Experimental Osteoarthritis in the Rabbit: A Study of 133Xenon Washout Rates from the Synovial Cavity

Synovial perfusion in 6 rabbit knees, with experimentally induced osteoarthritis (joint instability), was studied by recording the initial ¹³³Xe washout rates from the joint space. The Unstable, osteoarthritic knee was compared with the contralateral sham operated control knee at intervals of 6 to 96 weeks postoperatively. Within the first half year the ratio between the ¹³³Xe washout rates in the osteoarthritic and control joints was significantly increased. These findings were supported by the increased blood flow to the joint region, visualized by scintigraphy of osteoarthritic rabbits given ^99mTc-microspheres intracardially. However, methodological sources of error do not allow any conclusions regarding the much less increased ¹³³Xe washout rates found in advanced osteoarthritis.

The initially increased synovial blood flow coincided with the existence of joint effusion and the early development of osteophytes, all conditions supposed to be a consequence of posttraumatic synovitis. Attention is drawn to these pathogenic phenomena in studies dealing with the initial changes in experimental models of osteoarthritis and to a possible etiological significance.     

Changes in 35S-Sulphate Uptake in Different Tissues in the Knee and Hip Regions of Rabbits During Immobilization, Remobilization and the Development of Osteoarthritis

In order to observe phenomena accompanying the development of osteoarthritis of the rabbit knee, consistently appearing within 4—6 weeks as a consequence of immobilization of this joint, studies on ³⁵S-sulphate (Na₂³⁵SO₄) uptake were carried out. The investigation suggests that immobilization of the knee leads within 2-4 weeks to increased sulphate uptake in bone, cartilage and ligaments in the knee region and in the hip (not immobilized) of the same leg. The first marked changes in ssS-sulphate uptake were found in the ligaments and the articular cartilage as early as after 4 days of immobilization.     

Glycosaminoglycan Metabolism in Experimental Osteoarthritis Caused by Immobilization: The Effects of Different Periods of Immobilization and Follow-up

Glycosaminoglycan metabolism at different developmental stages of osteoarthritis provoked by immobilization was studied in rabbits. Tissue concentrations and the specific radioactivity of glycosaminoglycans (GAG) after a long immobilization (IZ) period (12 weeks) without follow-up, and a moderate IZ time (4-7 weeks) with a long follow-up period (average 22 weeks) were compared with the results obtained after a short IZ time (17 days). In tibial weight-bearing cartilage the specific ³⁵S-activity had increased and the galactosamine, glucosamine and uronic acid concentrations had decreased in all the IZ groups examined. In the marginal cartilage, the menisci and the collateral ligament, the specific radioactivity and GAG concentrations had decreased or were normal after a long IZ with or without follow-up, although they were markedly elevated after a short IZ time. The depletion of GAG correlated roughly with the IZ and follow-up times, except for periodically immobilized rabbits. In long-term “chronic” disease the specific radioactivities and GAG concentrations were depressed on the contralateral, non-IZ sides. The changes in the non-IZ knee are discussed. In studies of experimental osteoarthritis it is important to determine the developmental stage of the disease to be studied.     

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.     

Glycosaminoglycan Metabolism of the Medial Meniscus, the Medial Collateral Ligament and the Hip Joint Capsule in Experimental Osteoarthritis Caused by Immobilization of the Rabbit Knee

A study was made of glycosaminoglycan metabolism in experimental osteoarthritis caused by immobilization of the rabbit knee in extension. Samples from the medial meniscus, the medial collateral ligament of the knee and the hip joint capsule were obtained and analysed after 2, 6, 10, 17, 30 and 87 days of immobilization, samples from the mobile limb serving as controls.

The tissue concentrations of glycosaminoglycans were determined from measurements of hexosamine and uronic acid after prior papain proteolysis and subsequent purification. The uptake of ³¹S-sulphate (DPM/ug hexosamine) was used as an indicator of the synthesis rate of sulphated glycosaminoglycans. In both early and advanced immobilization osteoarthritis, the synthesis rate and the content of glycosaminoglycans were increased in all tissues.     

Histopathological Evaluation of Treatment with Chondroitin Sulphate for Osteoarthritis Induced by Continuous Immobilization in Rabbits

The aim of this study was to evaluate histologically the action of chondroitin sulphate in osteoarthritis experimentally induced by continuous immobilization. Fourteen young female Norfolk rabbits aged 2.5–3 months at the beginning of the experiment were divided into two equitable groups submitted to immobilization of the right knee for a period of 12 weeks. The treated group received 1.0 ml/animal/s.c. of 12% chondroitin sulphate, once a week for 12 weeks, and the untreated group did not receive any treatment. Two additional animals were not submitted to knee immobilization (sham group). Microscopical examination of knee preparations stained with haematoxylin–eosin and Masson trichrome showed lesions of both joints in treated and untreated groups, with no significant difference between the scores obtained for the right and left knees. Examination of preparations stained with picrosirius red showed collagen fibre alignment and misalignment in the right and left knees of the animals of all groups, but statistic analysis could not be performed. It was not possible to differentiate the proteoglycan concentration between limbs or groups (treated and untreated) by safranin O or toluidine blue staining. It was possible to conclude that the chondroitin sulphate was not able to reduce the histological changes induced by this osteoarthritis experimental model.     

