Articular cartilage after meniscectomy: Rabbit knees studied with the scanning electron microscope

Ten rabbits had a medial meniscectomy in both knees. The articular surface was studied with a scanning electron microscope 2, 4 or 12 weeks after the operation. Three sham operated and two unoperated rabbits served as controls.

Progressive articular cartilage damage was observed on the weight-bearing areas of the medial condyles. The findings are compatible with clinical observations after meniscectomy; they confirm that meniscectomy is not a harmless operation.     

Synovial response to intra-articular metal debris

Purpose: Intra-articular metal debris is occasionally seen after completion of an endoscopic anterior cruciate ligament reconstruction. The purpose of this study was to examine the tissue response to intra-articular stainless steel metal debris in order to better understand its role in the inflammatory process. Type of Study: Animal case control study. Methods: Metal filings created from a Beath pin were injected into 10 rabbit knees. The opposite knee of each rabbit was used as a control. The animals were killed at 4 weeks. The knee joints were fixed, sectioned, stained, and examined under light microscopy. Results: Mild to moderate levels of synovial proliferation were observed in all knees injected with metal particles. There was no evidence of phagocytosis of the metal particles, nor was there any evidence of an inflammatory response. No articular cartilage damage was observed as a result of the metal debris. Conclusions: We believe that the few particles of stainless steel that may be produced during the reaming process in anterior cruciate ligament reconstruction do not pose a significant risk of inciting an inflammatory reaction or cause articular cartilage damage within the knee joint.     

The effect of normal saline irrigation at different temperatures on the surface of articular cartilage: an experimental study in the rat

Purpose: To determine, with an electron microscope, the effects of irrigation fluid at different temperatures on the surface of articular cartilage. Type of Study: Experimental in vivo study. Methods: With a scanning electron microscope, we investigated the effect of 2 hours of irrigation with normal saline at 3 different temperatures (4°C, 25°C, and 37°C) on articular hyaline cartilage in an in vivo rat knee model study. The contralateral knees were used as controls. We then compared the results. Results: Specimens irrigated with cold (4°C) fluid seemed to show the most uneven surface and apparent fibril exposure. Those irrigated with fluid close to body temperature (37°C) showed the most even surface without fibril exposure. Conclusions: Although further work is needed to evaluate the long-term effects, using warm irrigation fluid to maintain the intra-articular environment at a more physiologically normal temperature may help reduce damage to cartilage during irrigation.     

Mechanical strength of osteochondral junction.

The junction between articular cartilage and bone in adult humans was tested in shear to determine their bonding strength in relation to the tensile strength of full thickness articular cartilage and shear strength of underlying subchondral bone. The site of failures was also studied under light microscope and scanning electron microscope. The junction failed at a shear strength of 7.25 +/- 1.35 MPa (n = 18). On the other hand, the tensile strength of cartilage and shear strength of subchondral bone were 3.75 +/- 0.75 MPa (n = 17) and 2.45 +/- 0.85 MPa (n = 15) respectively. Light and scanning electron microscopic examination revealed that in fact, failures occurred at the tidemark. The ultrastructure of the tidemark suggested that this region might have structural weakness against mechanical stresses. The relevance of the results were discussed in relation to the osteochondral fractures in adult humans.     

In vivo gene delivery to articular chondrocytes mediated by an adeno-associated virus vector.

