[Improved osseointegration of titanium implants of different surface characteristics by the use of bone morphogenetic protein (BMP-3): an animal study performed at the metaphyseal bone bed in dogs]. / Verbesserte Osseointegration von Titanprüfkörpern unterschiedlicher Oberflächenbeschaffenheit durch Beschichtung mit Bone Morphogenetic Protein (BMP-3)--eine tierexperimentelle Studie im metaphysären Knochenlager des Hundes.

AIM: Aim of this study was to determine whether coating of titanium implants of various surfaces with BMP-3 would improve the osseous integration of the implants into the orthotopic bony implant bed. METHOD: In this experimental study 190 micro g per implant of highly purified bone morphogenetic protein 3 (BMP-3) precipitate isolated from porcine bone were available for the coating of each of 24 cylindrical test implants (12 with hydroxyapatite and 12 with plasmapore surface). The remaining 24 test implants with the same surface makeup served as negative controls. Implantation sites were randomly assigned for the 4 versions of implants available and all implants were embedded into the medial or lateral femoral condyle of both legs of 12 German shepherds. The drilling holes were performed in such a matter that after embedding the cylindrical devices a gap of 1 mm surrounding the implants remained. A biomechanical testing and histological evaluation was performed on the explants 42 days after surgery. RESULTS: In biomechanical testing forces necessary to extract the implants from the explanted bones in BMP-3 coated devices were up to 70% higher compared to the ones in the non-coated reference groups. Quantitative histomorphometric examination showed in BMP-3-coated implants an increasing formation of new bone close to their own surface (gap-healing) which was higher than in the corresponding non-coated controls (hydroxyapatite + BMP-3 32.1%, hydroxyapatite controls 20.3%, plasmapore + BMP-3 30.2%, plasmapore controls 13.1%). The extent of direct bone implant contact as percentiles of the corresponding implants perimeter (ongrowth) was also significantly higher in the BMP-3-coated implants compared to the non-coated controls (hydroxyapatite + BMP-3 37.7%, hydroxyapatite controls 22.4%, plasmapore + BMP-3 15.3%, plasmapore controls 6.4%). CONCLUSION: In this study it was proven the first time that implants of various surface textures as used in endoprosthetics are able to be coated by the osteoinductive growth factor BMP-3. In that way metallic implants can achieve osteogenic properties which have positive effects in osseointegration.

[Histologic and histomorphometric follow-up observations of osseointegration of corundum-blasted BMP-3 coated titanium test implants (Ti6Al4V) at orthotopic site in the giant rabbit]. / Histologische und histomorphometrische Verlaufsbeobachtungen zur knöchernen Integration von korund-gestrahlten BMP-3-beschichteten Titanprüfkörpern (Ti-6Al-4V) im orthotopen Lager des Riesenkaninchens.

AIM: To establish whether the additional coating of titanium implants with Bone Morphogenetic Protein-3 (BMP-3) might enhance osseous integration. METHOD: Each of 15 cylindrical titanium test implants (Ti-6AI-4V) was coated using 230 micrograms porcine BMP-3. A further 15 implants with identical (corundium-blasted) surface served as negative controls. An uncoated and a BMP-3-coated test object were implanted into the femurs of 15 adult giant rabbits. New formation of bone around the implants was examined microscopically and histomorphometrically on postoperative days 14, 35 and 56. RESULTS: Coated implants revealed superior osseointegration with statistical evaluation using the t-test for matched samples showing a significantly higher volume of new bone 5 weeks after surgery. Microscopic examination revealed osseointegration with no connective tissue membrane around the surface of the implants. CONCLUSIONS: Our results indicate that composite metal implants are suitable carriers for BMP-3 and that improved fixation of the implants can be achieved.

Superior efficacy in suppression of heterotopic bone formation using fractionated irradiation of 5 x 2 Gy compared to a single dose of 7 Gy. An experimental study in rats.

