Internalization and intracellular processing of bone morphogenetic protein (BMP) in rat skeletal muscle myoblasts (L6).

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily capable of inducing bone and cartilage formation in ectopic extraskeletal sites and transducing their effects through binding to serine-threonine kinase receptors. In this study, the fate of 125I-labelled native BMP after binding to cell surface receptors on L6-myoblasts was examined with both continuous and intermittent exposure of the ligand. BMP was readily internalized in L6 cells at +37 degrees C, and the internalization reached a plateau in 2 h. Intracellular degradation of 125I-labelled BMP was established, and degradation products were also detected in binding buffer, indicating exocytosis of the processed ligands. BMP receptors were shown to be subject to acute down-regulation by the ligand, and receptors were completely recycled in 3 h. Hence, we conclude that BMP receptors, like receptors for various other polypeptide ligands, have the ability to mediate intracellular delivery and degradation of the ligand.

Time-related deviations of fibronectin and type I, II and III collagen on the interface between a hydroxyapatite disc and the rim of a calvarial trephine defect in rabbits.

To pursue the events around hydroxyapatite (HA) discs (diameter 9 mm), implanted in calvarial defects in rabbits (diameter 11 mm), immunohistochemical changes of fibronectin and type I, II and III collagen were quantitatively determined in the connective tissue-HA (CTHA) and host bone-HA (BHA) interface at 8, 12 and 16 wk postimplantation. A marked enhancement of type I collagen staining in the BHA interface was noted at the 12th and the 16th wk in comparison to the CTHA interface and connective tissue in the untreated control defect. However, one of the characteristics of the staining in the CTHA interface was the finding of exceptionally high fibronectin and type III collagen at the 8th and the 12th wk in contrast to the BHA interface and the untreated control defect. The change in these immunohistochemically determined compositions was probably due to an active proliferation of vascular components in the CTHA interface. Bone regeneration in the CTHA interface was parallel to an increase in type I collagen and a decrease in fibronectin and type III collagen from 8 to 16 wk. This study indicated that variances in composition or characteristics of connective tissue in CTHA interface have taken place ahead of bone morphogenesis. The time-related derivation of connective tissue matrix components in the BHA and CTHA interface was confined to the interaction of implanted HA with host tissues in contact with the implant.

Microscopic evaluation of bone-implant contact between hydroxyapatite, bioactive glass and tricalcium phosphate implanted in sheep diaphyseal defects.

In order to compare morphological discrepancies in bone-implant contact in vertebrates, cylinders of hydroxyapatite, bioactive glass and tricalcium phosphate were implanted in segmental defects of the tibia in sheep. Three types of visible bone-implant contact were observed microscopically at 4 months after implantation. The trabecular web-like bone-implant contact noted in tricalcium phosphate seemed superior to the disseminated patchy bone-implant contact in bioactive glass and the buttressed bone-implant contact in hydroxyapatite with respect to both bone ingrowth and bioresorption of the implant. Differences of physicochemical properties on the surface among the three kinds of bioceramic implants probably give rise to different types of bone-implant contact.

Morphological and biomechanical difference in healing in segmental tibial defects implanted with Biocoral or tricalcium phosphate cylinders.

To evaluate the effects of two bioceramics on bone regeneration during repair of segmental bone defects, Biocoral and tricalcium phosphate cylinders were implanted in osteotomized sheep tibial defects 16 mm in length and followed up for 16 weeks. In comparison with the TCP-implanted defect, a significant increment in area and density of external callus was quantified radiomorphometrically at 3 weeks, and a marked increase in maximal torque capacity, maximal angle of deformation and absorption of energy was demonstrated mechanically in the Biocoral-implanted tibia at 16 weeks after implantation. Better bone integration with the substratum was microscopically observed in Biocoral cylinders. With good osteointegration and biomechanical-performance, Biocoral seems to be superior to TCP in repair of segmental defects in weight-bearing limbs.

Permanent open shunt as a reason for impotence or reduced potency after surgical treatment of priapism in 26 patients.