PURPOSES: (1) To investigate the efficiency of direct in vivo adeno-associated virus (AAV) vector-mediated gene transduction to chondrocytes in relation to normal and injured articular cartilage. (2) To evaluate the effects of ultra-violet light-activated gene transduction (LAGT) in chondrocytes in vivo. (3) To determine dissemination of active rAAV vector after intra-articular administration. METHODS: Rabbit knees with either normal or injured cartilage received an intra-articular injection with 1.5x10(12) infectious rAAV-eGFP particles. The right knees received rAAV-eGFP alone, whereas the left knees were given LAGT-treatment. The transduction efficiencies were determined at 1 and 3 weeks after infection by fluorescence-activated cell scanning. The occurrence of active shedding was monitored in serum and various tissues. RESULTS: After 1 week, 7% of the chondrocytes in normal cartilage were transduced by direct rAAV transduction technique. Chondrocytes in cartilage defects demonstrated higher transduction rates compared to chondrocytes in normal cartilage. LAGT increased the cellular eGFP expression in the internal zones to 12%, but did not have any effect in the external zones in defects. Finally, infectious particles were not detected in either serum or tissue samples. CONCLUSIONS: Direct rAAV-mediated gene transfer in vivo to articular chondrocytes is possible. LAGT improves rAAV transduction of chondrocytes in vivo but appears to have a very limited range of effect induction. Expression of eGFP was not determined in other tissues than synovium and cartilage in the treated joints.     

Intra‐articular injections of bone morphogenetic protein‐7 retard progression of existing cartilage degeneration

We investigated the effect of weekly intra‐articular injections of bone morphogenetic protein‐7 (BMP‐7) on prevention of progression of existing cartilage degeneration in an osteoarthritis model in rabbits. An anterior cruciate ligament transection (ACLT) model was used to create a progressive osteoarthritis model. BMP‐7 was intra‐articular injected weekly into the right knee and PBS into the left knee from 4 weeks after ACLT. Both sides of the knees were compared macroscopically, histologically, immunohistochemically, and by micro CT. Macroscopically, fibrillation in the femoral condyle was observed 4 weeks after ACLT. In the control knees, cartilage degeneration further progressed throughout the 12‐week period. In the BMP‐7 treated knee, osteoarthritis progression was milder than in the control knees. Histologically, safranin‐O staining was decreased in the surgical knees at 4 weeks. Obvious erosions in both medial and lateral condyles were revealed in the control knees at 12 weeks, while cartilage matrix was predominantly retained in the BMP‐7 treated knees. The macroscopic and microscopic OA score in the BMP‐7 treated knee was better than that in the control in each rabbit. Immunohistochemical analysis demonstrated that both type II collagen and BMP‐7 were more expressed in cartilage treated with BMP‐7. Micro CT analysis showed that osteophytes were smaller in the BMP‐7 treated knee compared to that of the control. Weekly intra‐articular injections of BMP‐7 inhibited progression of existing cartilage degeneration. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1502–1506, 2010     

Arthroscopy in spontaneous osteonecrosis of the knee.

Eight knees with spontaneous osteonecrosis were investigated through arthroscopy, and in seven the intra-articular findings were rechecked at arthrotomy performed one to two weeks later. Anbormalities in the articular cartilage were observed in detail under arthroscopy that could not be seen in roentgenograms. In the early stage of osteonecrosis, flattening and fissures in the articular surfaces, with and without formation of a cartilage flap, were useful findings in choosing intra-articular surgical procedures. In the late stage, free bodies such as cartilage plates in the joint and regeneration with fibro-cartilaginous tissue over the necrotic lesion were also important in selecting surgical treatment. In addition to roentgenographic classification of developmental stages in osteonecrosis, arthroscopy is necessary in differentiating osteoarthroses, in observing the articular surface of the femoral condyles, and in determining the stage of disease when roentgenography provided insufficient information to justify intra-articular procedures such as drilling and bone grafting.     

An experimental study on early changes of articular cartilages in subluxated patella of rabbits

The pathogenesis and clinical progress of chondromalacia patellae have not been well studied in experimental models. We, therefore, studied the patellar subluxation produced experimentally in rabbit knees. In this model, sequential changes of the patellar cartilage were followed by simple X-rays, CT, macroscopic, histologic and scanning electron microscopic examinations up to 32 weeks. Pathological changes extending from horizontal fissures in the middle to the deep layers of the patellar cartilage and thinning of the subchondral bone were found at about six weeks after operation. The early changes of chondromalacia patellae were mostly what is called the basal degeneration, which was presumably attributed to the shearing stress on the articular cartilage. The sequential changes were not very remarkable; none of the rabbits developed OA changes by thirty-two weeks after operation. From these findings, we have that the cartilaginous changes in chondromalacia patellae progress very slowly which probably result from the vigorous repair by the cartilage and also from the acquisition of adaptability on the patellofemoral joint.     