Postoperative irradiation of the operative field is an established method to prevent heterotopic ossification in total hip arthroplasty. In this study two theoretical dose-equivalent regimens of radiation therapy were compared. Allogenic bone matrix was implanted in both thighs of 50 adult male Wistar rats to induce heterotopic ossification. Immediately after operation the implants of 40 animals were irradiated using a single-dose of 7 Gy or 5 fractions of 2 Gy each. Ten rats served as a controlgroup and did not undergo irradiation. Radiation therapy with 5 x 2 Gy led to a highly significantly better suppression of heterotopic ossification than irradiation with 1 x 7 Gy (p < 0.001; paired-t-test). Single-dose irradiation reduced the mean calcium contents to 138.87 +/- 22.84 micrograms Ca2+/mg implanted bone matrix; fractionated irradiation obtained a reduction to 63.35 +/- 21.16 micrograms Ca2+/mg implanted bone matrix. In thigh implants not exposed to irradiation the mean calcium content was 191.50 +/- 11.46 micrograms Ca2+/mg implanted bone matrix. Radiographically better suppression of bone formation could be documented after irradiation with 5 x 2 Gy compared to 1 x 7 Gy and non-irradiated implants. The histological aspect of the explanted specimens showed quantitatively more new bone formation in the non-irradiated controls than in both irradiation groups. In view of experimentally demonstrated better effects, as well as the reduced side effects, fractionated irradiation appears preferable.

Autologous chondrocyte implantation and osteochondral cylinder transplantation in cartilage repair of the knee joint. A prospective, comparative trial.

BACKGROUND: Current methods used to restore the joint surface in patients with localized articular cartilage defects include transplantation of an autologous osteochondral cylinder and implantation of autologous chondrocytes. The purpose of this study was to evaluate the clinical and histological outcomes of these two techniques. METHODS: We performed a prospective clinical study to investigate the two-year outcomes in forty patients with an articular cartilage lesion of the femoral condyle who had been randomly treated with either transplantation of an autologous osteochondral cylinder or implantation of autologous chondrocytes. Biopsy specimens from representative patients of both groups were evaluated with histological staining, immunohistochemistry, and scanning electron microscopy. RESULTS: According to the postoperative Lysholm score, the recovery after autologous chondrocyte implantation was slower than that after osteochondral transplantation at six months (p < or = 0.015), twelve months (p < or = 0.001), and twenty-four months (p < or = 0.012). On the basis of the Meyers score and the Tegner activity score, the results were equally good with the two methods two years after treatment. Histomorphological evaluation of biopsy specimens within two years after autologous chondrocyte implantation demonstrated a complete, mechanically stable resurfacing of the defect in all patients. The tissue consisted mainly of fibrocartilage, while localized areas of hyaline-like regenerative cartilage could be detected close to the subchondral bone. Although a gap remained at the site of the transplantation in all five biopsy specimens examined as long as two years after osteochondral cylinder transplantation, histomorphological analysis and scanning electron microscopy revealed no differences between the osteochondral transplants and the surrounding original cartilage. CONCLUSIONS: Both treatments resulted in a decrease in symptoms. However, the improvement provided by the autologous chondrocyte implantation lagged behind that provided by the osteochondral cylinder transplantation. Histologically, the defects treated with autologous chondrocyte implantation were primarily filled with fibrocartilage, whereas the osteochondral cylinder transplants retained their hyaline character, although there was a persistent interface between the transplant and the surrounding original cartilage. Limitations of our study included the small number of patients, the relatively short (two-year) follow-up, and the absence of a control group.

[Osteogenetic activity of BMP-3-coated titanium specimens of different surface texture at the orthotopic implant bed of giant rabbits]. / Osteogene Aktivität BMP-3-besichichteter Titanprüfkörper unterschiedlicher Oberflächenbeschaffenheit im orthotopen Lagen des Riesenkaninchens.

INTRODUCTION: The successful combination of osteoinductive factors with current materials used in both endoprosthetics and implantology improves bony ingrowth and long-term stability of the chosen implants. The aim of the present experimental animal study was to clarify in what way faster bony integration can be achieved through additional BMP-3-coating of titanium test implants of different surface textures (hydroxy-apatite-coated or corundum-blasted). METHODS: Thirty of 60 cylindrical titanium test implants with a hydroxy-apatite or corundum-blasted surface were coated with 230 microg porcine, high-purified BMP-3-precipitate per implant to check their osteoinductive potential in a bioassay. In each case a BMP-3-coated and an uncoated control-device were implanted with a gap formation of 1 mm into the femoral part of the patellofemoral joint of the right and left leg of 30 adult giant rabbits. Serial saw slices of all explanted specimens were prepared, and the osseous integration of the implant and time-dependent bone neoformation were analyzed microscopically and histomorphometrically 14, 35, and 56 days after implantation. RESULTS: Coating of TiAl4V6-test devices with BMP-3 led in both groups after gap implantation to an improved osseointegration, that was histomorphological and histomorphometrical verifiable. Statistical evaluation using the t-test for matched samples showed 5 weeks after surgery a significant higher volume of new formed bone of the BMP-3-coated corundum-blasted or hydroxy-apatite-coated TiAl4V6 test devices compared to the non-coated controls of the same type (P < 0.01). Light microscopy demonstrated osseointegration without connective tissue membrane around the surface of the implants after 2, 5, and 8 weeks. Better osseointegration was achieved in the hydroxy-apatite-coated implants than in the corundum-blasted implants. CONCLUSIONS: Our results indicate that composite metal implants, as used in endoprosthetics and implantology, are suitable carriers for BMP-3 and improved fixation of the implants can be achieved.