A permanent open shunt as a cause of impotence or impaired potency after a shunt operation for priapism is an unusual situation. In this series we studied the persistence of an open shunt in 26 patients who had developed impotence or impaired potency after operative treatment for priapism. All patients had been examined by cavernosography on the suspicion of an open shunt, giving a positive finding in five of 26 cases, in all of which impotence was cured by closure of the shunt. In five patients without a permanent open shunt potency returned to normal only after 6-12 months.

Use of myoblasts in assaying the osteoinductivity of bone morphogenetic proteins.

A novel, time- and BMP-saving in vitro method for the detection and quantitation of bone morphogenetic protein (BMP) activity was developed based on the measurable effects of BMP on rat skeletal muscle myoblasts (L6). Calcium incorporation, stimulation of alkaline phosphatase activity and production of osteocalcin were used as markers of bone cell metabolism and on-going morphogenesis. The morphological change was confirmed by Chlorantine fast red and von Kossa staining. The response of various BMPs was purity-dependent and consistent with intramuscular implantations of the same materials. Neither TGF-beta1 nor insulin could induce the same actions. The data from this study indicate that at least in part in vivo implantations of BMP extracts can be replaced by in vitro measurement of osteoinductivity. Considerable saving of time, BMP and experimental animals can be achieved using cell culture conditions for the determination of bone-forming activity.

The role of transforming growth factor-beta on retarded osteoblastic differentiation in vitro.

Various matrix growth factors play important roles in the development and growth of cartilage and bone. Among them transforming growth factor-beta superfamily and especially bone morphogenetic proteins are known to be important factors, since they induce bone and cartilage formation in ectopic sites in vivo. We have previously shown that the human osteosarcoma cell line Saos-2 expresses molecules that in vivo induce new bone formation with asymmetric bone maturation. In this study we examined the role of Saos-2-conditioned medium in prolonged cultures of mesenchymal C3H/10T1/2 cells. The C3H/10T1/2 cells were cultured with Saos-2-conditioned medium for 28 days. We show that Saos-2-treated C3H/10T1/2 cells performed retarded osteoblastic differentiation when compared to recombinant BMP-2 and -4 induced differentiation. We further show that this retardation is due to excessive amounts of transforming growth factor-beta in Saos-2-conditioned medium. Our results also suggest that this model can well be used to study additional cofactors involved in retarded osteogenesis.

Image processing for femoral endosteal anatomy detection: description and testing of a computed tomography based program.

A computed tomography (CT)-based image processing computer program was developed for three-dimensional (3D) femoral endosteal cavity shape modelling. For the examinations 50 cadaver femora were used. In the CT imaging 30 axial slices were taken above and below the lesser trochanter area from each femur. Different image analysis methods were used for femoral cavity detection depending on the structure of the processed slice. In the femoral shaft area simple thresholding methods succeeded, but in the problem areas of the metaphyseal femur edge, detection operators and local thresholding were required. In contour tracking several criteria were used to check the validity of the border pixels. The results were saved as four output data files: (i) a file for the longest anteroposterior (ap), mediolateral (ml) and oblique diameters computed by a Euclidian method, (ii) and (iii) files for 2D and 3D data respectively, and (iv) a file for centre points of each slice. Finally, testing of the results and dimensions obtained from the image analysis were carried out manually by sawing the femora into 10 stipulated horizontal slices. The ap and ml dimensions were measured with a caliper ruler. The CT-based image processing yielded a peak distribution of dimensions with a negative difference to those obtained in manual measurements. The mean difference between the image processing and the manual measurements was 1.1 mm (+/-0.7 mm, +/-1 SD). The difference was highest in the proximal slices of the femora of group I (with lowest cortical thickness), i.e. 1.3 mm (+/-0.8 mm) and lowest in the distal slices of the femora from group III (with highest cortical thickness), i.e. 0.9 mm (+/-0.6 mm). The results are acceptable for further use of the program to study endosteal anatomy for individual femoral component selection and designing basis.

Aggressive granulomatous lesions associated with hip arthroplasty. Immunopathological studies.