碱性成纤维细胞生长因子调节兔关节软骨细胞与包埋后聚乳酸体外培养的实验研究

目的 通过碱性成纤维细胞生长因子（bFGF）调节卵磷脂和多聚赖氨酸共同包埋的聚乳酸三维支架与兔关节软骨细胞的体外培养,观察bFGF对组织工程软骨细胞生长的调节作用,探寻软骨组织工程的适宜方法。方法将体外培养的第3代兔关节软骨细胞种植于卵磷脂和多聚赖氨酸包埋的聚乳酸三维支架,加入bFGF共同培养,行大体、倒置显微镜、扫描电镜及免疫组织化学观察,分析软骨组织的形成,并行统计学分析。结果通过bFGF调节的卵磷脂和多聚赖氨酸共同包埋的聚乳酸三维支架一关节软骨细胞复合物在培养过程中不仅能够保持其初始外形,而且能保持种植细胞稳定的三维均相分布,无细胞脱落现象。同时脆性亦逐渐降低,韧性增加,有弹性,表面湿润、光滑,培养2周后,逐渐形成富含Ⅱ型胶原,具有典型软骨组织结构的成熟工程化软骨。其细胞生长及胶原分泌量明显强于对照组,统计学分析有显著性差异。结论bFGF能够促进组织工程软骨细胞的增殖,并具有增强软骨细胞功能的作用。     

Induction of arthritis in healthy knee joints after intra-articular injection of the proteolytic enzyme elastase - An experimental investigation in the rabbit

AIM: Humoral aspects are being discussed in the initiation of arthritis. Therefore, the effects of the proteolytic enzyme elastase on the cartilage of knee joints in rabbits have been investigated. The enzyme was evaluated using activities comparable to those in post-traumatic knee joint hemarthrosis in humans. METHOD: Polymorphonuclear leukocyte elastase was injected into one of the knee joints of 10 rabbits. In 5 animals (first study group), joints were then immobilized with a cast for 6 weeks. In the other 5 (second study group), no immobilization was applied. In the first zero group (2 animals), 0.9 % NaCl was injected intra-articularly without immobilization, whereas in the second zero group (2 anmals) knees were immobilized for 6 weeks without prior injection. Thus, the effect of immobilization could be evaluated additionally. Joint specimens were then examined histologically and electron microscopically. RESULTS: There was clear evidence of elastase having severe destructive effects on cartilage regardless of additional joint-immobilization. In neither zero group was there prearthritic damage to the cartilage.CONCLUSION: To prevent the initiation of cartilage damage by humoral factors, early elimination of the pathological intra-articular effusion is necessary.     

不同浓度木瓜蛋白酶建立兔膝骨关节炎模型的比较研究

目的:比较不同浓度木瓜蛋白酶建立的兔膝骨关节炎模型,为研究骨关节炎病理及诊疗方法提供数据支持。方法:将60只新西兰大耳白兔随机分为4组,分别在1、3、5d右膝关节腔注射2%(低剂量组)、5%(中剂量组)、10%(高剂量组)(w/v)木瓜蛋白酶和0.03mol/L的L-半胱氨酸混合溶液0.1ml/kg(实验组),以及等量生理盐水(对照组)。整个实验过程观察兔状态并记录体重和膝关节宽度。首次注射后第2、4、6周将兔分批处死,取股骨髁、胫骨平台及滑膜做大体及组织病理学观察,并进行评分。结果:实验组均出现关节宽度增大、关节软骨变薄、基质分解破坏、滑膜增生等炎症及退行性改变,严重程度随浓度增高而增高,也随时间延长而发展;对照组无上述改变,二者比较差异有统计学意义。结论:兔膝骨关节腔注射2%、5%、10%木瓜蛋白酶和0.03mol/LL-半胱氨酸混合溶液0.1ml/kg可制备不同严重程度的骨关节炎模型,周期短,可重复且成功率高。     

