Phase III study in stage IV non-small-cell lung cancer patients treated with two courses of cisplatin/gemcitabine followed by a randomization to three additional courses of the same combination or gemcitabine alone.

BACKGROUND: This randomised phase III study investigated if in responsive and stable disease (SD) stage IV patients after two courses of cisplatin and gemcitabine, single-agent gemcitabine (experimental arm) was not inferior in terms of overall survival (OS) to cisplatin-gemcitabine (standard arm). PATIENTS AND METHODS: Noninferiority was defined as an increase in the hazard of death (HR) < or = 1.33 in the experimental arm. From January 2001 to February 2004, 340 patients were registered and 250 were randomised. Cisplatin was administered on day 1 at 75 mg/m2 and Gemcitabine on days 1 and 8 at 1250 mg/m2 every 3 weeks. RESULTS: Response rate after two courses was 29%. The 1-year progression-free survival was 13% in both arms. One-year survival was 52% in the standard and 42% in the experimental arm for an HR of 1.21 [90% confidence interval (CI) 0.97-1.51]. Postprogression survival was in favour of the standard arm (HR 1.30, 95% CI 0.99-1.70, P = 0.051). Grades 3-4 toxicity favoured in the experimental arm. CONCLUSION: In responsive and SD patients with stage IV non-small-cell lung cancer it was not possible to demonstrate that three courses of gemcitabine alone are not inferior, in terms of OS, to the standard approach of three courses of cisplatin-gemcitabine.

The use of small titanium screws for orthodontic anchorage.

The use of conventional dental implants for orthodontic anchorage is limited by their large size. The purpose of this study was to quantify the histomorphometric properties of the bone-implant interface to analyze the use of small titanium screws as an orthodontic anchorage and to establish an adequate healing period. Overall, successful rigid osseous fixation was achieved by 97% of the 96 implants placed in 8 dogs and 100% of the elastomeric chain-loaded implants. All of the loaded implants remained integrated. Mandibular implants had significantly higher bone-implant contact than maxillary implants. Within each arch, the significant histomorphometric indices noted for the "three-week unloaded" healing group were: increased labeling incidence, higher woven-to-lamellar-bone ratio, and increased osseous contact. Analysis of these data indicates that small titanium screws were able to function as rigid osseous anchorage against orthodontic load for 3 months with a minimal (under 3 weeks) healing period.

Animal housing influences the response of bone to spaceflight in juvenile rats.

The rat has been used extensively as an animal model to study the effects of spaceflight on bone metabolism. The results of these studies have been inconsistent. On some missions, bone formation at the periosteal bone surface of weight-bearing bones is impaired and on others it is not, suggesting that experimental conditions may be an important determinant of bone responsiveness to spaceflight. To determine whether animal housing can affect the response of bone to spaceflight, we studied young growing (juvenile) rats group housed in the animal enclosure module and singly housed in the research animal holding facility under otherwise identical flight conditions (Spacelab Life Science 1). Spaceflight reduced periosteal bone formation by 30% (P < 0.001) and bone mass by 7% in single-housed animals but had little or no effect on formation (-6%) or mass (-3%) in group-housed animals. Group housing reduced the response of bone to spaceflight by as much as 80%. The data suggest that housing can dramatically affect the skeletal response of juvenile rats to spaceflight. These observations explain many of the discrepancies in previous flight studies and emphasize the need to study more closely the effects of housing (physical-social interaction) on the response of bone to the weightlessness of spaceflight.

Development of a fluorescent light technique for evaluating microdamage in bone subjected to fatigue loading.