The local immunopathological response was analyzed in six patients who had a revision of a total hip prosthesis because of an aggressive granulomatous lesion and in six patients who had a revision because of common loosening of the prosthetic stem. All twelve patients had had a total replacement arthroplasty for primary osteoarthrosis. All of the prostheses had been cemented. The aggressive granulomas consisted of well organized connective tissue containing histiocytic-monocytic and fibroblastic reactive zones. The granulomas were highly vascularized, and villous structures were observed at many sites. In contrast, the areas around the loose cemented stems were characterized by dense connective tissue. Immunohistological evaluation revealed that most of the cells in the aggressive granulomatous tissue were multinucleated giant cells and C3bi-receptor and nonspecific esterase-positive monocyte-macrophages. This cytological finding suggests a foreign-body-type reaction, compatible with the rapidly progressive lytic nature of the lesion that was shown radiographically. There was a clear-cut difference between aggressive granulomatosis and the more common lesion accompanying prosthetic loosening--namely, the relative lack of activated fibroblasts in granulomatosis. We suggest that granulomatosis involves an uncoupling of the normal sequence of monocyte-macrophage-mediated clearance of foreign material and tissue debris that is normally followed by fibroblast-mediated synthesis and remodeling of the extracellular matrix. We also suggest that aggressive granulomatosis in association with a cemented hip prosthesis is a distinct entity, not only clinically and radiographically, but also histopathologically.

Induction of new-bone formation in the host bed by human bone-tumor transplants in athymic nude mice.

UNLABELLED: Specimens of twelve osteosarcomas, five chondrosarcomas, one giant-cell tumor, and five extraskeletal soft-tissue sarcomas were transplanted into male athymic nude (nu/nu) mice. Survival of the transplant was determined by the volume-doubling time and the sex chromatin of the tumor cells obtained from two female patients. By these criteria and the similarity of the histological composition of the original tumor and the transplant, survival occurred in four of twelve osteosarcomas and four of five chondrosarcomas. Without any local infiltration of lymphocytes or plasma cells, or other evidence of cell-mediated immunity, the surviving tumors regressed by the fourth week after the operation. Transformed osteoblasts and osteoprogenitor cells were replaced by fibrous connective tissue or fibrogenic tumor-tissue cells. Osteocytes degenerated and disappeared from the lacunae. The one giant-cell tumor transplant survived, growing very slowly, but by the end of the first week after transplantation whorls of mononucleated cells appeared in sites previously occupied by multinucleated cells. Transplants of leiomyosarcoma, liposarcoma, and synovioma (one tumor of each) degenerated. One of two fibrosarcomas survived transplantation. The most striking reaction of the mouse host bed was to encompass six of twelve osteosarcomas and four of five chondrosarcomas in deposits of normal living cartilage, bone, and bone marrow. The incidence of new bone inducedy by living transplants was only slightly greater than by implants of freeze-dried killed osteosarcoma tissue. Not one of five extraskeletal sarcomas, living or dead, induced bone formation. These observations suggest that an osteoinductive agent is transmitted by some osteosarcomas and chondrosarcomas. This agent initiates differentiation of host mesenchymal cells into normal non-tumorous cartilage and bone, which later colonized by bone marrow. CLINICAL RELEVANCE: Our observations present experimental evidence of the origin of the envelope of normal non-tumorous bone that may surround tumorous bone. In 1926, Phemister recognized the clinical significane of this envelope as a pitfall in the differential diagnosis of malignant bone tumors, chondrosarcoma, myositis ossificans circumscripta, and other neoplasms. He emphasized the importance of examining the entire specimen for the distribution of deposits of tumorous and normal bone. The induction of normal bone formation in the host bed surrounding transplants of osteosarcomas and some chondrosarcomas (but not transplants of fibrosarcoma, liposarcoma, or leiomyosarcoma) is evidence of a specific tumor-cell characteristic. Thus, the bone inductive response is not an unspecific reaction to injury from expansion or of tumor growth but a biological response to tumor-cell products. Transplants of human malignant tumors growing in the thymus-deficient mouse can be treated by combinations of radiation, amputation, and new chemotherapeutic agents...

Radiographic changes of the revascularized femoral head.

Twenty-two necrotic femoral heads in young adults were radiologically followed-up after grafting with vascularized bone by comparing the preoperative and the postoperative state of the hip joint. Three parameters were observed and followed; the flattening of the femoral head; the degree of osteoarthrosis of the joint; and the degree of incorporation of the graft into the recipient bone. The results, expressed by index figures, showed that the femoral head flattened during the first two years postoperatively; that the maximal incorporation occurred during the same period of time; and that the appearance of postoperative osteoarthrosis was slow during the first year and increased subsequently. This numerical characterization of radiological findings allows systematic individual analysis after revascularization of the femoral head with bone grafts. It is also suited for comparisons between patients, between series of patients and of various treatment techniques. Furthermore, this quantification provides a numerical index that seems to correlate with the outcome of the treated hip joint.