Maturation-dependent change and regional variations in acoustic stiffness of rabbit articular cartilage: an examination of the superficial collagen-rich zone of cartilage

OBJECTIVE: The purpose of the study was to investigate maturation-dependent changes of acoustic (ultrasound) stiffness and other ultrasound features of articular cartilage in healthy rabbit knees. METHODS: Five groups of rabbits of various ages (3 weeks, 8 weeks, 6 months, 1 year, 2.5 years) consisting of five rabbits per group were examined. Signal intensity (index of stiffness), signal duration (index of surface irregularity) and interval between signals (index of thickness) of the ultrasound reflection from articular cartilage were examined at four sites: posterior lateral femoral condyle, posterior medial femoral condyle, lateral tibial plateau, and medial tibial plateau. The sites were observed macroscopically and microscopically with a light microscope and a polarized light microscope. RESULTS: At the lateral and medial condyles and the lateral tibial plateau, signal intensity was least in 3-week-old specimens. The intensity increased until 6 months or 1 year of age. At these sites, the signal durations and intervals between signals were least at the ages of 6 months or 1 year. At the medial tibial plateau, the intensity was the least at 2.5 years of age and the interval between signals was least at 3 weeks of age; there was no effect of age on signal duration. Cartilage surfaces of all specimens were smooth and no degenerative changes were macroscopically or microscopically evident. The surface brightness of cartilage under the polarized light microscope was consistent with signal intensity values. CONCLUSIONS: The response of articular cartilage to ultrasound was maturation-dependent. Acoustic properties differed from mechanical stiffness properties, which were determined using indentation. Ultrasound may detect properties of the surface collagen of the articular cartilage.     

Pathology of synovitis and hemophilic arthropathy

Hemophilia is an X chromosome linked disease characterized by an increased tendency to hemorrhage. Due to recurrent haemarthroses specific changes occur in synovium and cartilage. This process is called haemophilic arthropathy. The pathogenetic mechanisms involved are not precisely known. Current concepts, which are based on experimental in vitro studies and clinical experience, hold that the synovium becomes catabolically active because of the exposure to blood components and as a result induces cartilage destruction. A considerable amount of reports concerning blood induced joint damage suggest that synovial changes have a leading role in the development of the joint damage and therefore precede the changes in cartilage. However, there are also observations that question whether this is the only and the initiating mechanism of joint damage in hemophilia they hold that intra-articular blood has a direct harmful effect on cartilage before synovial changes and suggest that joint damage may occur before synovial inflammation is evident. Primarily there may be damage of articular cartilage with synovitis as a consequence. These studies show that synovitis is involved, but that it is not the only mechanism in the joint damage caused by intra-articular bleeding. These findings do not contradict the current concept of blood-induced cartilage damage in which synovial changes are thought to play an important role. Several pathological processes are possibly involved, some of them occurring in parallel and others sequentially. Possibly intra-articular blood first has an direct effect on cartilage, and then it affects the synovium. Thus, both processes occur in parallel, and while they influence each other they probably do not depend on each other. This concept resembles degenerative joint damage as found in osteoarthritis.     

