The effect of different mineral frames on ectopic bone formation in mouse hind leg muscles induced by native reindeer bone morphogenetic protein.

INTRODUCTION: Bone morphogenetic proteins (BMPs) require carrier material for slow release and framing material for osteoconduction. MATERIALS AND METHODS: The effect of a frame on early bone formation induced by partially purified native reindeer BMP in composite implants containing 3 mg of BMP, type IV collagen and tricalcium phosphate (TCP/Col/BMP) or hydroxyapatite (HA/Col/BMP) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA/Col/BMP) or biocoral (NC/Col/BMP) was evaluated using a mouse hind leg muscle pouch model. Collagen with native reindeer BMP (Col/BMP) and corresponding implants without native reindeer BMP served as controls. Evaluation was done by incorporation of 45Ca, radiographically and histologically 3 weeks after the implantation. RESULTS: None of the implants without native reindeer BMP were able to induce new bone visible on radiographs. The area of new bone formation in the Col/BMP (p=0.026) and TCP/HA/Col/BMP (p=0.012) groups was significantly greater than in the TCP/Col/BMP group. The optical density of the new bone area was significantly greater in the TCP/HA/Col/BMP group than in the TCP/Col/BMP (p=0.036) or Col/BMP (p=0.02) groups. 45Ca incorporation was many times greater in all the groups containing native reindeer BMP than in the corresponding groups without BMP. In the Col/BMP (p=0.046) and TCP/HA/Col/BMP (p=0.046) groups, 45Ca incorporation was significantly greater than in the TCP/Col/BMP group. No significant differences were found in any parameters between HA/Col/BMP and NC/Col/BMP groups and the other BMP-containing groups. CONCLUSIONS: Hydroxyapatite, biocoral and biphasic tricalciumphosphate-hydroxyapatite are equally good as framing material for native reindeer BMP, while tricalciumphosphate is somewhat worse. Osteoinduction of native reindeer BMP works well with collagen alone.

Influence of ethylene oxide sterilization on the activity of native reindeer bone morphogenetic protein.

We studied the effects of ethylene oxide sterilization (Steri-Vac 4XL, temperature 29 degrees C, exposure time 4 h 10 min, ethylene oxide concentration 860 mg/l) on the osteoinductivity of partially purified native reindeer bone morphogenetic protein (BMP) in a hind leg muscle pouch model of male NMRI mice. BMP was administered in implants containing 3 mg in a collagen carrier. Implants without sterilization and without BMP served as controls. New bone formation was evaluated based on the calcium yield, radiographic and histological examination 3 weeks after implantation. The implants without BMP were not able to induce new bone visible in radiographs. In the sterilized BMP group, the mean area of new bone was 35% ( p=0.004) and density 32% ( p=0.000) smaller than in the nonsterilized group. Calcium yield was 20% lower in the sterilized group than in the nonsterilized group, but this difference was not significant ( p=0.22). It was many times lower in the group without BMP than in the above-mentioned groups ( p=0,001). We conclude that ethylene oxide gas sterilization reduces the bone-forming activity of native reindeer BMP by one third.

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.

Composite implant composed of hydroxyapatite and bone morphogenetic protein in the healing of a canine ulnar defect.

BACKGROUND AND AIMS: Hydroxyapatite (HA) has been considered as a carrier material for bone morphogenetic proteins (BMP). The aim of this study was to evaluate the capacity of a composite implant of HA and native bovine BMP to heal a 2 cm segmental defect in the canine ulna. MATERIALS AND METHODS: A composite HA+BMP implant was compared with plain HA implants and cortical autografts. The fixation was accomplished with an intramedullary Kirschner wire. The bone union was evaluated by X-rays taken at operation and after 3, 6, 9, 12, 16, 25, 35 weeks and by histology and mechanical torsion tests. RESULTS: HA implants were not able to produce complete bone union even with BMP. There was some bridging between the implant and the bone in the defects treated with either plain HA or HA+BMP implant, the bridging being slightly more pronounced with HA+BMP. The autografts showed a significantly better capacity to heal the defect. The HA implant did not resorb markedly during the study. There was no significant difference in mechanical strength between the HA and HA+BMP groups. CONCLUSIONS: HA was not an adequate bone substitute material in this study model, and BMP was not able to enhance sufficiently the poor capacity of HA to heal canine ulnar defects.

Bovine bone implant with bovine bone morphogenetic protein in healing a canine ulnar defect.