Discitis following removal of intervertebral disc.

Thirty-three patients with discitis following removal of an intervertebral disc and treated at the Orthopaedic Hospital of the Invalid Foundation in Helsinki, Finland, are reported. The incidence of discitis in this series extending over a period of 16 years (1960-1975) was 0.75%. The appearance of the disease paralleled mainly the frequency of postoperative infections at the hospital in general. Two-thirds of the patients were treated conservatively, while additional surgical treatment was needed for the others. At average follow-up 7.5 years later, most patients showed advanced symptoms from the back, and increased degenerative changes were observed on roentgenograms. About one-half of the patients had retired from regular work. Preoperative prophylaxis, early diagnosis, and appropriate management of the disease is especially urgent to overcome and inhibit the consequences of postoperative discitis.

Lumbar isthmic spondylolisthesis in children and adolescents. Radiologic evaluation and results of operative treatment.

A clinical and radiologic follow-up study of a group of 75 children and adolescents, comprised of both boys and girls, who underwent spondylodesis for spondylolisthesis between the years 1979 and 1984 is reported. Sagittal rotation, lumbosacral joint angle, lumbar lordosis, wedging of olisthetic vertebrae, and the rounding of the upper sacrum showed considerable statistical correlation to the amount of slipping and accordingly should be noted when estimating the risk of progression of the spondylolisthesis. When the spondylolisthesis was accompanied by scoliosis, it was noted that seriousness of the former was closely correlated to that of the latter. Most patients profited by the operation, and solid fusion was achieved in almost all cases. The posterolateral spondylolysis performed using graft from the iliac crest, the interbody fusion technique, or their combination turned out to be the most reliable surgical methods. The combined technique was especially required in cases with a high degree of slipping.

Internal fixation of the fragment of osteochondritis dissecans in the hip using bone transplants. A report of two cases.

Two patients with osteochondritis dissecans of the hip were operated on and their case histories are presented. The relatively large foci were situated on the weight-bearing surface of the joint and the fragments were fixed using transplants of cortical bone. In both cases the focus healed, and the symptoms were relieved. At follow-up six and nine years later respectively, the clinical results were good and no signs of osteoarthritis had as yet developed.

Clinical outcome and survival of Link RS total hip prosthesis.

We report the clinical outcome and survival of 100 total hip arthroplasties with Link RS cementless hip prostheses after a mean of 63 months (51 to 93). The average preoperative Merle d'Aubigné hip score was 9.4 points and the average score at the latest follow-up was 15.3 points. Thirteen prostheses have been revised and seven await revision. Survivorship analysis based on intention to revise showed only 68% survival at seven years (95% CI 53% to 81%). The survivals of femoral and acetabular components verified to be loose at surgery were 78% and 89% respectively at seven years. A number of other patients have pain and it seems likely that there will be substantial reduction in these survival figures in the future.

Cementless revision of aggressive granulomatous lesions in hip replacements.

In 16 patients we used uncemented Lord prostheses at revision operations for aggressive granulomatosis after cemented hip arthroplasties; in 12 bone grafts also were used. In 13 hips the granulomatous lesions were multifocal, and in one the acetabular component was involved. There was no evidence of infection in any case: all the patients had normal ESR and CRP levels. The revision operation was performed on average 9.4 years after the primary replacement; the mean age at revision was 64 years. On radiographs, the bone around the prosthesis had consolidated by an average of 16 months. At follow-up, two to six years later (mean 3.5 years) there had been no recurrences, nine patients had an excellent Mayo hip score, five were good and two fair.

Aggressive granulomatous lesions after hip arthroplasty.