A new method using fluorescent light microscopy has been developed to visualize and evaluate bone microdamage. We report the findings of two different experiments with a common aim of comparing the fluorescent light technique to the brightfield method for quantifying microdamage in bone. In Experiment 1, 36 canine femurs were tested in four-point cyclic bending until they had lost between 5 and 43% of their stiffness. The loaded portion of the bone was stained en bloc with basic fuchsin for the presence of damage. Standard point counting techniques were used to calculate fractional damaged area (Dm.Ar = Cr.Ar/B.Ar, mm2/mm2) under brightfield and fluorescent microscopy. In Experiment 2, bone microdamage adjacent to endosseous implants, subjected to fatigue loading (150,000 cycles, 2 Hz and 37 degrees C) ex vivo was examined. The bone around the implant was either allowed to heal (adapted specimen) for 12 weeks after placement in dog mid-femoral diaphyses prior to testing or was loaded immediately to simulate non-healed bone surrounding endosseous implants (non-adapted). Crack numerical density (Cr.Dn = Cr.N/B.Ar, #/mm2), crack surface density (Cr.S.Dn = Tt.Cr.Le/B.Ar, mm/mm2) and fractional damaged area were calculated separately by both techniques in the adapted and non-adapted specimens. In both Experiments 1 and 2, significantly more microdamage was detected by the fluorescent technique than by the brightfield method. Also, there was a trend towards higher intraobserver repeatability when using the fluorescent method. These results suggest that the brightfield technique underestimates microdamage accumulation and that the fluorescent technique better represents the actual amounts of microdamage present. The results demonstrate that the fluorescent method provides an accurate and precise approach for bone microdamage evaluation, and that it improves the prediction of stiffness loss from damage accumulation.

Microdamage adjacent to endosseous implants.

Intense remodeling occurs in lamellar bone adjacent to osseointegrated endosseous implants. The purpose of this study was to compare microdamage accumulation subsequent to ex vivo fatigue loading of bone that surrounds an endosseous implant, (a) immediately after placement (nonadapted bone) and (b) following a 12 week healing period after placement (adapted bone). We hypothesize that there is less microdamage in the more compliant adapted bone than in the older nonadapted bone. Nonthreaded titanium plasma sprayed (TPS)-coated endosseous implants were placed into dog mid-femoral diaphyses and allowed to heal for 12 weeks. Block sections of bone, each containing one implant, were cut anteroposteriorly, resulting in an implant containing lateral cortex, and a medial cortex that was used for testing the nonadapted specimens. Control specimens (n = 14 each for adapted and nonadapted) were loaded at 0 N. Experimental specimens (n = 13, adapted; n = 14, nonadapted) were loaded at 100 N in cantilever bending for 150,000 cycles at 2 Hz, at 37 degrees C on a Bionix 858 testing machine. Specimens were bulk stained with basic fuchsin and 120-140 microm sections were obtained. Crack numerical density (Cr.Dn = Cr.N/ B.Ar, #/mm2), crack surface density (Cr.S.Dn = Tt.Cr.Le/ B.Ar, mm/mm2), and percent damage area (Dm.Ar = Cr.Ar x 100/B.Ar, mm2/mm2) were measured at x 250. Statistically significant differences (p < 0.0001) were seen for Cr.Dn, Cr.S.Dn, and Dm.Ar on the compressed cortices suggesting that adapted bone near the implant accumulated significantly less microdamage than nonadapted bone. Also, the adapted nonloaded control specimens had approximately 20-fold less damage than the respective nonadapted specimens. This study suggests that the compliant adapted bone adjacent to endosseous implants is relatively resistant to fatigue loads. The high success rates of endosseous implants may be due to the presence of a rapidly remodeling region that maintains tissue compliance and limits microdamage initiation.

Implant-borne suture expansion in rabbits: a histomorphometric study of the supporting bone.

Osseointegrated implants have a large potential for diverse clinical applications, including support for sutural expansion and facial prostheses. The objectives of this study were to evaluate: (1) the histomorphometric response of thin cortical bone to implant placement and (2) whether loading of the bone surrounding these implants affects osseointegration as evaluated by histomorphometry. Eighteen New Zealand White rabbits had two titanium implants placed bilaterally in the anterior surface of their nasal bones. The rabbits were divided into an unloaded control group, one experimental group loaded at 1 Newton (N), and another loaded at 3 N. Fluorescent labels were used to mark areas of active bone formation. All rabbits were euthanized after 12 weeks of loading. Stereological point-hit and line-intercept methods were used to measure bone volume, direct bone-implant contact, new bone volume, and bone turnover rate in the bone surrounding the implants. All the implants remained stable during the loading period. A factorial ANOVA with repeated measures was used to compare the variables. The only significant difference among the three groups was a higher bone volume in the lateral coronal far region in the control group (p < 0. 05). Within all groups, bone volume (p < 0.002), turnover rate (p < 0.001), and percent of new bone (p < 0.05) were higher within 1 mm of the implant compared to 1-3 mm away. This may be due to the increased stress and strain in the bone adjacent to the implant. This study indicates that there are no detrimental effects of loading on osseointegration when implants placed in the thin facial cortices are used as anchors for sutural expansion.