Deformation of loaded articular cartilage prepared for scanning electron microscopy with rapid freezing and freeze-substitution fixation

To investigate the effect of joint loading on collagen fibers in articular cartilage, 45 knees of adult rabbits were examined by scaning electron microscopy. The knees were loaded at the patella with a simulated “quadriceps force” of 0.5-4 times body weight for 0.5 or 25 minutes, plunge-frozen, and fixed by freezesubstitution with aldehydes. Six knees were loaded for 3 hours and then fixed conventionally. Fixed tibial plateaus were examined and then freeze-fractured through the area of tibiofemoral contact, dried, coated, and examined by scanning electron microscopy to assess the overall deformation of the tibial articular surface and matrix collagen fibers. With tissue prepared by conventional fixation used as a standard, the quality of fixation was graded by light and transmission electron microscopy of patellar cartilage taken from half of the freeze-fixed knees. In loaded specimens, an indentation was present where the femur contacted the tibial plateau. The diameter and apparent depth of the dent were proportional to the magnitude and duration of the load; no dent was seen in the controls. The thickness of the cartilage at the center of the indentation was reduced 15-80%. Meniscectomy always produced larger deformations in otherwise equivalent conditions. Icecrystal damage to cells was evident by transmission electron microscopy and scanning electron microscopy, but at magnifications as high as ×30,000 the collagen fibrils prepared by freeze-substitution and conventional aqueous methods were identical. In loaded regions, the collagen matrix of the tibial cartilage was deformed in two ways: (a) radial collagen fibers exhibited a periodic crimp, and (b) in regions where an indentation was created by the femoral condyle, the radial fibers were bent, in effect creating tangential zone where none had existed before. The radial fibers apparently are loaded axially and buckle under normal loads.     

Articular cartilage adjacent to experimental defects is subject to atypical strains

We tested the hypothesis that articular cartilage adjacent to experimental osteochondral defects is not subject to unusual strains under load. A 2.5-mm drill hole was made in the medial femoral condyle of 15 knees from 10 adult rabbits. Experimental joints were loaded with simulated quadriceps force, then frozen under load and preserved by freeze-substitution fixation. Deformation in the region of the defect was evaluated by scanning electron and light microscopy and compared with nondrilled and nonloaded control knees. To simulate blood clot, alginate was placed into some defects before loading. In loaded knees, articular cartilage at the edge of the drill hole was abnormally flattened and folded into the defect. Opposing tibial cartilage or meniscus intruded into the femoral defect beyond the cement line. Alginate did not prevent incursion of opposing cartilage. In this standard drill-hole model, the articular cartilage defect is occupied by the opposing surface when a joint is loaded. Any tissue growing or surgically implanted in the defect is subject to loading and displacement, therefore complicating attempts to characterize the healing or regenerative potential in similar drill-hole models. Deformation of cartilage at the defect edge suggests load concentration or increased compliance. Either phenomenon would contribute to subsequent degeneration of the cartilage adjacent to defects.     

Early changes in the ground substance of articular cartilage in experimental hemarthrosis in dogs,measured by the fixed-charge density method

Summary This work presents early findings in the articular cartilage of dog knees in experimental hemarthrosis produced by injections of whole blood, red blood cells, white blood cells, serum, and iron citrate. The effect of coagulation and synovectomy was also studied. The fixed-charge density determination has proved to be a reliable, easy, and rapid method for finding—very early in the process—quantitative changes in cartilage ground substance. Changes in the proteoglycan (PG) content of articular cartilage matrix take place after a certain duration and intensity of hemarthrosis, after which loss of ground substance is repeatedly and consistently observed. After some time (6 months, and culminating after 14 months in our study) a reparative reaction takes place. In experimental hemarthrosis of longer duration this healing reaction seems to be overwhelmed by the degrading forces, with probable further evolution to the fullblown pathology. The cellular components of the blood seem to play a major role. Prophylactic synovectomy seems to be effective only in preventing further bleeding. Coagulation of the intra-articular blood does not alter its deleterious effect. Finally, a small iron ligand alone, although proven in vitro to penetrate into cartilage, is not capable of producing matrix changes.     

