Which drugs should we test?
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Biomedical subjects
Publications and source records attributed to M W Elves.
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The effect of Indomethacin upon the process of fracture repair and osteogenesis in bone isografts has been studied in the rat. It was found that the drug had no significant effect upon new bone formation in heterotopic bone grafts. It had no effect upon the osteogenesis in repairing drill holes in 2 month old rats. A significant impairment of osteogenesis was detected, however, in older (6--9 months) animals given continuous treatment with Indomethacin from the week before fracture. This effect was not apparent if treatment was terminated on the day after induction of the lesion. There is histological evidence of increased fibrogenesis and decreased osteogenesis and remodelling in fractures in old rats given high dosages of Indomethacin.
Osteogenesis in subcutaneous, cancellous bone isografts in the rat was assessed at 16 days from the uptake of 85Sr. by comparing the uptake in grafts devoid of one or more cellular components, the contribution of each to osteogenesis was estimated. The per cent contribution of each of the osteogenetic elements was: endosteal lining cells together with marrow stroma, 60%; periosteal cells, 30%; free, hemopoietic marrow cells and osteocytes, no significant contribution. These findings are compared with those previously reported for cortical bone isografts and it appears that the rate of osteogenesis is 2.2 times higher in cancellous grafts than in cortical grafts of the same weight, but only 1.7 times higher when grafts of the same volume are compared. Osteogenetic and hemopoietic activities associated with marrow appear to arise from different groups of cells. It is shown how, with iliac bone marrow, these two groups of cells may be separated physically, the hemopoietic cells being removed by means of a jet of saline, and the osteogenetic cells by subsequent enzyme treatment plus a second jet of saline. The osteogenetic and hemopoietic elements were difficult to separate from bone marrow within the femoral diaphysis.
It has been shown in experimental animals that the living cells in a bone autograft can make an important contribution to osteogenesis. However, some common clinical techniques, such as the topical use of antibiotic powders on grafts or on the graft bed, are likely to damage or kill the cells. In this experimental study in rats, bone isografts dusted with chloramphenicol or methicillin powder or with Polybactrin spray before subcutaneous implantation produced little or no new bone over a period of two weeks whereas untreated, control grafts showed abundant osteogenesis, as did grafts pretreated with solutions of antibiotics. The effect of short-term storage of the grafts for 3 to 24 hours in air, saline or culture medium before implantation was also examined. Grafts stored in culture medium generally did as well as, or better than, fresh control grafts whereas immersion in saline inhibited osteogenesis. The importance of these results for clinical bone grafting is discussed.
Isografts of cortical bone were tranplanted subcutaneously in the rat and the rate of osteogenesis 12 to 14 days later was assessed by measurement of 85Sr uptake and by histology. Some grafts were implanted complete whereas others had had one or more of their cellular components (viz. periosteum, endosteum, osteocytes, marrow) removed by mechanical or enzymatic pretreatment. From an analysis of the differences in osteogenesis between grafts devoid of different combinations of cellular components, the contribution of each component to osteogenesis was determined. The results indicate that the endosteal lining cells and marrow stroma together produce more than half of the new bone, the periosteal cells contribute about 30%, the osteocytes possibly make a small (10%) contribution, and the free, hemopoietic cells of the marrow make no significant contribution. Evidence about the relative contributions to osteogenesis of graft and host cells is reviewed and the possible osteogenetic role of bone marrow is discussed.
Estimations were made of the amount of bone (histologically) and the rate of bone formation (85Sr incorporation) in the epiphyses of the knees of osteoarthrosis-prone (STR/ORT) and normal (CBA/ORT) mice. Though the bone was significantly thicker in the STR/ORT mice, this was not the cause of the articular degeneration. Bone sclerosis and cartilage breakdown were chronologically very closely related with perhaps the cartilage changes occurring initially. In male STR/ORT mice bone formation was depressed in the cancellous bone of the epiphyses as, unlike the normal mice, it was at the same level as the compact bone of the femoral shaft. As there was no elevation of the osteoblastic activity in knee joints with developing osteoarthrosis, it would appear that bone sclerosis associated with the disease was due to decreased osteoclasis.
A lymphocyte transformation test has been used to study metal sensitivity in patients receiving total joint replacements. In the case of nickel, very significant levels of transformation were obtained with lymphocytes from patients with positive patch tests for this metal. In the case of patch test negative recipients of total joint prosthesis an elevated index of transformation was found in seven out of 15. Transformation in the presence of cobalt was less striking and there was evidence of toxicity of the metal in doses used to obtain stimulation. In eight out of 12 patch test positive individuals a significant level of transformation was obtained. Two of the seven patients carrying joint prostheses who wwere also patch test negative gave an elevated index of transformation. The clinical implications of these results are discussed.
The effect of two doses of X-irradiation upon the osteogenesis in isografts or allografts of cancellous bone has been studied. Exposure of rats to 500 rads of X-irradiation caused a transient depression in osteogenesis in the skeleton. This depression was more marked, however, after 600 rads of X-ray. Five hundred rads of X-ray had no effect upon the level of osteogenesis in isografts 2 weeks after grafting, but did depress new bone formation in 3-week-old grafts. The higher dose of X-rays caused a more profound depression in new bone formation at both 2 and 3 weeks. Both 500 rads and 600 rads of X-ray had little effect upon the degree of first phase osteogenesis in H-1 disparate allografts. However, there was some improvement in late phase new bone formation in recipients treated with 600 rads. Both doses of X-rays markedly improved the amount of first phase osteogenesis in H-1 identical allografts. There was also a significant improvement in the new bone formation in grafts in the second phase. The significance of these findings is discussed.
