Effects of mechanical compression of a fibrous tissue interface on bone with or without high-density polyethylene particles in a rabbit model of prosthetic loosening.

BACKGROUND: The mechanisms leading to aseptic loosening of a total hip replacement are not fully understood. A fibrous tissue interface can be present around the implant. Hypothetically, component micromovements can compress this interface and cause increased fluid pressure according to biphasic models. We tested the hypothesis that compression of a fibrous membrane with or without the presence of high-density polyethylene particles leads to bone degradation. METHODS: A titanium implant was inserted in forty-five rabbit tibiae, and, after osseous integration was achieved, a fibrous tissue interface was generated. The animals were randomized to undergo a sham operation, treatment with compression of the fibrous membrane, treatment with high-density polyethylene particles, or treatment with both compression and particles. Morphometric analysis of the surrounding bone was performed on cryostat sections after Giemsa staining and staining of tartrate-resistant acid phosphatase activity. RESULTS: Forty specimens were available for analysis; five tibiae with an infection were excluded. After nine weeks, the controls showed vital bone, whereas the specimens treated with compression showed necrosis of bone and replacement of bone by cartilage in a discontinuous layer (p < 0.05 for both) but not fibrous tissue. Treatment with high-density polyethylene particles caused replacement of bone by fibrous tissue (p < 0.05) but not necrosis or cartilage formation. Compression combined with the presence of high-density polyethylene particles caused bone necrosis and loss of bone with replacement by cartilage and fibrous tissue (p < 0.05). CONCLUSIONS: In this in vivo study in rabbits, fibrous membrane compression led to bone necrosis and cartilage formation, possibly because of fluid pressure or fluid flow, whereas the presence of high-density polyethylene particles led to the loss of bone with replacement of bone by fibrous tissue. Cartilage formation may be a protective response to fluid pressure and/or fluid flow. Fibrous membrane compression may play an important role in the early stages of loosening of a total hip replacement.

Cathepsin K deficiency in pycnodysostosis results in accumulation of non-digested phagocytosed collagen in fibroblasts.

The rare osteosclerotic disease, pycnodysostosis, is characterized by decreased osteoclastic bone collagen degradation due to the absence of active cathepsin K. Although this enzyme is primarily expressed by osteoclasts, there is increasing evidence that it may also be present in other cells, including fibroblasts. Since fibroblasts are known to degrade collagen intracellularly following phagocytosis, we analyzed various soft connective tissues (periosteum, perichondrium, tendon, and synovial membrane) from a 13-week-old human fetus with pycnodysostosis for changes in this collagen digestion pathway. In addition, the same tissues from cathepsin K-deficient and control mice were analyzed. Microscopic examination of the human fetal tissues showed that cross-banded collagen fibrils had accumulated in lysosomal vacuoles of fibroblasts. Morphometric analysis of periosteal fibroblasts revealed that the volume density of collagen-containing vacuoles was 18 times higher than in fibroblasts of control patients. A similar accumulation was seen in periosteal fibroblasts of three children with pycnodysostosis. In contrast to the findings in humans, an accumulation of internalized collagen was not apparent in fibroblasts of mice with cathepsin K deficiency. Our observations indicate that the intracellular digestion of phagocytosed collagen by fibroblasts is inhibited in humans with pycnodysostosis, but probably not in the mouse model mimicking this disease. The data strongly suggest that cathepsin K is a crucial protease for this process in human fibroblasts. Murine fibroblasts may have other proteolytic activities that are expressed constitutively or up regulated in response to a deficiency of cathepsin K. This may explain why cathepsin K-deficient mice lack the dysostotic features that are prominent in patients with pycnodysostosis.

Mechanical compression of a fibrous membrane surrounding bone causes bone resorption.

Early micromovement and migration of a prosthesis of a hip or knee predicts late clinical loosening of the prosthesis. Such migration is likely to be associated with mechanical compression of the fibrous membrane interpositioned between bone and prosthesis during movement. Compression of the fibrous membrane by loading may lead to locally high fluid pressures reaching the underlying bone tissue. It has been established that high fluid pressures can lead to bone resorption. This resorption may eventually lead to clinical loosening of the prosthesis. We developed an experimental model to study the effects of compression of a soft tissue layer located between a titanium implant and cortical bone. In twelve rabbits, this device was implanted in the proximal tibia and allowed to osseointegrate. Next, a layer of soft tissue was allowed to form between titanium and bone. Subsequently, in six rabbits a cyclic load of 60 times in 2 min per day during 2 weeks was applied, leading to compression of the interpositioned soft tissue layer only. In the other six rabbits no load was applied. In all six loaded specimens, osteocyte death and bone resorption was observed underneath the area where compression of the fibrous membrane was exerted to a depth exceeding the amplitude of the loading device. Furthermore, formation of fibrocartilage was observed in the loaded areas. Formation of fibrocartilage, osteocyte death or bone resorption did not occur in the controls. Our results indicate that compression of a fibrous membrane surrounding bone can lead to resorption of the underlying bone primarily because of osteocyte death and subsequent resorption of dead bone tissue. This may explain the observation that early migration of a hip or knee prosthesis is predictive of clinical loosening of the prosthesis.