Ex vivo expansion of cord blood progenitors.

Human umbilical cord blood contains abundant primitive and committed hematopoietic progenitors; in addition, the general availability and the ease of procurement make cord blood a very attractive alternative source of transplantable hematopoietic tissue. However, the major limitation to a widespread use of cord blood for transplantation lays in its limited volume. For such a reason, until now, cord blood transplant has been mainly restricted to children and small size adults. Ex vivo expansion of cord blood stem cells could make the use of cord blood transplant feasible also for adult patients. Recently we developed a stroma-free culture system in which a progressive, increasingly greater production of hemopoietic progenitors belonging to all the hematopoietic lineages was sustained for over six months. A similar sustained and prolonged expansion of the most primitive stem cells that can be detected in vitro (LTC-IC), was also documented. The extremely prolonged maintenance and the massive expansions suggest that extensive self-renewal and little differentiation can be triggered in vitro by FLT3/FLK2 ligand (FL) plus c-mpl ligand (Thrombopoietin) and this could represent a first step towards the implementation of clinical expansion-transplantation strategies.

Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation.

Cord blood (CB) is an attractive alternative to bone marrow or peripheral blood as a source of transplantable hematopoietic tissue. However, because of the reduced volume, the stem cell content is limited; therefore its use as a graft for adult patients might require ex vivo manipulations. Two systems have been described that identify these stem cell populations in vitro in both mice and humans: (1) the long-term culture-initiating cells (LTC-IC), thus named because of their ability to support the growth of hematopoietic colonies (colony-forming cell (CFC)) for 5-6 weeks when co-cultured on stromal layers; (2) the generation of hematopoietic progenitors (CFC) from stroma-free liquid cultures for extended periods of time, which provides further indirect evidence of the presence of primitive stem cells. Both systems detect largely overlapping but not identical populations of stem cells. Thus the identification of the growth factor requirements for the maintenance and amplification of both systems is relevant. On this basis, analysis of the effects of 18 cytokine combinations on stroma-free liquid cultures of CB CD34+ cells, showed that: (1) after 7- and 14 day-incubation periods, several growth factor combinations expanded the LTC-IC pool to a similar extent; as compared to the LTC-IC, the generation of CFC was not impressive; (2) time-course analysis of the LTC-IC expansion demonstrated that, by extending the incubation period, only a few growth factor combinations, containing FL, TPO, KL and IL6, could support a further, increasingly greater LTC-IC expansion (up to 270000-fold of the initial value). In similar culture conditions, CFC production underwent continuous expansion, which persisted for over 7 months and reached values of one million-fold of the initial value. The simultaneous presence of FL and TPO was both necessary and sufficient to support this phenomenon. The addition of KL+/-IL6 did not appear to substantially modify the extent of LTC-IC expansion; nevertheless, it played an important role in sustaining an even more massive and prolonged output of CFU-GM, CFU-Mk and BFU/CFU-GEMM (up to 100 million-fold); (3) the presence of IL3 was found to be negative, in that it inhibited both the extent of LTC-IC expansion and the long-term generation of CFC. Thus, FL and TPO appear as two unique growth factors that preferentially support the self-renewal of primitive stem cells; the additional presence of KL and IL6 seems to enhance the proliferative potential of at least one subpopulation of daughter stem cells, which may follow three differentiation pathways. Far from being definitive, our data demonstrated that massive stem cell expansion, in cord blood, can be obtained in reasonably well-defined culture conditions. This could represent an initial step towards larger scale cultures for transplantation and gene therapy protocols.

The role of c-Mpl ligands in the expansion of cord blood hematopoietic progenitors.