The cell population within and around allografts of fresh, marrow-containing cortical bone has been studied. The grafts were implanted into intramuscular sites in rats and removed at intervals over a 4 week period for examination by light and electron microscopy to establish the sequence of changes at both the tissue and cellular levels. The tissue organization and the ultrastructural appearances of the cells found at different times after grafting are described; the possible relationships of the different cell types to each other and their likely roles in both the immunological and histogenic events are postulated and discussed. A comparison of the observations in the allograft and in the previously studied autograft suggests that the cells from the granulationtissue that become osteoblasts in the autograft differentiate into fibrocytes and giant cells in the allograft. Furthermore, the increased period of initial degeneration observed in the allograft (2 weeks) compared with the autograft (less than 1 week) may represent the destruction of early, graft-derived attempts at osteogenesis. This, together with the relative paucity of new bone in the allograft, suggests that some cells of graft origin may contribute to the early osteogenesis observed in the autograft.
By the use of modern techniques, the nature of the immunological response to bone and cartilage grafts is becoming clear. Fresh bone, whether cancellous or cortical, will elicit a cell-mediated immunological response; removal of the bone marrow has little effect in reducing immunogenicity. Antibodies against cellular components of the graft are detectable in the recipient only when host and donor have a disparity for the major histocompatibility (H) antigen. Treatment of bone grafts, for the bone bank, by freezing removes their immunogenicity with regard to antibody production but leaves them capable of stimulating the cellmediated immune response. Freeze drying, on the other hand, impairs immunogenicity for both types of responses. Cartilage, grafted alone, is probably non-antigenic as far as both immune responses are concerned and, although there have been a few reports of stimulation of CMI and antibody production by cartilage, these have not been confirmed. Cartilage cells do, however, possess antigens of the major H-antigen system. The cartilage graft is therefore antigenic but only feebly immunogenic, as the matrix proteoglycans protect the cells from the afferent arm of the immune response. Osteoarticular allografts, consisting of both bone and cartilage, sensitize the host due to their bone components. The effect of the immune response upon the bone allograft is to destroy the graft-derived first phase of osteogenesis which, in turn, leads to a poor or non-existent host phase of new bone formation in most allografts. The exact effector mechanism by means of which this destruction is brought about is not known. Bone grafts may be protected from the immune response by use of immunosuppressive measures. Cartilage enjoys a considerable measure of protection from immunological effectors by virtue of its matrix. If this breaks down then the cartilage can become permeable to antibodies. It is suggested that "lymphokines," produced by sensitized lymphocytes, may play some role in destroying the cartilage graft.
Sensitivity to chromium, cobalt, nickel, molybdenum, vanadium, and titanium was studied by patch tests in 50 patients who had received total joint replacements. Nineteen (38%) were sensitive to one or more of the metals. In 23 patients non-traumatic failure of the prosthesis had occurred, and 15 of these patients were sensitive to metal. Out of 27 patients with no evidence of prosthesis loosening, four were sensitive to nickel and cobalt or nickel only. Dermatological reactions occurred in 13 patients after surgery; in only eight, however, was there evidence of metal sensitivity. These findings indicate that metal-on-metal total joint replacements may sensitise the patient to metals contained in the prosthesis. Although there is a high incidence of prosthesis failure among metal-sensitive patients it remains uncertain whether the loosening causes the sensitisation or vice versa.
The degree of new bone formation in isografts and allografts has been assessed by a radiotracer technique. Allografts used have been either H-1-identical or H-1-disparate with the recipient. It was found that new bone formation is curtailed within the first 3 weeks after grafting. This curtailment was significantly greater in those donor-recipient combinations which were H-1-identical but disparate for many weak locus antigens than those in which H-1 disparity existed with only a few minor locus differences. There was no late phase (i.e., host-derived) osteogenesis in the combination with H-1 identity, but multiple minor antigen disparities. In isografts and H-1-disparate grafts significant late phase osteogenesis was found in about 30% of recipients. Resorption of the graft was found to be significantly impaired in both H-1-identical and disparate grafts in comparison with isografts. There was a significant negative correlation between weight of the graft and degree of osteogenesis in isografts during the first 2 weeks after grafting, and also during the later phase (6-8 weeks). A similar negative correlation was found during the late phase in H-1-disparate grafts with few minor antigen disparities, but no correlation was found at any time in H-1-identical grafts with multiple minor antigen disparities.
The pattern of new bone formation has been studied in isografts of fresh iliac bone and also in isografts of dead or irradiated iliac bone. Two phases of osteogenesis have been found in some fresh grafts. An early phase occurs during the first 3 weeks after transplantation, whilst the second phase is found after 8 weeks. The first phase is absent from non-viable grafts and it is therefore concluded that cells of the grafts are largely responsible for this early osteogenesis. The second phase, it is suggested, has a major host component, and may be due to induction of osteogenic potential in host mesenchymal cells. There may be some dependence of the second phase on the first but the extent is not clear. Removal of the bone marrow from the graft has little effect upon the first phase of new bone formation and it is suggested that surviving endosteal cells are the main participants in early osteogenesis.
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The influence of the presence of "passenger leucocytes" on the production of anti-H2 antibodies has been studied in mice receiving allografts of foetal small intestine, adult skin or intradermally injected spleen cells. It was found that the humoral immune response to foetal intestine (a tissue without passenger leucocytes) was identical temporarily to that elicited by skin allografts and these responses differed from that following injection of allogeneic spleen cells in that antibodies to solid grafts took longer to appear. The humoral immune response to small intestine grafts was not evident until several days after the onset of graft rejection as assessed morphologicallymanti H2 antibody production was not observed in thymus deprived recipients of foetal small intestine allografts or allogeneic spleen cells, and this suggests that the humoral immune response to transplantation antigens is thymus dependent.
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