We reviewed 19 patients who presented with aggressive granulomatosis around the femoral stem after hip replacement. All had experienced stress pain and had required revision arthroplasty on average 8.8 years after the primary operation. Fifteen patients were men and four were women; none had rheumatoid arthritis. One patient had an uncemented Moore hemiprosthesis; the others all had cemented total hip replacements. When first detected, the granulomatous lesions were multifocal in 13 patients. The first granuloma was in the region of the lesser trochanter in 10, and near the tip of the stem in only two. Speed of growth varied but on average there was doubling of the area on anteroposterior films in 2.2 years (range 6 months to 4.6 years). Aggressive granulomatous lesions in replaced hips are a distinct condition, different from simple loosening or infection; the lesions may grow rapidly, so revision surgery is indicated soon after diagnosis.

New bone formation induced by injection of native reindeer bone morphogenetic protein extract.

BACKGROUND AND AIMS: Bone morphogenetic proteins (BMPs) are usually administered with a solid framing material during open surgery. In some instances, percutaneous administration of injectable BMP would be preferable. We tested the new bone-forming activity of injectable native reindeer BMP extract in the Balb/C mouse thigh muscle pouch model. MATERIALS AND METHODS: The injectable implants contained 6 mg of native reindeer BMP extract and either physiological saline (NaCl/BMP) or collagen (Gel/BMP). Corresponding implants without BMP served as controls. New bone formation was evaluated based on incorporation of Ca45 and radiographically three weeks after the injection into the mouse thigh muscles. RESULTS: None of the injections without BMP were able to induce new bone visible in radiographs, whereas the injections with BMP induced new bone effectively. There were no significant differences in the area of new bone (p = 0.247) and its density (p = 0.739) between the NaCl/BMP and Gel/BMP groups. Ca-45 incorporation was multifold in the NaCl/BMP and Gel/BMP groups compared to the controls (p = 0.000). No significant differences in Ca-45 incorporation (p = 0.739) between the NaCl/BMP and Gel/BMP groups were observed. CONCLUSION: Our results suggest that BMP can be administered percutaneously, and that collagen and physiological saline are equally good carriers of injectable implants of native reindeer BMP.

The role of autogeneic bone marrow in the repair of a skull trephine defect filled with hydroxyapatite granules in the rabbit.

For study of the effect of autogeneic bone marrow on the repair of skull defects filled with hydroxyapatite (HA) granules, 20 trephine defects 11 mm in diameter in 10 New Zealand rabbit skulls were made. Three defects were implanted with HA granules (HAg), and seven defects were implanted with HA granules mixed with autogeneic bone marrow (HAg/BM) from the femoral medullary canal. Autogeneic bone marrow (BM) was implanted in three defects, and seven defects were left unfilled. Histomorphometric quantitation of bone and connective-tissue ingrowth into defects showed that the area of new bone ingrowth in BM (70.3 +/- 8.4%) was significantly larger than that in HAg (34.4 +/- 3.9%) and in HAg/BM (24.0 +/- 5.1%) (P < 0.01 and P < 0.01, respectively). Immunohistologic staining detected fibronectin and collagen type III as the main components in the defects filled with HAg and HAg/BM. The osteoconductive capacity of HA granules was not stimulated by adding fresh autogeneic bone-marrow cells.

Low dosage of native allogeneic bone morphogenetic protein in repair of sheep calvarial defects.

A sheep skull trephine defect model was used to test the efficacy of allogeneic partially purified sheep bone morphogenetic protein (sBMP), extracted using a low-cost alternative technique based on 60% ammonium sulphate saturation of the guanethidine-HCI extract of pulverized bone matrix. Eight mg of partially purified sBMP was implanted in six 22-mm right-side sheep calvarial critical-size defects trephined in the diploë area using a midline incision; left-side defects implanted with an equal amount of type IV collagen served as controls. After 16 weeks the sheep were killed and the defects removed. Formation of new bone was evaluated using radiomorphometry and histomorphometry. The healing percentage in sBMP-implanted defects was 60.8 +/- 8.1% and in controls 49.8 +/- 6.7% (P < 0.05) as assessed by radiomorphometry. In cross-sectional histomorphometry, newly formed bone regenerated 50.9 +/- 15.1% in the defects with sBMP and 16.1 +/- 10.6% in controls (P < 0.01). The good result, considering the low dosage of sBMP, can be explained by the strong osteoinductivity and low immunogenicity of native allogeneic sBMP.