Xenograft is considered an alternative material for bone transplantation, but its bone healing capacity is inferior compared to that of autografts and allografts. Here, we tested whether bone morphogenetic protein (BMP) addition enhances the suitability of demineralized xenogeneic bovine bone for bone grafting in dogs, and whether xenogeneic bone is a suitable carrier material for BMPs. The capacity of demineralized bovine bone implants, with and without native partially purified bovine BMP, to heal a 2-cm ulnar defect was determined in six dogs over a follow-up time of 20 weeks. No instances of bone union were seen, but there was slightly more bone formation in the xenografts with BMP, though the difference was not statistically significant. The ulnas treated with an implant with BMP were also mechanically stronger, but the difference was not significant. Computed tomography scans showed no differences in the implant area in bone density, bone mineral content, or bone cross-sectional area. It is concluded that native, partially purified BMP does not sufficiently improve the suitability of bovine demineralized xenografts as a bone substitute material for dog. Demineralized xenogeneic bone does not seem to be a feasible carrier material for BMP.

Native bovine bone morphogenetic protein improves the potential of biocoral to heal segmental canine ulnar defects.

We studied the effect of a composite implant consisting of coral and native bovine bone morphogenetic protein (BMP) on the healing of 2 cm segmental defects in the canine ulna. Plain coral and cortical autograft bone implants were used as controls. The fixation was temporary for 9 weeks with an intramedullary Kirschner wire (6 ulnas with a composite implant of coral and BMP, 6 with plain coral and 6 with an autograft) or a plate and screws (3 ulnas with a composite implant and 3 with plain coral). X-rays were taken at 3, 6, 9, 12, 16, 26 and 36 weeks, and mechanical torsion tests were performed at the end of the study. The score for bone formation and bone union evaluated from radiographs was significantly higher in the composite implant group than in the plain coral group at 16 weeks, but the score was even higher with autografts. BMP accelerated the resorption of the coral implant. The mechanical strength of the composite implants was higher than that of the bones with a plain coral implant (P < 0.05), while the mechanical strength of the coral implants, even with BMP, was significantly lower than the strength of autografts (P < 0.01). In conclusion, BMP enhanced the capacity of a coral implant to heal a segmental ulnar defect by increasing bone formation, but the effect of this combination was not as good as that of a cortico-cancellous autograft.

Adequate blood flow is essential for reinnervation in neurovascular skin flaps.

The effect of arterial and venous ischaemia on reinnervation of skin flaps after transsection and resuture of the epigastric nerve was investigated in rat groin flaps. The results were compared with those in corresponding flaps with adequate blood flow. Arterial or venous ischaemia was induced by ligation of the epigastric artery or vein. The reinnervation of the flaps was studied after a 20-week healing period using specific antisera for calcitonin gene-related peptide (CGRP) in sensory nerves, and neuropeptide Y (NPY) in adrenergic nerves. Arterial ischaemia clearly and significantly hampered reinnervation. Venous ischaemia was even more harmful and practically no regenerated nerves were detected in the flaps. We conclude that adequate blood flow is critical for sensory and adrenergic reinnervation in skin flaps.

Intermediate-term clinical and radiographic results of Synatomic and AGC knee prostheses.

This study compared two total knee prostheses to determine whether the clinical and radiographic outcomes were different, focusing primarily on the patellofemoral articulation. The study group was comprised of 75 Synatomic (short-stemmed, anatomic VF type) and 79 AGC 2000 (universal, nonanatomic) prostheses. Patients underwent follow-up for an average of 63 and 50 months, respectively. At latest follow-up, the mean knee joint score was 84.4 in the Synatomic and 86.5 in the AGC group. Mean knee function scores were 63.5 and 63.4, respectively. No statistically significant difference was noted between the two prostheses.

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.

Extraction and characterization of native canine bone morphogenetic protein (cBMP) qualified with osteoinductivity.

Bone morphogenetic protein (BMP) was extracted from canine bone matrix, partially purified and tested for osteoinductivity. A radiographically and histologically detectable ectopic bone formation was induced by 6.0 mg canine (cBMP) in muscle pouch of BALB mouse at 21 days post implantation. Characterization of the cBMP preparation by a gel filtration chromatography defined that the material consisted of proteins or protein complexes with molecular weights from 4 to 120 kD. Isoelectric focusing showed that the molecules were acidic with isoelectric points of 4.6-5.6.

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.

Bone morphogenetic protein in bone neoplasms: comparison of different detection methods.

The presence of bone morphogenetic proteins (BMPs) in 13 primary bone neoplasms was investigated with conventional bioassay method and immunohistochemically using antiserum against highly purified mixture of bovine BMPs as antibody. In conventional bioassay after implantation of lyophilized bone tumor tissues into mouse muscle pouches 9/13 samples turned out positive by radiography and 10/13 histologically. By immunohistochemical staining, using the avidin-biotin-peroxidase method, signs of BMPs could be verified in all cases investigated. Microscopical scoring showed the local concentration of BMPs to be especially high in sections from giant cell tumors when compared to other bone neoplasms.

Reinnervation after nerve suture in rat groin flaps.