Effects of single-dose versus fractionated irradiation on the suppression of heterotopic bone formation--an animal model-based follow-up study in rats.

The histological and enzymatic effects of single-dose irradiation of 7 Gray (Gy) versus fractionated irradiation of 5 x 2 Gy on the suppression of heterotopic ossification were examined over a period of 60 days in adult male Wistar rats (n = 57). The standardized osteogenesis model system in rats 19, 10, 11, 16, 19] was used for this purpose. The course of developing ossifications was documented quantitatively and qualitatively by means of quantitative computed tomography/osteodensitometry and digital luminescence radiography. Assessment of the activities of the enzymes alkaline and acid phosphatase throughout the experiment as well as characterization of the isoenzyme of alkaline phosphatase (AP) in connection with histological observations displayed a metaplasia of the ingrowing connective tissue into bone-typical cells during osteoinduction. Thus, the increase of AP is the first sign of a functional transformation of mesenchymal stem cells into chondroid bone cells. The increase in the acid phosphatase level with a maximum of activity between the 15th and 30th day (according to the respective treatment group) is highly suggestive of a remodeling process paralleling incipient chondroclast and osteoclast activity. In the animal groups undergoing irradiation, the above-mentioned increase of enzymes occurred after a delay. Furthermore, the maximum values observed were lower than those in the group not undergoing irradiation. Both findings were more manifest in the animal group which underwent 5 x 2 Gy of radiation than in the group which underwent single-dose irradiation of 7 Gy. Radiation suppresses matrix-induced osteogenesis. The histological and enzymatic course of this process was unchanged in the animals which did not undergo irradiation. However, it was quantitatively reduced and accompanied by a retardation of osteogenesis. Both effects were again reduced with fractionated irradiation of 5 x 2 Gy, which is theoretically dose-equivalent to a 1 x 7 Gy application. Histological examinations revealed damage to the migratory, proliferating mesenchymal stem cell population by irradiation doses which had relatively small effects on preosteoblasts, osteoblasts, chondroblasts and other specialized cell forms. Therefore, it may be concluded that the smaller degree of heterotopic ossification in the irradiated groups was due to damage of and a decrease in the number of mesenchymal stem cells at the implant site. Our results stress the necessity of instituting postoperative irradiation therapy as early as possible to prevent heterotopic ossification. In view of experimentally proven better effects, fractionated irradiation has to be preferred to a dose-equivalent single-dose radiation, especially considering the fewer side-effects noted with fractionated irradiation.

[Osteochondral transplantation versus autogenous chondrocyte transplantation. A prospective comparative clinical study]. / Knorpelknochentransplantation versus autogene Chondrocytentransplantation. Eine prospektive vergleichende klinische Studie.

INTRODUCTION: The treatment of full-thickness cartilage defects still represents a problem that has not yet been solved satisfactorily. Current methods used to cover defects in the knee joint are osteochondral cylinder transplantation (OCT) and autologous chondrocyte transplantation (ACT). METHODS: With a prospective clinical investigation, at the time being with 2-year results, we have examined ACT in comparison to OCT in 20 patients with regard to clinical and histomorphological (histology, immunohistochemistry, RES) outcome. RESULTS: We found equally good results with both methods in Lysholm, Meyers and Tegner Activity Scores. Histomorphologic evaluation of biopsies obtained by arthroscopy after ACT showed a defect filling in all cases, mainly with fibrous cartilage, while localized areas of hyalinelike regenerative cartilage were documented near the base. We did not see any histomorphologically visible change in the transplants after OCT. CONCLUSION: At the time we prefer OCT instead of ACT given the correct indication.