The major limitations to the widespread use of high-dose chemotherapy or radiotherapy followed by autologous or allogeneic transplantation are the scarcity of stem cell donors and the depletion of the autologous stem cell reservoir. Cord blood is a readily available source of stem cells, which, however, might be limited in number. For this reason, up to now, cord blood transplantation has been restricted to children. Therefore, a major goal for experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. Two systems have been described to identify in vitro these progenitor cell populations in both mice and humans: A) long-term culture-initiating cells (LTC-IC), so named because of their ability to support the growth of hemopoietic colonies (colony-forming cell [CFC]) for five to six weeks when cocultured on stromal layers, and B) the generation of hematopoietic progenitors CFC from stroma-free liquid cultures for extended periods of time, which is another indirect evidence for the presence of primitive stem cells. The two systems detect largely overlapping but not identical cell populations of progenitor cells; thus, the identification of the growth factor requirements for the maintenance and amplification of both systems is relevant. The studies presented here demonstrate that CD34+ cord blood cells can be grown in stroma-free liquid cultures for extremely prolonged periods of time (up to six months). During such a period, hemopoietic precursors and committed progenitors belonging to all of the hematopoietic lineages are continuously and massively generated. Such a massive expansion is sustained by an increasingly larger expansion of primitive stem cells (CFU-BI and LTC-IC). The presence of both FL and thrombopoietin (TPO) was necessary and sufficient to support this phenomenon. The addition of KL +/- interleukin 6 (IL-6) does not appear to substantially modify the extent of LTC-IC expansion. FL and TPO appear to be two unique growth factors that preferentially support the self-renewal of primitive stem cells; the additional presence of KL and IL-6 seems to enhance the proliferative potential of at least a subpopulation of daughter stem cells which can undergo at least three differentiation pathways.

Fluoride treatment increased serum IGF-1, bone turnover, and bone mass, but not bone strength, in rabbits.

We hypothesized that fluoride partly acts by changing the levels of circulating calcium-regulating hormones and skeletal growth factors. The effects of oral fluoride on 24 female, Dutch-Belted, young adult rabbits were studied. The rabbits were divided into two study groups, one control and the other receiving about 16 mg fluoride/rabbit/day in their drinking water. After 6 months of fluoride dosing, all rabbits were euthanized and bone and blood samples were taken for analyses. Fluoride treatment increased serum and bone fluoride levels by over an order of magnitude (P < 0.001), but did not affect body weight or the following serum biochemical variables: urea, creatinine, phosphorus, total protein, albumin, bilirubin, SGOT, or total alkaline phosphatase. No skeletal fluorosis or osteomalacia was observed histologically, nor did fluoride affect serum PTH or Vitamin D metabolites (P > 0.4). BAP was increased 37% (P < 0.05) by fluoride; serum TRAP was increased 42% (P < 0.05); serum IGF-1 was increased 40% (P < 0.05). Fluoride increased the vertebral BV/TV by 35% (P < 0.05) and tibial ash weight by 10% (P < 0.05). However, the increases in bone mass and bone formation were not reflected in improved bone strength. Fluoride decreased bone strength by about 19% in the L5 vertebra (P < 0.01) and 25% in the femoral neck (P < 0. 05). X-ray diffraction showed altered mineral crystal thickness in fluoride-treated bones (P < 0.001), and there was a negative association between crystal width and fracture stress of the femur (P < 0.02). In conclusion, fluoride's effects on bone mass and bone turnover were not mediated by PTH. IGF-1 was increased by fluoride and was associated with increased bone turnover, but was not correlated with bone formation markers. High-dose fluoride treatment did not improve, but decreased, bone strength in rabbits, even in the absence of impaired mineralization.

Osteoporosis risk factors in female dental patients. A preliminary report.

Osteoporosis (OP) is a systemic condition that has dental implications. The purpose of this study was to compare OP risk among various dental specialty subpopulations at Indiana University School of Dentistry (IUSD). A survey was administered to 220 adult female dental patients assessing menstrual status and risk behaviors associated with low bone mass. The subjects' mean age was 48.2 +/- 1.1 years (mean +/- SEM). Overall, 38% of the surveyed patients exhibited high risk for OP. The orthodontic subpopulation (a dentate group with routine developmental malocclusions) was the youngest group and contained the lowest percentage at high-risk (6%). Conversely, the complete denture subpopulation was the oldest and contained the highest percentage of patients at high risk (53%). Postmenopausal women who had inadequate hormone replacement therapy exhibited a strong negative correlation for number of teeth retained with increasing years postmenopause (r = 0.6). Patients in the implant therapy group (many of which had adjunctive orthodontic care) had a mean age similar to the complete denture group, but a much lower risk for OP. This appears to be due to the extensive counseling these patients receive prior to treatment. It is concluded that risk factor analysis and patient counseling may be effective measures for reducing the osteoporosis risk of dental patients.

