidence for conversion of chondrocyte activity to mitosis which occurs, however, within the limit of "the point of irreversibility", analogous to the observations from the biochemical point of view. Biomechanical and nutritional factors are also discussed in relation to the results obtained from cartilages of the femoral heads in cases of femoral neck fracture and aseptic necrosis of the femoral head.     

补骨脂多糖局部填充修复家兔关节软骨全层缺损的实验研究

目的探讨中药提取物补骨脂多糖局部运用修复兔关节软骨全层态发生蛋白／明胶复合物组（B组）、空白组（C组）,每组24只,48个关节。分别给予A缺损的可行性和部分机理。方法将72只成年健康日本大耳白兔随机分为补骨脂多糖组（A组）、人骨形组补骨脂多糖凝胶、B组骨形态发生蛋白／明胶复合物填充膝关节股骨内髁滑车面软骨全层缺损区,c组只做钻孔处理,空白对照。术后取标本行大体观察、HE染色、改良Pinsda法组织学评分的方法进行评估。结果补骨脂多糖和rhBMP!明胶复合物在2、4、8周缺损区填充均优于空白组,为乳白色半透明质软类透明软骨组织;组织学评分与空白组存在极显著性差异（P〈0．01）,用药组之间没有统计学意义（P〉0．05）。结论补骨脂多糖局部运用可以修复家兔关节软骨缺损,经组织学观察修复组织以透明软骨为主,接近正常关节软骨。其修复关节软骨缺损是可行的,具有很好的实用价值。     

Ultrastructural study of articular cartilage in experimental pyogenic arthritis

Ultrastructural changes of the articular cartilage at the early stage of experimental pyogenic arthritis were studied by electron microscopy. The superficial zone of the cartilage at the site of the cartilage-synovium junction demonstrated far more changes than those at the site of the free surface of the articular cartilage. Changes were observed at the cartilage-synovium junction as early as four hours after the intraarticular injection of the infecting organism. The most interesting finding was migration of erythrocytes and polymorphonuclear leucocytes into the superficial zone of the cartilage at the early stage. The pericellular matrix in the middle zone became so loose in some areas that collagen fibrils were partially exposed after three days. The naked collagen fibrils were tapered, nicked and irregular in size after seven days.     

“自组装”工程化软骨修复体外关节软骨缺损的实验研究

[目的]探讨应用"自组装"工程化软骨修复体外关节软骨缺损的可行性及其转归。[方法]密度梯度离心法分离培养成人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,h MSCs)。将第3代h MSCs用含100 ng/ml GDF-5的软骨诱导液定向诱导,2周后重悬细胞,以5×10~6/ml的细胞密度接种于2%琼脂糖包被的24孔板,行自组装培养,2周后对标本行大体和组织学观察并采用生物化学法检测软骨相关的生物学特性。然后将部分标本植入到体外全层软骨缺损模型内,培养4周观察软骨缺损的修复效果,并比较修复前与修复后标本的软骨相关生物学特性变化情况。[结果]体外培养2周时预分化的h MSCs"自组装"形成一软骨样组织,组织学检测到软骨特异性成分阳性表达。全层软骨缺损修复模型体外培养4周后,大体和组织学可见缺损由工程化软骨所修复,交界面呈紧密黏附状,Ⅱ型胶原和蛋白多糖呈阳性表达,但弱于未移植组。生化结果显示移植组标本的细胞数、GAG和总胶原含量均低于未移植组(P0.05)。RT-PCR结果也反映出这一变化。[结论]"自组装"工程化软骨能有效地修复体外关节软骨缺损,并且能较稳定地维持软骨表型。     

Ultrastructural changes in the transitional zone between articular cartilage and synovial membrane during the development of experimental osteoarthrosis

We studied the ultrastructural changes that take place in the transitional zone between the articular cartilage and the synovial membrane during the development of experimental osteoarthrosis. We focused special attention on changes involving the proteoglycan complexes within the matrix of articular cartilage. We observed that changes in the transitional zone resemble those seen in articular cartilage during the development of osteoarthrosis. We also found transient cellular forms with fibroblast phenotype regulating the demands of both cartilage and synovial matrix. The transient nature of these elements determines the pronounced lability of this zone, and this may be related to the early development of osteoarthrosis.     

Trypsin-induced mitosis in the articular cartilage of adult rabbits.

Full-grown rabbits were injected in the knee joints with solutions of trypsin of various concentrations. The animals were sacrificed 2 weeks after the trypsin injection. Twenty-four hours before sacrifice they received 40 muCi 3H-thymidine intra-articularly. The changes in the knee joints were then studied by histological and autoradiographical methods. The injection of trypsin did not result in the development of osteoarthritis. However, autoradiography revealed that the chondrocytes started to divide after the injection. The mitosis of the chondrocytes can thus not be due to degeneration of the cartilage. The explanation put forward is that the mitosis of the chondrocytes may be the result of a decrease in the concentration of a growth controlling factor (chalone) initiated by the administration of trypsin.