In vivo treatment of rats with unfractionated heparin (UFH) or low molecular weight heparin (LMWH) does not affect experimentally induced colon carcinoma metastasis.

Recent randomized trials have suggested that treatment with low molecular weight heparin (LMWH) improves survival of cancer patients with venous thromboembolism, as compared to treatment with unfractionated heparin (UFH). Experimental studies have shown that UFH has activities besides its anticoagulant function which may affect progression of malignancy, including stimulation of new blood vessel formation. In contrast, LMWH has been suggested to inhibit angiogenesis. In the present study, we compared quantitatively the effects of treatment with UFH, LMWH or placebo on the development of experimentally induced colon carcinoma metastases in rat liver and on tumor-associated angiogenesis. It is shown that UFH and LMWH in therapeutic dosages neither affect development of metastases nor tumor blood vessel formation in this animal model. These results indicate that heparins do not affect colon cancer metastasis in liver. Further studies in other animal models are required to establish the mechanisms by which heparins potentially affect cancer.

Dietary omega-3 polyunsaturated fatty acids promote colon carcinoma metastasis in rat liver.

The effects of omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs on the development of experimentally induced colon carcinoma metastasis in rat liver were investigated quantitatively in vivo. Rats were kept on either a low-fat diet or on a fish oil (omega-3 PUFAs) or safflower oil (omega-6 PUFAs) diet for 3 weeks before the administration of colon cancer cells to the portal vein, until they were sacrificed at 1 or 3 weeks after tumor transplantation. At 1 week after transplantation, the fish oil diet had induced 7-fold more metastases (in terms of number and size) than had the low-fat diet, whereas the safflower oil diet had not affected the number and total volume of metastases. At 3 weeks after tumor transplantation, the fish oil diet and the safflower oil diet had induced, respectively, 10- and 4-fold more metastases (number) and over 1000- and 500-fold more metastases (size) than were found in the livers of rats on the low-fat diet. These differences were sex independent. Immunohistochemical analysis revealed that the immune system in the liver (Kupffer cells, pit cells, T cells, newly recruited macrophages, and the activation state of macrophages) did not play a significant role in this diet-dependent outgrowth of tumors. In conclusion, omega-3 and omega-6 PUFAs promote colon cancer metastasis in the liver without down-regulating the immune system. This finding has serious implications for the treatment of cancer patients with fish oil diet to fight cachexia.

Fluid pressure causes bone resorption in a rabbit model of prosthetic loosening.

High fluid pressures can be present throughout the effective joint space after total hip replacement and can extend locally to the bone implant interface. A new experimental implant model was developed to study whether local fluid pressure can induce osteolysis at a bone implant interface. Twelve mature rabbits received a titanium implant that was allowed to osseointegrate before a fluid pressure of 150 mm Hg was applied to a specific area of the titanium bone interface in six of the animals. Osteolysis occurred in all six implants exposed to the fluid pressure. In two of these animals, no measurable flow occurred. In both animals a localized osteolytic lesion developed with evidence of osteocyte death in the surrounding cortical bone. In four animals, fluid flow away from the lesion occurred after several days that corresponded to either the formation of a canal through the cortical bone or extended resorption of the surrounding cortical bone. In some of these specimens, the microscopic appearance was similar to that of prosthetic loosening: high numbers of macrophages were observed containing intracellular bone particles. No resorption was found in any of the six control animals. The authors' results point to the possibility that all features of prosthetic loosening that are ascribed to particle activation can be explained alternatively by effects of fluid pressure.

Short periods of oscillating fluid pressure directed at a titanium-bone interface in rabbits lead to bone lysis.

Fluctuating high fluid pressures have been reported in pseudojoints after total hip arthroplasty, and may be present throughout the effective joint space. When the pressure extends locally to the bone implant interface, we hypothesized that it might have led to bone resorption. We developed an experimental implant model to study whether oscillating fluid pressure, applied during 2 hours a day, can lead to osteolysis at the bone implant interface. 12 mature rabbits received a titanium implant, which was allowed to osseointegrate. Thereafter, fluid pressure was applied to a specific area of the titanium bone interface at the periosteal side of the cortex in 6 of the rabbits. The pressure, applied during 2 hours a day for 14 days, oscillated between 70 and 150 mm Hg, with a frequency of 0.1 Hz. Bone resorption was not found in any of the control animals, but it occurred under 4 implants exposed to fluid pressure (p = 0.03; Fisher's exact test). Localized osteolytic lesions had developed, with evidence of osteocyte death in the surrounding cortical bone. In 1 of the 2 specimens without osteolysis, there was evidence of fluid leakage into the soft tissues. In 4 specimens (3 with and 1 without osteolysis), bone formation was observed at the endosteal side opposite to the pressure zone. This did not occur in the controls. No signs of infection were observed. Our findings indicate that oscillating fluid pressure, even when present only during short periods, can lead to osteolysis and may be a cause of prosthetic loosening. Endosteal bone apposition may be a result of the interstitial flow that was created, giving false signals of mechanical load to the osteocytes.