Effects of granulocyte-colony-stimulating factor on potential normal granulocyte donors.

BACKGROUND: The use of granulocyte-colony-stimulating factor (G-CSF) to increase the granulocyte count and the yield from leukapheresis in normal donors is leading to renewed interest in granulocyte transfusion. Therefore, it is important to understand the side effects of G-CSF. STUDY DESIGN AND METHODS: We studied the effect of G-CSF on peripheral blood counts and recorded the side effects experienced 24 hours after an injection of G-CSF in normal subjects donating peripheral blood progenitor cells for research. RESULTS: Following administration of G-CSF to 261 donors, the neutrophil count increased to 20.6 to 24.5 x 10(9) per microL depending on the dose of G-CSF. This represented a 6.2 to 7.4-fold increase over the neutrophil count before G-CSF administration. Of all donors, 69 percent experienced one or more side effects. The most common effects were: muscle and bone pain, headache, fatigue, and nausea. There was a relationship between the dose of G-CSF and the likelihood of experiencing a side effect. Most side effects were mild, but about 75 percent of donors took analgesics because of them. CONCLUSIONS: In a granulocyte donation program involving G-CSF stimulation, about two-thirds of donors would experience one or more side effects, but these would usually be mild and well tolerated.

Expression of neutrophil antigens after 10 days of granulocyte-colony-stimulating factor.

BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF) is becoming the standard agent for mobilizing granulocytes. Most granulocyte donors are given a single dose of G-CSF, but in some cases they are given G-CSF for several days, and multiple granulocyte concentrates are collected. The administration of a single dose of G-CSF induces several changes in the expression of neutrophil antigens, but the effects of multiple daily doses of G-CSF are not known. STUDY DESIGN AND METHODS: Seven healthy people received 5 microg per kg of G-CSF for 10 days. Their expression of several neutrophil antigens before, during, and after the administration of G-CSF was analyzed through the use of flow cytometry. RESULTS: The expression of L-selectin (CD62L), Fcgamma receptor (FcgammaR) III (FcgammaRIII, CD16), and the leukocyte function antigen (CD11a) decreased throughout the course of G-CSF administration, while the expression of FgammaR I (FcgammaRI, CD64) and lipopolysaccharide-binding protein receptor (CD14) increased. The expression of FcgammaR II (FcgammaRII, CD32) also increased, but not until the fourth day of G-CSF administration. The expression of amino peptidase N (CD13), C3bi receptor (CD11b), and the neutrophil beta2 integrin unit (CD18) did not change during the administration of G-CSF, but that of both CD13 and CD18 increased 3 days after the last dose. The expression of neutrophil-specific antigen NB1 initially increased, returned to pre-G-CSF levels after 4 days, and then increased again after 10 days of G-CSF administration. CONCLUSION: Changes in the expression of several neutrophil antigens occurred throughout a 10-day course of G-CSF Most of the changes occurred after one dose, but additional changes occurred later in the 10-day course and after its completion. These changes may affect the function of G-CSF-mobilized granulocytes.

[Defect reconstruction using demineralized bone matrix. Experimental studies on piglets]. / Defektaufbau mit demineralisierter Knochenmatrix. Experimentelle Untersuchungen beim Minischwein.

The aim of this study was to evaluate the bone stimulation forced by Demineralized Bone Matrix (DBM)-Chips and-Gel in comparison to the bone-ingrowth into a porous hydroxylapatite ceramic (Endobon) in mini pigs. The following results were obtained: 1. DBM-Chips and DBM-Gel did not stimulate bone healing when filled into cancellous bone defects. The defect did not heal within 12 weeks. 2. Up to 35 days the least amount of new bone formation was observed within porous hydroxylapatite ceramic. Up to 12 weeks complete bone ingrowth in to the ceramic has been seen with close bonding between new formed bone and the ceramic trabeculae. 3. By continuous labelling with fluorochromes the new bone formation could be analysed by fluorescence microscopy and the dynamics could be related to time after implantation.

Different behavior of human osteoblast-like cells isolated from normal and heterotopic bone In vitro.

In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM beta-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3-6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells.

Leigh syndrome, cytochrome C oxidase deficiency and hypsarrhythmia with infantile spasms.

A rare patient with infantile spasms, hypsarrhythmia, cytochrome c oxidase deficiency and Leigh syndrome is reported. Although rare, infantile spasms and Leigh syndrome may occur simultaneously. Leigh syndrome should be included in the differential diagnosis of infantile spasms.