The effect of hyaluronic acid on cartilage in the immobilized rabbit knee

Out of 30 adult rabbits, 20 had one knee immobilized with a plaster of Paris cast for 6 or 12 weeks, and 10 rabbits were used as untreated controls. Prior to immobilization, 10 knees were injected with high-molecular weight hyaluronic acid. The articular cartilage of the femoral condyles was studied by light microscopy, whereas that of the patella and tibia was analyzed biochemically. Degenerative changes of the articular cartilage similar to those seen in arthrosis were observed after 6 weeks. The intraarticular injection of hyaluronic acid did not prevent these changes; instead, the reparative processes seemed inhibited.     

The effect of hyaluronic acid on cartilage in the immobilized rabbit knee

Out of 30 adult rabbits, 20 had one knee immobilized with a plaster of Paris cast for 6 or 12 weeks, and 10 rabbits were used as untreated controls. Prior to immobilization, 10 knees were injected with high-molecular weight hyaluronic acid. The articular cartilage of the femoral condyles was studied by light microscopy, whereas that of the patella and tibia was analyzed biochemically. Degenerative changes of the articular cartilage similar to those seen in arthrosis were observed after 6 weeks. The intraarticular injection of hyaluronic acid did not prevent these changes; instead, the reparative processes seemed inhibited.     

Adverse effects on rabbit anterior cruciate ligament after knee immobilization: changes in permeability of horseradish peroxidase

Changes in the permeability of horseradih peroxidase (HRP) and the histology of the rabbit anterior cruciate ligament (ACL) caused by knee immobilization were studied. The knees of adult rabbits were immobilized for 2–8 weeks by a long leg cast with the knee flexed at 120°, and some joints were allowed to be remobilized for 4 weeks after 6 or 8 weeks’ immobilization. After 1 ml of HRP was injected into the knee joint and allowed to permeate into the ACL, the degree of permeation at the mid-portion of the ACL between femur and tibia was visualized with diaminobenzidine tetrahydrochloride. The ACL was examined histologically by light and transmission electron microscopy. Although the HRP permeated into almost the entire area of the transverse section of the non-immobilized normal ACL, as the period of immobilization increased, the extent of permeation gradually decreased. The decrease was significant from week 4 to week 6 and from week 6 to week 8. Degenerative change with necrosis of cells at the central area of the ACL was noted in the 6-week immobilization group, and the 8-week period of immobilization increased the extent of this degeneration. Although the permeation recovered in both the 6-week and 8-week immobilization groups 4 weeks after remobilization, there were fewer cells with a normal morphology in the central area in the 8-week immobilization group than in the control. These results suggest that long-term immobilization adversely affects the ACL. Received: 4 April 1997     

The Effect of Osteotomy and Cartilage Damage on Mitotic Activity: An Experimental Study in Rabbits

In 15 rabbits, osteotomy and osteotomy including cartilage damage were performed. With autoradiography (³H-thymidine) it was shown that only one knee in the osteotomy group had labeled chondrocytes in the tibial cartilage. in the knees with articular damage, labeled chondrocytes were found in the femur as well, which could be the result of a factor liberated from the damaged cartilage, a factor stimulating mitotic activity.     

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     

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 μCi 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.     

Effect of calcitonin in early and late stages of experimentally induced osteoarthritis. A histomorphometric study