Benign cellular responses in rats to different wear particles in intra-articular and intramedullary environments.

We examined the cellular responses to various particles injected into the knees and the intramedullary femoral cavities of rats in the presence of polymethylmethacrylate (PMMA) plugs. The intra-articular particles were mainly ingested by synovial fibroblasts. Increased numbers of macrophages were not detected and there was only a slight increase in synovial thickness. Cellular responses in the intramedullary space were similarly mild and bone resorption around the PMMA plug did not occur. Bone formation was inhibited only by polyethylene particles. In contrast to current views, our study shows that wear particles per se do not initiate bone resorption.

Kupffer cells and pit cells are not effective in the defense against experimentally induced colon carcinoma metastasis in rat liver.

The present study was performed to investigate processes involved in circumvention of the immune system by advanced stages of tumor growth in the liver. The efficacy of Kupffer cells and pit cells against cancer cells was tested in vivo in an experimental model of colon carcinoma metastasis in rat liver. Liver tumors were induced by administration of CC531 colon cancer cells into the vena portae. After 3 weeks, livers were obtained and partly fixed for electron microscopic procedures or frozen in liquid nitrogen for enzyme and immunohistochemistry at the light microscope level. The activation status of Kupffer cells was studied by expression of Ia-antigen (MHC class II) and by measurement of glucose-6-phosphate dehydrogenase (G6PDH) activity in the cells in situ as a measure of production of reactive oxygen species. Large numbers of Kupffer cells were found in liver parenchyma surrounding colon carcinomas when compared with levels in control livers, but these cells were not activated. Large numbers of activated monocytes and macrophages, cytotoxic T cells but only a few pit cells were found to be recruited to the boundary between liver parenchyma and tumors or their stroma. In those areas where cancer cells invaded liver parenchyma, only newly recruited macrophages and some Kupffer cells were present but few cytotoxic T cells or pit cells were found. The low activation status of Kupffer cells both in terms of production of reactive oxygen species and Ia-antigen expression and the absence of significant numbers of pit cells at tumor sites suggest that Kupffer cells and pit cells do not play a significant role in advanced stages of tumor growth. High levels of prostaglandin E2 were detected in the parenchyma of livers containing tumors and transforming growth factor beta was detected in the stroma of the tumors, therefore suggest that cytotoxicity of newly recruited monocytes, macrophages and cytotoxic T cells may be limited in these stages because of local production of these immunosuppressive factors.

Ultrastructural localization of activity of phosphatases by low temperature incubation of unfixed cryostat sections.

In the present study, we demonstrate the activity of several phosphatases ultrastructurally in long-term (up to 24 months) cold-stored (-80 degrees C) rat tissues. Phosphatase activity was histochemically studied with the use of unfixed cryostat sections in combination with low temperature (4 degrees C) incubation conditions in order to prevent inactivation of enzyme activity and to limit the loss of ultrastructure. 5'-Nucleotidase activity was observed at plasma membranes, mainly at bile canalicular membranes of hepatocytes in liver. Thiamine pyrophosphatase activity was detected not only in trans side cisternae but also in medial and cis side cisternae of Golgi complexes in the parotid gland. Glucose-6-phosphatase activity was localized in endoplasmic reticulum as well as at the outer membrane of the nuclear envelope. Acid phosphatase reaction product was found in lysosomes. Furthermore, the localization patterns of 5'-nucleotidase and thiamine pyrophosphatase activity were compared with those obtained after different fixation procedures such as immediate chemical fixation of tissues or fixation of tissues after freezing and thawing. The results showed similar localization patterns of these enzymes after the different pretreatments. However, with respect to the ultrastructural morphology, some damage was observed in unfixed material after incubation. It can be concluded that the procedure described here enables ultrastructural localization of activity of phosphatases in long-term cold-stored tissues. This procedure will be useful for a retrospective study on archival material when histochemical parameters are needed.

Demonstration of 5'-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure.

In the present study a technique was developed to demonstrate 5'-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4 degrees C rather than at 37 degrees C. The optimized incubation medium contained 300 mM Tris-maleate buffer, pH 7.2, 5 mM adenosine monophosphate as substrate, 30 mM cerium chloride as capturing agent for liberated phosphate, 10 mM magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5'-nucleotidase activity were obtained when incubations were performed at 37 degrees C and 4 degrees C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37 degrees C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8 micrograms. 5'-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37 degrees C than at 4 degrees C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37 degrees C, in comparison with that at 4 degrees C, were the appearance of multi-lamellar structures at bile canaliculi at 37 degrees C. We conclude that the present method is valid for the demonstration of 5'-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.