Comparison of two blood cell separators in collecting peripheral blood stem cell components.

To ensure that a sufficient number of CD34+ cells are collected for an allogeneic blood progenitor cell transplant, the most effective blood cell separator should be used to collect peripheral blood stem cell (PBSC) components. We compared the effectiveness of two blood cell separators. We gave 29 healthy people 7.5 or 10 micrograms kg-1 of granulocyte colony stimulating factor (G-CSF) daily for 5 days and collected one PBSC component with either a Fenwal CS3000 (n = 15) or a Cobe Spectra (n = 14) blood cell separator. The volume of blood processed was the same for each machine (8.4 +/- 1.0 L; range = 4.9-9.4 L for the CS3000 and 8.9 +/- 1.0 L; range 6.7-10.9 L; P = 0.71). The components collected with the CS3000 contained more mononuclear cells (39.6 +/- 21.9 x 10(9) compared with 26.9 +/- 5.6 x 10(9), P = 0.02) and fewer neutrophils (1.38 +/- 1.88 x 10(9) compared with 5.53 +/- 8.71 x 10(9), P = 0.001). The total number of CD34+ cells collected with the two instruments was the same (470 +/- 353 x 10(6) for the CS3000 and 419 +/- 351 x 10(6) for the Spectra; P = 0.64) as was the number of CD34+ cells collected per litre of whole blood processed (55.9 +/- 42.0 x 10(6) L-1 compared with 45.9 +/- 37.9 x 10(6) L-1; P = 0.59). The mononuclear cell collection efficiency was greater for the CS3000 (82.4 +/- 54.9% compared with 53.3 +/- 14.1; P = 0.04) but the CD34+ cell collection efficiencies were the same (87.4 +/- 61.1% for the CS3000 compared with 56.3 +/- 23.5% for the Spectra; P = 0.07). In conclusion, both blood cell separators collected components which contained large numbers of CD34+ cells, but those collected with the CS3000 contained fewer neutrophils and the CS3000 was more efficient at collecting mononuclear cells.

Composition of peripheral blood progenitor cell components collected from healthy donors.

BACKGROUND: Peripheral blood progenitor cell (PBPC) components are being collected from healthy donors for allogeneic transplantation, but the quantity, quality, composition, and variability of PBPCs collected from healthy people given granulocyte-colony-stimulating factor (G-CSF) have not been evaluated. STUDY DESIGN AND METHODS: PBPC components were collected from 150 healthy people who were given G-CSF (5, 7.5, or 10 microg/kg/day) for 5 days. The components were evaluated for white cell (WBC), mononuclear cell, CD34+ cell, neutrophil, platelet, and red cell (RBC) composition. RESULTS: The quantities collected were: WBCs, 35.0 +/- 16.4 x 10(9) (range, 11.9-163.3 x 10(9)); mononuclear cells, 33.3 +/- 14.4 x 10(9) (range, 11.9-139.6 x 10(9)); CD34+ cells, 412 +/- 287 x 10(6) (range, 70-1658 x 10(6)); neutrophils, 1.71 +/- 3.59 x 10(9) (range, 0-27.6 x 10(9)); RBCs, 7.2 +/- 4.0 mL (range, 0-22.1 mL); and platelets, 480 +/- 110 x 10(9) (range, 250-920 x 10(9)). PBPC components collected from people given G-CSF at 7.5 or 10 microg per kg per day contained significantly more CD34+ cells (respectively, 428 +/- 300 x 10(6); range, 70-1658 x 10(6) and 452 +/- 294 x 10(6); range, 78-1380 x 10(6)) than those from people given G-CSF at 5 microg per kg per day (276 +/- 186 x 10(6); range, 91-767 x 10(6)) (p = 0.007 and p = 0.002). When 10 microg per kg per day of G-CSF was given, 50 percent of the components contained enough CD34+ cells for transplantation to a 75-kg recipient (375 x 10(6) CD34+ cells), but 10.6 percent of the components contained less than 150 x 10(6) CD34+ cells and thus would provide a transplantable dose only for a 30-kg patient. CONCLUSION: One PBPC component collected from a healthy donor given 7.5 or 10 microg per kg per day of G-CSF should contain 70 to 1660 x 10(6) CD34+ cells, with 0 to 22 mL of RBCs. Because of the great variability in the number of CD34+ cells collected, the quantity of CD34+ cells in each component should be measured after each procedure to ensure that sufficient quantities of cells are present for a successful transplant.