Histopathological study of the aging process in the human sacroiliac joint

It has not been well defined the morphologic characteristics of the aging process of the sacroiliac joints in man. We have, therefore, studied the sacroiliac joints of human subjects by light and scanning electron microscopy. The joints were collected from 55 Japanese fetuses and adult cadavers. The articular cartilages on both sacral and iliac sides were composed of hyaline cartilage in the fetuses and adults. In fetuses, loose fibrous strands were found in the articular space and appeared to represent a residue of the intermediate layer during formation of the articular space. In adults who were older than 30, the joints showed definite degeneration which was more severe on the cartilage at the iliac side than that of sacral side. Scanning electron microscopic observations supported the light microscopic findings. Fusion of the two articular surfaces was observed in 24% of the adult joints. These findings strongly suggest that the buffering capacity of the sacroiliac joints decreases with aging which appears to relate to the clinical symptom of low back pain.     

Effects of hyaluronan on periosteal grafts for large full-thickness defects in rabbit articular cartilage

We studied the effects of hyaluronan (HA) on chondrogenesis in periosteal grafts in rabbit knees to elucidate the effects of this agent in the repair of articular cartilage. Large full-thickness defects of the articular cartilage were created in the anteromedial part of the femoral articular surface of bilateral knee joints. Periosteal grafts were then harvested and sutured onto the defects. HA was injected in the right knee immediately after the operation and then once a week for 4 weeks (HA group). The same volume of saline was injected in the left knee in the control group. The animals were killed 2, 5, 8, and 12 weeks after the operation. Macroscopic and histological findings of the regenerated tissue were evaluated with a semiquantitative histological grading system. The total histological scores of the HA group were better than those in the control group at each time examination point. At 12 weeks, in particular, the scores for surface regularity and integration to adjacent articular cartilage were significantly better in the HA group than in the control group (P < 0.05). No significant differences were observed between the two groups in regard to the area healed (%). HA may have beneficial effects on the repair of large full-thickness defects of the articular cartilage with autologous periosteal grafts. Received for publication on Feb. 18, 1998; accepted on Oct. 20, 1998     

Hemoglobin stimulates the expression of matrix metalloproteinases, MMP-2 and MMP-9 by synovial cells: a possible cause of joint damage after intra-articular hemorrhage.

Intra-articular bleeding causes degradation of articular cartilage leading to joint disorders, but the mechanisms is not well understood. The present study examined the effect of hemoglobin on the ability of synovial tissues to produce plasminogen activators and matrix metalloproteinases that play important roles in the degradation of articular cartilage. Human Hb added to primary cultures of human knee synovial cells markedly increased fibrinolytic activity and gelatinolytic activity. The fibrinolytic activity was due to an increase in uPA activity. Western blot analysis and gelatin zymography indicated that the increased gelatinolytic activity was due to increased MMP-2 and -9. In order to know whether the effect of Hb on cultured synovial tissue is also true in in vivo system or not, rabbit hemoglobin was injected into rabbit knee joints. Coinciding with in vitro study, hemoglobin elicited considerable increase in fibrinolytic and gelatinolytic activity. The level of proteoglycan fragments in the hemoglobin-treated joint fluid was significantly elevated, indicating cartilage matrix degradation. Cartilage damage after hemoglobin treatment was also confirmed by histological study. These findings suggest that hemoglobin stimulates the secretion of uPA, MMP-2 and MMP-9 by synovial tissues, and raise a possible role of hemoglobin in joint damage after intra-articular bleeding.     

Survival of articular cartilage after controlled impact

Survival characteristics of forty-three specimens of living human bone and articular cartilage from the knees of eight renal-transplant donors were studied, using a drop-tower device. Autoradiography and light and scanning electron microscopy revealed no evidence of chondrocyte death or structural damage until stress levels of twenty-five newtons per square millimeter were reached, corresponding to strains on the order of 20 to 30 per cent and involving energy absorption of one millijoule per cubic millimeter. The data for strain rates of 500 and 1000 s-1 suggest that impact loads sufficient to fracture a femoral shaft of an automobile occupant are nearly sufficient to cause chondrocyte death and fissuring in the articular cartilage of either the knee or the hip if the load-bearing areas measure less than 500 square millimeters.