OBJECTIVE: To investigate both prophylactic and therapeutic roles of salmon calcitonin on the articular cartilage of rabbit's knees. METHODS: Right knee instability was produced in 30 New Zealand white rabbits by sectioning the cranial cruciate ligament (CCL). Animals were separated into four groups: placebo prophylactic-stage group (n=6), killed 8 weeks post surgery, calcitonin prophylactic-stage group (n=6), treated immediately after surgery with salmon calcitonin and killed at 8 weeks, placebo therapeutic-stage group (n=9) killed at 16 weeks post surgery and calcitonin therapeutic-stage group (n=9), treated with salmon calcitonin from 8th to 16th week and killed at 16 weeks post surgery. A histomorphometric study was based on the morphological changes of the articular cartilage and subchondral bone (degeneration indexes), as well as the articular cartilage thickness, chondrocytes' arrangement and their metabolic activity (regeneration indexes). RESULTS: Calcitonin groups showed smoother articular surface, no or minimal signs of ulceration, smaller osteophytes, and less subchondral cystic formation than placebo groups. Normal distribution of chondrocytes or hypercellularity was noticed in areas of mild osteoarthritic (OA) changes in the calcitonin groups indicating regeneration activity. Periodic Acid Schiff's and Alcian blue staining were negative in the placebo groups while increased absorption in the calcitonin groups revealed high anabolic activity. CONCLUSIONS: In prophylactic stages salmon calcitonin seemed to inhibit the progression of osteoarthritis by increasing the layers of hyaline cartilage, restoring the cellular metabolism, and decreasing the volume of osteophytes. In therapeutic stages, the hormone had a healing effect by decreasing the subchondral cysts, regenerating the hyaline cartilage and restoring cellular metabolism. Both macroscopic and histological findings of this study supported the biochemical results of previous studies showing the therapeutic effect of calcitonin on osteoarthritis.     

Suture and immobilization of acute peripheral injuries of the meniscus in rabbits

A comparative study of suturing or nonsuturing with or without immobilization of acute peripheral injuries to the medial meniscus of rabbits is reported. Artificial injuries to both knees of 44 male China Blue-Violet rabbits were sutured or left unsutured. One knee joint of each rabbit was immobilized postoperatively for 6 weeks. The maximum breaking strength was measured, pathologic examinations were performed, and healed areas were measured. The results showed that there was greater healed area, higher maximum breaking strength, earlier matured scar tissue formation, and more regular fiber alignment in the treatment groups with simple immobilization of the knee joint. These results were superior to suturing plus immobilization, suggesting that external immobilization of the knee joint is the key to the treatment of acute peripheral injuries of the meniscus. It may be the procedure of choice for clinical orthopedists dealing with such injuries. The measurement of the meniscus healed area and maximum breaking strength and the use of new material with this method of immobilization were introduced by the authors.     

The effect of temporary exercising of a joint during an immobilization period: an experimental study on rabbits

The right hind limbs of adult rabbits were immobilized for 5 weeks. The rabbits were then allowed to move freely for 8 weeks. The rabbits of the control group were immobilized uninterruptedly. In a second group of rabbits the plaster was removed during the immobilization period 6 days a week for 5 minutes for careful exercise and in a third group of rabbits, for violent exercise. The temporary careful exercise was of no benefit and was sometimes harmful, and the violent exercise was usually injurious for the immobilized knees as regards the stiffening, thickening and the development of radiologically detectable degenerative changes.     

The early effects of joint immobilization on medial collateral ligament healing in an ACL-deficient knee: a gross anatomic and biomechanical investigation in the adult rabbit model.

In this study, the short-term effects of immobilization on joint damage and medial collateral ligament (MCL) healing were investigated in unstable, anterior cruciate ligament (ACL)-deficient knees in rabbits. Forty-six 12-month-old female New Zealand white rabbits were separated into three groups. Animals from each group had surgery on their right knees: group I, sham controls (n = 9); group II, complete transection of the ACL and removal of a 4 mm segment (gap injury) of MCL midsubstance with no immobilization of the limb (n = 19); and group III, same injuries to the ACL and MCL (as group II) but with immobilization of the limb (n = 18). No surgical repair of disrupted ligaments was performed. Left knees served as unoperated contralateral controls. All animals were allowed unrestricted cage activity until sacrifice in subgroups at 3, 6, and 14 weeks of healing when biomechanical properties of all MCLs were measured. All knee joints were systematically examined for gross evidence of damage to articular cartilage, menisci, and periarticular soft tissues. To monitor relative in vivo loads on injured limbs during healing, hindlimb weight bearing was assessed at biweekly intervals. Results indicated that animals in both groups II and III bore relatively lower loads (compared to preinjury values) on their injured hindlimbs. Mechanical testing of MCLs showed only minor changes in sham controls, while group II and III healing MCLs demonstrated significantly lower force and stress at MCL complex failure compared to contralateral controls. In specific comparisons of group III to group II animals, we noted that immobilization prevented joint damage over the early intervals studied. In addition, immobilization resulted in MCL laxity similar to contralateral control values but inhibited development of structural strength and stiffness in healing MCLs. These results suggest that in the rabbit, short-term immobilization of an ACL-deficient knee offers some advantages to the joint and to certain low load behaviors of the healing MCL, but it also results in a smaller quantity of scar tissue that is less able to resist higher loads. Longer-term studies involving remobilization are necessary before the effects of brief immobilization on joint damage and MCL healing in this ACL-deficient model can be fully defined.     