Blood counts in healthy donors 1 year after the collection of granulocyte-colony-stimulating factor-mobilized progenitor cells and the results of a second mobilization and collection.

BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF)-mobilized blood cells are being used for allogeneic transplants, but the long-term effects of G-CSF on healthy individuals are not known. Furthermore, it is not certain how many CD34+ cells can be collected in a second mobilization and collection procedure. STUDY DESIGN AND METHODS: Nineteen people were given 2, 5, 7.5, or 10 micrograms of G-CSF per kg per day for 5 days, and blood progenitor cells were collected by apheresis on the sixth day; this was done on two occasions separated by at least 12 months. Blood counts obtained before and after each course of G-CSF and the quantity of cells collected were compared. RESULTS: There were no differences in white cell (WBC), platelet, red cell, and WBC differential counts measured before each course of G-CSF, and all the values were in the normal range. In a subset of 12 people who received 7.5 or 10 micrograms of G-CSF per kg per day for both courses, the numbers of neutrophils, mononuclear cells, and CD34+ cells in the blood after each course were similar (34.1 +/- 7.31 x 10(9)/L vs. 36.4 +/- 12.3 x 10(9)/L, p = 0.24; 6.59 +/- 2.28 x 10(9)/L vs. 5.63 +/- 2.11 x 10(9)/L, p = 0.24; and 92.0 +/- 55.6 x 10(5)/L vs. 119.2 +/- 104.6 x 10(6)/L; p = 0.48, respectively), as were the quantities of mononuclear cells (31.0 +/- 8.4 x 10(9) vs. 31.0 +/- 6.1 x 10(9); p = 0.64) and CD34+ cells (417 +/- 353 x 10(6) vs. 449 +/- 286 x 10(6); p = 0.53) collected in the two apheresis procedures. Furthermore, there was a positive correlation between the quantity of CD34+ cells collected from each of the 12 people per liter of whole blood processed in the two procedures (r2 = 0.86, p < 0.001). CONCLUSION: One year after the administration of G-CSF to healthy people, their blood counts were normal and unchanged from pretreatment counts. If healthy people donate blood progenitor cells after a second G-CSF course the quantity of CD34+ cells collected will be similar to that obtained in the first collection.

The kinetics of G-CSF mobilization of CD34+ cells in healthy people.

When healthy people are given granulocyte colony stimulating factor (G-CSF) for 10 days the number of CD34+ cells in the peripheral blood begins to increase on the fourth day, reaches a maximum on the sixth day and then decreases. In this study, we further define the time and variability of peak mobilization of CD34+ cells. Twenty-two healthy people were given G-CSF (7.5 or 10 micrograms kg-1 day-1) subcutaneously each morning for 5 days and peripheral blood CD34+ cell counts were analysed immediately prior to the fourth (day 4) and fifth (day 5) G-CSF injection and 24 h after the fifth injection (Day 6). White blood cell (WBC) and neutrophil counts were greatest on day 6 [WBC = 43.8 +/- 13.9 x 10(9) L-1 (mean +/- 1 SD) and neutrophils = 36.6 +/- 12.8 x 10(9) L-1]. In contrast the CD34+ cell counts on day 6 (107 +/- 104 x 10(6) L-1) were less than on day 5 (128 +/- 136 x 10(6) L-1) (P = 0.048) but still greater than on day 4 (60.7 +/- 40.2 x 10(6) L-1) (P < 0.0001). The CD34+ cell counts of 10 donors were measured 2, 4 and 6 h after the fifth injection to determine if the counts increased further between days 5 and 6. The number of CD34+ cells in the blood on day 5 2 h after the fifth injection (193 +/- 277 x 10(6) L-1) was greater than the number prior to the injection (158 +/- 190 x 10(6) L-1), 4 h post-injection (139 +/- 158 x 10(6) L-1) and 6 h post-injection (170 +/- 236 x 10(6) L-1), but the differences were not significant (P = 0.29, 0.25 and 0.45). The number of CD34+ cells in the blood of 12 people were measured before and after the fourth G-CSF dose. Prior to the day 4 injection the CD34+ count was 61 +/- 40 x 10(6) L-1. At 2, 4 and 6 h the counts were 60 +/- 40, 61 +/- 29 and 64 +/- 30 x 10(6) L-1, respectively, and the differences were not significant (P = 0.99, P = 0.98, and P = 0.73). In conclusion, when healthy volunteers are given daily G-CSF injections, the number of mobilized CD34+ cells was the greatest on day 5, slightly less on day 6 and the least on day 4. If only one PBSC component is needed, PBSCs can be collected on day 5 after only 4 days of G-CSF. If PBSC components are collected on both days 5 and 6, the fifth dose can be given either before or after the collection of the first PBSC component.