Angiogenesis and osteogenesis in an orthopedically expanded suture.

The purpose of this study was to examine the angiogenic and the subsequent osteogenic responses during a 96-hour time-course after sutural expansion. Fifty rats were divided into: (1) a control group that received only angiogenic induction through injection of 5 ng/gm recombinant human endothelial cell growth factor (rhECGF); (2) an experimental group that received orthopedic expansion and rhECGF; (3) a sham group that received expansion and sodium chloride (NaCl) injection; and (4) a baseline group that received no expansion or injection. All rats were injected with 3H-thymidine (1.0 microCi/gm) 1 hour before death to label the DNA of S-phase cells. Demineralized sections (4 microm thick) were stained with hematoxylin and eosin. Angiogenesis and cell migration were analyzed with a previously established cell kinetics model. Analysis of variance was used to test the hypothesis that enhancement of angiogenesis stimulates reestablishment of osteogenic capability. Blood vessel number, area, and endothelial cell-labeled index significantly increased in experimental groups, but no difference was found between control and baseline groups. Labeled-pericyte index and activated pericyte numbers in the experimental group were also higher than in the sham groups. These results show that supplemental rhECGF enhances angiogenesis in expanded sutures but not in nonexpanded sutures. Data also suggest that pericytes are the source of osteoblasts in an orthopedically expanded suture.

Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood.

The use of umbilical cord blood as a source of marrow repopulating cells for the treatment of pediatric malignancies has been established. Given the general availability, the ease of procurement, and progenitor content, cord blood is an attractive alternative to bone marrow or growth factor mobilized peripheral blood cells as a source of transplantable hematopoietic tissue. However, there is a major potential limitation to the widespread use of cord blood as a source of hematopoietic stem cells for marrow replacement and gene therapy. There may be enough hematopoietic stem cells to reconstitute children, but the ability to engraft an adult might require ex vivo manipulations. We describe an in vitro system in which the growth of cord blood CD34+ cells is sustained and greatly expanded for more than 6 months by the simple combination of two hematopoietic growth factors. Progenitors and cells belonging to all hematopoietic lineages are continuously and increasingly generated (the number of colony-forming unit-granulocyte-macrophage [CFU-GM] present at the end of 6 months of culture are well over 2,000,000-fold the CFU-GM present at the beginning of the culture). Very primitive hematopoietic progenitors, including long-term culture-initiating cells (LTC-ICs) and blast cell colony-forming units, are also greatly expanded (after 20 weeks of liquid culture, LTC-IC number is over 200,000-fold the initial number). The extremely prolonged maintenance and the massive expansion of these progenitors, which share many similarities with murine long-term repopulating cells, suggest that extensive renewal and little differentiation take place. This system might prove useful in diverse clinical settings involving treatment of grown-up children and adults with transplantation of normal or genetically manipulated hematopoietic stem cells.

Megakaryocyte growth and development factor (MGDF)-induced acute leukemia cell proliferation and clonal growth is associated with functional c-mpl.

The effects of human recombinant megakaryocyte growth and development factor (MGDF) (also known as thrombopoietin (TPO)), alone or in combination with other growth factors, on the proliferation and on the clonal growth of clonogenic progenitors from 24 acute myeloblastic leukemia (AML) patients were evaluated. A significant proliferative response to MGDF alone (proliferation index > 1.5) was observed in nine of 23 cases; the responding cases belonged to all FAB subtypes. However, the greatest response (proliferation index > 7) was found in one M6 and in one M7 case. MGDF also enhanced interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), c-kit ligand (KL) and FLT3 ligand (FL) stimulated blast cell proliferation. MGDF as a single factor induced or significantly enhanced colony formation by clonogenic precursor cells in 12 of 14 AML cases. MGDF strongly increased KL-induced leukemic colony growth in seven cases, whereas it only moderately enhanced IL-3- or GM-CSF-induced colony growth. The analysis of tyrosine phosphorylated protein(s) upon MGDF stimulation in fresh AML cells was also performed. The results demonstrated a band of approximately 90 kDa phosphorylated protein(s) upon MGDF stimulation in AML responsive cases, but not in unresponsive ones. Taken together the present findings suggest that, in a consistent proportion of AML cases, MGDF stimulates blast cell growth and induces tyrosine protein phosphorylation.