Osteoporosis increases the severity of cartilage damage in an experimental model of osteoarthritis in rabbits

OBJECTIVE: To evaluate the effect of osteoporosis (OP) in cartilage damage developed in an experimental model of osteoarthritis (OA) in mature female rabbits in order to investigate the relationship between OP and OA. METHODS: OA was experimentally induced by anterior cruciate section and partial medial meniscectomy in the left knee of 12 rabbits. OP was experimentally induced prior to OA in six rabbits by bilateral ovariectomy (OVX) and systemic corticosteroid administration during 4 weeks. Knees were evaluated with high resolution magnetic resonance imaging (MRI) before knee surgery to rule out any detrimental effect of corticosteroids on cartilage. Gross and microscopic cartilage changes were assessed 16 weeks after surgery in bilateral knees. Left knees were considered osteoarthritic or osteoarthritic plus osteoporotic. Right knees were used as osteoporotic and healthy controls, respectively. Bone mineral density (BMD) was measured with dual energy X-ray absorptiometry (DXA) at the lumbar spine, global knee and subchondral knee bone, and its variations correlated with cartilage abnormalities. RESULTS: MRI before knee surgery disclosed no cartilage or bone abnormalities in any of the studied groups. OP increased the severity of cartilage abnormalities in experimental knee OA significantly (P<0.05). Cartilage damage was inversely correlated with BMD variations measured at the lumbar spine (r=-0.74; P=0.015). BMD changes in global and subchondral knee bone also showed a trend to correlate inversely with cartilage damage. CONCLUSIONS: Prior induction of OP increases the severity of cartilage damage in experimental OA. Increase in cartilage damage correlates with bone loss. These findings suggest a direct relationship between OP and OA.     

Microvascular morphology of bone in arthrosis: Scanning electron microscopy in rabbits

The morphology of the bone microvasculature in rabbit arthrosis was studied by scanning electron microscopy (SEM) and related to changes in intraosseous pressure (IOP) and phlebographic appearance.

Unilateral gonarthrosis was induced in 34 rabbits by immobilization of one knee in extension with a plastic splint for 5 weeks. Bilateral IOP recording in a subgroup of 12 rabbits demonstrated hypertension in arthrosis of 22 ± 2.9 mmHg compared with 11 ± 4.7 mrnHg in control knees. lntraosseous phlebography in 13 rabbits showed a dilated vascular bed with prolonged clearance of contrast media. The morphology of normal microvasculature by SEM of intravascular methylmethacrylate casts in 9 rabbits was characterized by well-defined and well-demarcated sinusoids with only a few arteriovenous shunts, whereas in arthrosis substantial changes included fusion of sinusoids, leakage of cast material through the sinusoid walls, and development of numerous shunts.

The study demonstrated profound microvascular morphologic changes that may reflect a vascular genesis for some of the bone changes in arthrosis.     

Microvascular morphology of bone in arthrosis: Scanning electron microscopy in rabbits

The morphology of the bone microvasculature in rabbit arthrosis was studied by scanning electron microscopy (SEM) and related to changes in intraosseous pressure (IOP) and phlebographic appearance.

Unilateral gonarthrosis was induced in 34 rabbits by immobilization of one knee in extension with a plastic splint for 5 weeks. Bilateral IOP recording in a subgroup of 12 rabbits demonstrated hypertension in arthrosis of 22 ± 2.9 mmHg compared with 11 ± 4.7 mrnHg in control knees. lntraosseous phlebography in 13 rabbits showed a dilated vascular bed with prolonged clearance of contrast media. The morphology of normal microvasculature by SEM of intravascular methylmethacrylate casts in 9 rabbits was characterized by well-defined and well-demarcated sinusoids with only a few arteriovenous shunts, whereas in arthrosis substantial changes included fusion of sinusoids, leakage of cast material through the sinusoid walls, and development of numerous shunts.

The study demonstrated profound microvascular morphologic changes that may reflect a vascular genesis for some of the bone changes in arthrosis.     

Synovitis causes hypoxia and acidity in synovial fluid and subchondral bone

The purpose of the present study was to evaluate the environmental changes in synovial fluid and subchondral bone during synovitis in rabbits in which the knee joint on one side was subjected to a procedure causing instability; a traumatic synovitis rapidly developed. Three weeks following the procedure, partial pressure of oxygen (Po₂), partial pressure of carbon dioxide (Pco₂) and hydrogen ion concentration (pH) were measured in vivo in the juxta-articular bone and in the synovial fluid of both the normal and the affected sides. Mass spectrometry was used for simultaneous registration of Po₂ and Pco₂, while a monocrystalline antimony pH electrode was used for simultaneous measurement of pH in vivo. Hypoxia, hypercapnia and relative acidity were found in synovial fluid and subchondral bone of knees with synovitis. The metabolic environment of synovial fluid and subchondral bone was considerably changed at the 3-week stage of experimental osteoarthritis and this was probably secondary to regional venous congestion.