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.

Comparison of native xenogeneic and allogeneic bone morphogenetic proteins in the sheep skull defect assay model.

BACKGROUND AND AIMS: The purpose of this study was to determine, if there is any difference in the osteoinductive response between xenogeneic and allogeneic bone morphogenetic protein (BMP) in the skull defect assay model. MATERIAL AND METHODS: The skull samples of two experimental series were chosen for uniform radiomorphometric measurement. In the xenogeneic BMP group (X-group), 52 mg of native moose (Alces alces) BMP (mBMP) was implanted in six 22-mm sheep midline skull defects, and in the allogeneic BMP (A-group), 8 mg of native sheep BMP (sBMP) was used in six same size parietal skull defects. The formation of new bone was confirmed histologically and the quantity of it was measured radiomorphometrically at 16 weeks after implantation from the standardized radiographs using a common flatbed scanner and image processing software, the mean grey scale intensity (radiopacity) multiplied by the original defect area giving the new bone volume. This was divided by the milligram amount of implanted BMP to get the value for comparison of BMP activities. RESULTS: The control defects in both groups, implanted with 13 mg of type IV collagen, showed that the defects did not heal spontaneously during the follow-up time. In the X-group, the healing percentage was 92.4 +/- 3.0%, the calculated average bone volume (ABV)/mg being 12.4 +/- 0.5 units, whereas in the A-group the healing percentage was 62.9 +/- 8.5% and ABV/mg 60.9 +/- 6.7. Hence the activity per milligram proved to be more than four-fold with allogeneic compared to xenogeneic BMP. However, when the difference in spontaneous healing of the defects in the two series was taken into consideration, only slight but not significant difference (P = 0.077) of activity could be demonstrated between xenogeneic (5.7 +/- 1.7) and allogeneic BMP (9.05 +/- 3.7). CONCLUSIONS: The result is in concordance with the previous published results showing that no significant immune inhibition is seen after a single implantation of xenogeneic BMP in an orthotopic site.

Xenogeneic moose (Alces alces) bone morphogenetic protein (mBMP)-induced repair of critical-size skull defects in sheep.

A standardized skull defect in adult sheep was used to test the healing capacity of xenogeneic, partially purified, moose-derived bone morphogenetic protein (mBMP) extracted from the fresh long bones of moose (Alces alces) calves. An amount of 52 mg of mBMP mixed with 13 mg of purified type IV collagen (5:1) (mBMP/COL) in gelatin capsules was implanted into six 22-mm-diameter skull defects in adult sheep for comparison with six defects implanted with fresh autogenous bone marrow (BM) and six other controls implanted with a gelatin capsule containing 13 mg of type IV collagen (C). The amount of new bone formed was quantified from radiographs by computerized image analysis and histology. The healing percentage in the mBMP/COL group was significantly higher (93.18 +/- 4.51%) than in the BM (33.17 +/- 20.05%) or C group (31.32 +/- 17.41%) at 16 weeks after implantation. The difference between BM and C was not statistically significant. The level of anti-BMP antibody in the serum showed a significant increase in the group implanted with mBMP, but returned to normal after 6 weeks. The experiment demonstrated that xenogeneic mBMP possesses a strong osteoinductive capacity and weak immunogenicity.

Partial purification and characterization of bone morphogenetic protein from bone matrix of the premature moose (Alces alces): degradation of bone-inducing activity during storage.

In spite of the advances in recombinant techniques in the production of bone morphogenetic proteins (BMPs), the best clinical results so far have been obtained with human and animal source-extracted BMPs. Also, the poor availability of recombinant products gives rise to continued research with different extracted and purified proteins. In a search for a new source of bone-matrix-derived BMP with high osteoinductive activity, BMP was extracted from fresh bone matrix of the premature moose (Alces alces). Bone-inducing activity was investigated by implanting 0.5-20 mg of BMP into thigh muscle pouches of BALB mice. Radiologically detectable formation of new bone required 2.0 mg of partially purified BMP. Immediately after the extraction, an analytic chromatogram with known molecular weight (MW) markers showed three fractions with different MWs. After 15 months of storage at +1 degree C lyophilized and desiccated, BMP was fractionated by HPLC gel filtration and bioassayed. New bone formation was evaluated qualitatively by histology and quantitatively by radiomorphometry, the quantity of calcified tissue per milligram of implanted agent being determined. Fractions I and III, with high (100-700 kD) and low MW (15-25 kD), respectively, were apparently more effective inducers of new bone than the second-time-tested partially purified BMP complex, the activity of which had significantly (p < 0.05) decreased during 15 months of storage compared to initial results after extraction. However, the bone-inducing activity of fractions I and III corresponded closely to the initial activity of the BMP complex. Fraction II, with medium MW (25-55 kD), caused an apparent inflammatory reaction and no bone formation, and was though to be immunogeneic. Fraction III was considered to include the dominant BMP component with MW 18.5 and fraction I an association of BMP with other non-collagenous bone matrix proteins after one-step gel filtration. The results suggest that BMP from the premature moose has high bone-forming activity. With identification and removal of apparently immunogenic protein fractions, the inflammatory reaction and inhibitory effect on bone induction could be eliminated, and still higher bone-forming activity was attained. Acid protease enzymes were assumed to be responsible for the observed decline in the inductive activity of semi-purified BMP after 15 months of storage, as both osteoinductive fractions proved to be acidic in isoelectric focusing.

Heterotopic osteoinduction in a rat membrane-isolated latissimus dorsi island flap. A pilot study.

Applying our knowledge of heterotopic osteoinduction by bone morphogenetic protein (BMP) and demineralized bone matrix (DBM), we sought to induce ossification of membrane-isolated latissimus dorsi flaps in the rat. Our aim was to produce an animal model for a versatile "custom-made" bone island flap which could be used as a substitute for bone. Ten latissimus dorsi island flaps in nine Wistar rats, 5-6 weeks of age, were prepared using microsurgical techniques in aseptic conditions. The flaps were isolated from other tissues with silicone, Gortex or OpSite membranes. We applied 3-9 mg partially purified bovine BMP or 0.1 -0.25mg BMP bound covalently to type IV collagen with 15mg DBM inside the flaps. We have five animals with eight implants of BMP and DBM in latissimus muscle pouches as rat bioassay controls. The results were evaluated after a period of three weeks using soft X-ray radiography and histology with hematoxylin-eosin-azure II and Alcian blue stains. Positive radiological results were observed in 10/10 flaps (100%), in controls in 7/8 (87.5%). Positive histological results comprised 8/10 (80%) and in controls 7/8 (87.5%). Two flaps (20%) showed partial necrosis. These did not lower the percentage of either positive histological or radiological findings, but exemplified some of the problems which are faced in this kind of tissue engineering.