Sutural expansion using rigidly integrated endosseous implants: an experimental study in rabbits.

Rigidly integrated implants offer great promise for orthodontic and orthopedic anchorage in the oral and midfacial regions. Rigid anchorage can be used to control unwanted tooth movement, provide abutments in edentulous arches, and open the vertical dimension of occlusion. To evaluate the use of endosseous implants in the midface region, two flanged titanium implants were placed on either side of the midnasal suture of 18 New Zealand White rabbits. The rabbits were divided into an unloaded control and two experimental groups. One experimental group was loaded at 1 Newton (N) and the other at 3 N. All rabbits were euthanized after 12 weeks of loading. Stereologic point-hit and line-intercept methods were used to analyze microradiographic and multiple fluorochrome histology of the suture. All implants remained stable during the loading period. The distance between the implants increased significantly in the loaded groups compared with the control, and was significantly higher in the 3 N group than in the 1 N group. Percent bone volume was significantly decreased, while the percent suture volume tended to be increased in the loaded groups. Mineral apposition and bone formation rates at the sutural surfaces were increased in the loaded groups (P < 0.05), but did not differ between loaded groups. These results indicate that relatively low loads (1 or 3 N) applied to rigidly integrated endosseous implants across an unfused suture are satisfactory for achieving expansion under the conditions of this study. The 3 N load resulted in slightly more expansion, but did not affect the rate of bone formation at the suture.

Histomorphometrical analysis of the bone-implant interface: comparison of microradiography and brightfield microscopy.

Section thickness has been shown to affect the histomorphometrical measurement of bone-implant contact when analysed under brightfield microscopy. This study investigated whether microradiography of the bone-implant interface eliminated the errors associated with thick section analysis. Seven implant containing sections were utilized. Microadiographs of the thick (approximately 100 microns) sections were taken and the sections were subsequently ground to thicknesses of 50 microns and 25 microns. Photomicrographs were taken of the microradiographs and of the sections at each thickness (100, 50 and 25 microns) under brightfield microscopy. The photomicrographs were analysed for direct bone-implant contact in the cortical passage region and along the total length of the implant. The effect of section thickness on multiple fluorochrome labelling in 10 rabbit femur specimens was also examined. Centre-to-centre interlabel distance was measured for each label pair at a thickness of 100 microns and then again after the sections were ground to 50 microns and 25 microns. The thick (100 microns) sections showed a significantly greater amount of bone-implant contact than either the thin sections or the microradiographs. There was no difference in direct bone-implant contact measured by microradiography or thin sections. However, the microradiographic analysis showed a much lower variability of the bone-implant contact than the sections evaluated under brightfield microscopy. In addition, they have the added benefit of providing information on bone mineral density. Centre-to-centre interlabel distance was not significantly different for any label pair owing to section thickness. Data from this study provides evidence that the use of microradiographs for histomorphometrical analysis of the bone-implant interface is superior to brightfield analysis of thin sections owing to the lower variability of microradiographical data and the ability to obtain bone mineral density measures. Additionally, given that interlabel distance was not significantly affected by section thickness, the use of 100 microns thick sections for analysis of fluorochrome labels in cortical bone is supported.

Angiogenic induction and cell migration in an orthopaedically expanded maxillary suture in the rat.