Ectopic bone formation by composites of BMP and metal implants in rats.

Disc-shaped implants of titanium alloy (Ti-6Al-4V) were treated on one side by corundum-blasting (CB) or by coating with hydroxyapatite (HA) or pure titanium (Ti) using plasma spraying. Half of the implants were additionally coated with purified swine BMP-3. The composites and the uncoated controls were implanted into abdominal wall-muscle pouches of rats. 25 days after implantation, ectopic bone formation could be observed macroscopically and histologically in a high frequency in all 3 groups of BMP-coated implants, whereas the controls were constantly inactive. The volumes of induced bone were similar for BMP-3-coated pure Ti and HA implants, while CB implants were significantly less active. Our findings indicate that the bone formation process is influenced by the chemical composition and by the structure of the implant surface.

[Fundamental aspects of osteoinductive activity of bone tissue and potential clinical application]. / Grundsätzliche Aspekte der osteoinduktiven Aktivität von Knochengewebe und Möglichkeiten ihrer klinischen Anwendung.

Osteoinductive polypeptide factors such as the various isoforms of Bone Morphogenetic Protein described are of increasing scientific and clinical interest because of the great progress in this field of skeletal biology recently achieved. In a review some fundamental chemical and biological aspects of bone and its endogenous osteoinductive activity including isolation and assay of osteoinductive factors are described. Through combination of osteoinductive factors with appropriate carrier materials it seems possible to create osteoinductive implant materials and the results of such investigations and some basic aspects of carrier materials are summarized. The perspectives of the possible clinical applications of such composite are presented.

Analysis of the expression of NB1 antigen using two monoclonal antibodies.

BACKGROUND: Neutrophil-specific antigen NB1 is expressed on neutrophil subpopulations in 97 percent of healthy individuals and is located on 56- to 64-kDa glycoprotein. While the molecule carrying NB1 has been identified, the nature of the NB1 epitope has not been well characterized. STUDY DESIGN AND METHODS: Two monoclonal antibodies (MoAbs), 1B5 and the recently produced 7D8, and four alloantibodies, all specific for NB1, were used to investigate the expression of NB1 on neutrophils from several donors. RESULTS: MoAb 7D8 was shown to be specific for NB1. It reacted with NB1-positive neutrophils from 52 donors in the granulocyte immunofluorescence assay and did not react with NB1-negative neutrophils from 8 donors. MoAb 7D8 immunoblotted a 56- to 64-kDa molecule on neutrophils from eight NB1-positive donors and did not react with this molecule on NB1-negative neutrophils from two donors. When 7D8 was tested in the monoclonal antibody immobilization of granulocyte antigens assay, it reacted with two NB1 alloantibodies, but not with NA1 or NA2 alloantibodies. To determine if MoAbs 7D8 and 1B5 recognized the same epitope, both were tested against the same NB1-positive neutrophils and the cells were analyzed by two-color flow cytometry. Both antibodies bound independently to neutrophils, which indicated that the antibodies recognized different epitopes. When similar studies were performed with MoAb 7D8 and three NB1 alloantibodies, 7D8 partially inhibited the binding of two of the alloantibodies. The size of the NB1-positive subpopulation was analyzed in 25 people using flow cytometry with both MoAbs and three alloantibodies. The subpopulation of antigen-positive cells was similar in all donors when 7D8 and the three NB1 alloantibodies were tested; however, the subpopulation recognized by MoAb 1B5 was smaller in two of the donors. Neutrophils from one of these people were analyzed by immunoblotting, and no differences were detected in the molecule carrying NB1 in those neutrophils and that molecule in control neutrophils. CONCLUSION: NB1 specificity is made up of at least two separate epitopes. The expression of NB1 varied among antigen-positive individuals. While NB1 is expressed by a 56- to 64-kDa glycoprotein, the structure of this protein on antigen-negative cells has not been determined.