Regeneration of sensory and adrenergic nerves in the skin was studied in rats. The aim was to investigate the effect of reanastomosing the cut nerve ends of the nerve trunk leading to the microvascular groin flap. Reinnervation was demonstrated immunohistochemically using calcitonin gene-related peptide (CGRP) as marker for sensory nerves, neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers for adrenergic nerves and Protein Gene Product 9.5 (PGP 9.5) as general neuronal marker. It was demonstrated that reanastomosing of the nerve trunk was favourable for both the sensory and sympathetic reinnervation of microsurgical flaps.

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.

Producing vascularized bone by heterotopic bone induction and guided tissue regeneration: a silicone membrane-isolated latissimus dorsi island flap in a rat model.

Transformation of mesenchymal-type tissue into cartilage and bone can be induced by bone morphogenetic protein, and by its parent substratum, demineralized bone matrix. The authors were interested in transforming muscle island flaps into vascularized bone that could be used as autogeneic skeletal replacement parts. In Wistar rats, tubular latissimus dorsi muscle island flaps were created, using microsurgical techniques. The flaps were inserted by a cylinder of demineralized bone matrix (DBM) and enclosed in silicone rubber membrane tubes. The animals were followed-up for 10, 21, or 35 days. Rats with DBM implanted in muscle pouches served as controls. Quantitative radiomorphometry and qualitative histology were performed. A statistically significant linear time-related increase in radiomorphometrically-measured calcified tissue was found in the flaps with DBM from 10 days to 5 weeks. At 3 and 5 weeks, lamellar and cancellous bone with fully developed marrow was detected microscopically. There was no significant difference in bone quantity in the island flaps after 35 days, compared with the muscle pouches implanted with DBM, although the difference at 21 days was still significantly in favor of the island flaps. Using allogeneic DBM in rat muscle island flaps surrounded with a silicone membrane, it was possible to generate in vivo autogenous new bone with a good vascular supply and good mobility, allowing later transfer to another site. The experiment provided a basic technique that can be used as a standard in testing various osteoinductive substances for the production of vascular-pedicled new bone.

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.

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.

Stabilization of an inserted tricalcium phosphate spacer enhances the healing of a segmental tibial defect in sheep.

The effect of inserting a tricalcium phosphate (TCP) spacer stabilized by a rigid or non-rigid fixation technique on the healing of segmental tibial defects of critical size was established. The osteotomized tibiae, 11 with and 8 without TCP spacers, were fixed by an external circular device in 11 mature sheep and by plates in 8 mature sheep, respectively. Healing was evaluated roentgenographically 16 weeks after the operation. Compared with the defects without TCP spacers, enhanced stability and healing were observed in the defects with TCP spacers under an identical external fixation. Furthermore, a significantly higher incidence of healing was obtained with plate fixation than with external device fixation in the TCP-implanted defects (P < 0.04). An abundant bridging callus was roentgenograpically demonstrated in most of the healed defects, but none in the unhealed defects. The TCP spacer with its mechanical integrity enhances the stability of external fixation, and the stable immobilization provided by rigid fixation is essential for osteoconduction of an inserted TCP spacer in the healing of segmental diaphyseal defects in sheep.

Survival of Lubinus straight (IP) and curved (SP) total hip prostheses in 543 patients after 4-13 years.

The clinical outcome and survival of 543 total hip arthroplasties with Lubinus cemented total hip prostheses after a mean of 7.9 years are reported. Clinical evaluation revealed no significant differences between patients with a straight stem and those with a curved anatomic stem. Loosening was statistically associated with young age, male sex, heavy weight, diagnosis other than osteoarthritis and year of operation. Survivorship analysis of the Lubinus IP implant at 8 years revealed a 95% chance of survival. The survival of the SP implant was 96%. After this time point the curve for SP implants began to fall at a faster rate than that for IP implants.

Measurement of total and local bone morphogenetic protein concentration in bone tumours.

Bone morphogenetic protein (BMP) has been shown to be one of the significant factors in the prognosis of bone tumours. In normal development BMP induces new bone formation and later takes part in fracture healing, but its function in malignant tumours is not known. In this study the concentration of bone morphogenetic protein was measured in primary bone tumours by two methods. Local staining intensity was detected immunohistologically by the avidin-biotin-peroxidase method determining the highest dilution of anti-serum against bovine bone morphogenetic protein. The total amount of BMP in a tumour sample was measured by an enzyme-linked immunosorbent assay technique after digesting the tissue with collagenase to remove proteins from the connective tissue. Immunohistochemical staining showed that bone morphogenetic protein was present in the cytoplasm and in reactive bone formed by malignant cells. The local concentration was highest in the tissue of giant cell tumours compared to chondrosarcoma, osteosarcoma and benign bone tumours. The total amount in malignant bone tumours was 2.4 times higher compared to benign bone tumours.