The purpose was to examine the effect of an angiogenic factor on cell migration patterns and osteoblast histogenesis during the 96 h following orthopaedic expansion of the anterior maxillary suture. Fifty rats were divided into four groups: (1) a control group that received only angiogenic induction via injection of 5 ng/g body wt recombinant human endothelial-cell growth factor; (2) an experimental group that received orthopaedic expansion and angiogenic induction; (3) a sham group that received orthopaedic expansion and normal saline injection; and (4) a baseline group that received no expansion or injection. The experimental and sham groups were subdivided to conduct experiments over 1, 2, 3 or 4 days. The anterior portion of each maxilla was dissected free and demineralized. Sections (4 microns thick) were cut from every block and stained with Mayer's haematoxylin and eosin. Cell migration was analysed using a previously established cell-kinetics model. The osteoprogenitor cells were divided into four categories according to nuclear volume: A cells (40-79 microns3), B cells (80-119 microns3), C cells (120-169 microns3) and D cells (> or = 169 microns3 A' cells are the portion of the A cell population that responds to osteogenic stimulus. As previously defined in periodontal ligament, the reciprocal association of a decreasing number of less differentiated (A + A) cells and an increasing number of C + D cells, as a function of distance from the nearest major blood vessel, was consistently found in all groups. This suggests a vascularly oriented gradient of progressively more differentiated osteoprogenitor cells. Also, A + A' cells were predominately located within 20 microns of the nearest major blood vessel whereas the C + D cells were found at a distance > 30 microns from the nearest major blood vessel. These results suggest that the A'-->C shift occurs 20-30 microns from the nearest major blood vessel. In the angiogenic induction groups, the numbers of committed osteoprogenitors (A + A') were significantly higher than in the sham group at day 1. At day 3, the numbers of preosteoblasts (C + D) in angiogenic sutures were significantly higher than in the sham groups. This enhancement of preosteoblast population strongly suggests the possible role of activated pericytes in expanded sutures as a source of osteoprogenitor cells.

The effects of human FLT3 ligand on in vitro human megakaryocytopoiesis.

The human homolog of the murine flt3/flk2 gene product is a tyrosine kinase receptor that plays a role in regulating the proliferation and differentiation of cells in the hematopoietic system. Using a plasma-clot clonal assay and a long-term bone marrow culture (LTBMC) system, we studied the effects of the recently cloned human flt3 ligand (FL) alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or stem cell factor (c-kit ligand [KL]) on human megakaryocytopoiesis. The effects of FL on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit-megakaryocyte (BFU-MK), and the more differentiated colony-forming unit-megakaryocyte (CFU-MK) were determined. FL alone had no megakaryocytic colony-stimulating activity (MK-CSA), but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. FL synergized with IL-3 at the level of both CFU-MK and BFU-MK and with GM-CSF and KL at the level of CFU-MK. Although FL alone exhibited a limited potential in sustaining long-term megakaryocytopoiesis in vitro, it synergistically augmented the ability of IL-3 and KL, alone or in association, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that FL plays a significant role in this process by amplifying the MK-CSA of GM-CSF, IL-3, and KL.

Effects of human FLT3 ligand on myeloid leukemia cell growth: heterogeneity in response and synergy with other hematopoietic growth factors.

A novel hematopoietic growth factor for primitive hematopoietic progenitor cells, the ligand for the flt3/flk2 receptor, (FL), has been recently purified and its gene has been cloned. In the present study, we investigated the effects of FL on the proliferation and differentiation of normal and leukemic myeloid progenitor cells. We demonstrate that FL is a potent stimulator of the in vitro growth of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or G-CSF-dependent granulocyte-macrophage committed precursors from Lin- CD34+ bone marrow cells of normal donors. By contrast, FL does not affect the growth of erythroid-committed progenitors even in the presence of erythropoietin. The effect of FL on the proliferation and on the in vitro growth of clonogenic leukemic precursor cells was studied in 54 acute myeloid leukemia (AML) cases. Fresh leukemia blasts from 36 of 45 patients with AML significantly responded to FL without any relation to the French-American-British (FAB) subtype. FL stimulated the proliferation of leukemic blasts in a dose-dependent fashion. Synergistic activities were seen when FL was combined with G-CSF, GM-CSF, IL-3, or stem cell factor (SCF). FL as a single factor induced or increased significantly colony formation by clonogenic precursor cells from 21 of 24 patients with AML. In the presence of suboptimal and optimal concentrations of G-CSF, GM-CSF, IL3, SCF, or a combination of all factors, FL strongly enhanced the number of leukemic colonies (up to 18-fold). We also evaluated the induction of tyrosine phosphorylated protein on FL stimulation in fresh AML cells. We demonstrate that, on FL stimulation, a band of phosphorylated protein(s) of about 90 kD can be detected in FL-responsive, but not in FL-unresponsive cases. This study suggests that FL may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor with other cytokines.