Tissue engineered bone repair of calvarial defects using cultured periosteal cells.

Periosteum has been demonstrated to have cell populations, including chondroprogenitor and osteoprogenitor cells, that can form both cartilage and bone under appropriate conditions. In the present study, periosteum was harvested, expanded in cell culture, and used to repair critical size calvarial defects in a rabbit model. Periosteum was isolated from New Zealand White rabbits, grown in cell culture, labeled with the thymidine analog bromodeoxyuridine for later localization, and seeded into resorbable polyglycolic acid scaffold matrices. Thirty adult New Zealand White rabbits were divided into groups, and a single 15-mm diameter full-thickness calvarial defect was made in each animal. In group I, defects were repaired using resorbable polyglycolic acid implants seeded with periosteal cells. In group II, defects were repaired using untreated polyglycolic acid implants. In group III, the defects were left unrepaired. Rabbits were killed at 4 and 12 weeks postoperatively. Defect sites were then studied histologically, biochemically, and radiographically. In vitro analysis of the cultured periosteal cells indicated an osteoblastic phenotype, with production of osteocalcin upon 1,25(OH)2 vitamin D3 induction. In vivo results at 4 weeks showed islands of bone in the defects repaired with polyglycolic acid implants with periosteal cells (group I), whereas the defects repaired with untreated polyglycolic acid implants (group II) were filled with fibrous tissue. Collagen content was significantly increased in group I compared with group II (2.90 +/- 0.80 microg/mg dry weight versus 0.08 +/- 0.11 microg/mg dry weight, p < 0.006), as was the ash weight (0.58 +/- 0.11 mg/mg dry weight versus 0.35 +/- 0.06 mg/mg dry weight, p < 0.015). At 12 weeks there were large amounts of bone in group I, whereas there were scattered islands of bone in groups II and III. Radiodensitometry demonstrated significantly increased radiodensity of the defect sites in group I, compared with groups II and III (0.740 +/- 0.250 OD/mm2 versus 0.404 +/- 0.100 OD/mm2 and 0.266 +/- 0.150 OD/mm2, respectively, p < 0.05). Bromodeoxyuridine label, as detected by immunofluorescence, was identified in the newly formed bone in group I at both 4 and 12 weeks, confirming the contribution of the cultured periosteal cells to this bone formation. This study thus demonstrates a tissue-engineering approach to the repair of bone defects, which may have clinical applications in craniofacial and orthopedic surgery.

Evaluation of signal transduction mechanisms for the mitogenic effects of prostaglandin E2 in normal human bone cells in vitro.

Prostaglandin E2 (PGE2) is one of the most potent stimulators of bone formation in vivo. In these studies, we investigated the mechanism(s) underlying PGE2 effects on human bone formation by evaluating the effects of PGE2 on normal human bone cell (HBC) proliferation in vitro. Cell proliferation of normal HBCs was increased by PGE2 as measured by increased [3H]thymidine incorporation after 18 h and increased cell number after 48 h of treatment. The effect of PGE2 to stimulate cell proliferation was biphasic, with a maximum stimulation between 0.01 and 1.0 nM PGE2 in different experiments. At higher concentrations of PGE2 (0.1 microM), HBC proliferation was inhibited. Signal transduction for PGE2 has been reported to include both protein kinase A (PKA) and protein kinase C (PKC) pathways. In these studies, concentrations of PGE2 which stimulated cell proliferation did not increase cyclic adenosine monophosphate (cAMP) production. However, higher concentrations of PGE2 increased cAMP production (7- to 12-fold at 1-10 microM) and inhibited cell proliferation. Because stimulators of PKC, such as phorbol esters, have been reported to stimulate cell proliferation, the action of PKC inhibitors were tested. Both staurosporine and sangivamysin (PKC inhibitors) totally abrogated the effect of PGE2 to stimulate cell proliferation. Additional studies revealed that PGE2 increased 45Ca uptake in a dose-dependent manner with a peak response occurring between 1 and 10 nM PGE2 concentrations in different experiments. Furthermore, when the calcium channel blocker, verapamil, was added to HBC cultures treated with PGE2, the stimulation of 45Ca uptake and cell proliferation by PGE2 was completely blocked. These data suggest that PGE2 increases cell proliferation through activation of a verapamil-sensitive calcium channel. In conclusion, these data are consistent with a model in which stimulation of HBC proliferation by low doses of PGE2 is mediated by an enhancement of phospholipase C, which results in both an increase in PKC activity and an increase in intracellular calcium influx.

Effects of extracellular calcium on insulin-like growth factor II in human bone cells.

Extracellular calcium concentration is critically important for normal function of the body. Recently, reports have shown that cells derived from parathyroid glands contain an extracellular calcium receptor that is responsive to changes in extracellular calcium. Bone is intimately involved in calcium homeostasis; therefore, we sought to test the hypothesis that extracellular calcium has direct effects on bone cells. Extracellular calcium was increased by the addition of varying concentrations of CaCl2 (0.4-2.0 mM) to the control medium. An increase in extracellular calcium increased cell proliferation, as assessed by 3H-thymidine incorporation, in a number of cell types including normal human bone cells derived from vertebrae (HBV155) and a number of human osteosarcoma cell lines. The increase in cell proliferation by elevated CaCl2 was dose dependent, whereas MgCl2 was not effective at the doses tested (up to 2 mM added MgCl2). To test the hypothesis that the mitogenic activity of elevated extracellular calcium involved a growth factor, levels of insulin-like growth factor II (IGF-II) were measured in the conditioned medium of HBV155 cells by radioimmunoassay after removal of binding proteins by size exclusion chromatography. The effects of an increase in extracellular calcium by 1 mM were: 1) increased culture media levels of IGF-II within 1 h of treatment, 2) the increase in IGF-II levels reached a maximum after 8 h of treatment, and 3) IGF-II levels were still elevated after 24 h of treatment. Furthermore, a blocking monoclonal antibody against IGF-II abolished the increased cell proliferation in HBV155 cells following elevation of extracellular calcium. Taken together, these findings suggest that an increase in extracellular calcium results in an increase in IGF-II which is required for the subsequent increase in cell proliferation.

Combined magnetic fields increase insulin-like growth factor-II in TE-85 human osteosarcoma bone cell cultures.

In vitro exposure to low-energy, combined magnetic fields (CMF) increased the release of insulin-like growth factor (IGF)-II from human TE-85 osteosarcoma cells. Short-term CMF exposure of only 10 min increased IGF-II levels in conditioned medium 1 h post CMF exposure. IGF-II levels were measured with a radioreceptor assay using H-35 cells that contain abundant IGF-II but not IGF-I receptors. This assay also uses a recently validated BioGel P-10 acid gel filtration method to remove IGF binding protein before quantitation of either IGF-I or IGF-II. In addition to an increase in IGF-II levels, DNA synthesis, as an index of cell proliferation, was increased during the 24-h period post CMF exposure. A monoclonal antibody against IGF-II blocked the increase in cell proliferation following CMF exposure, whereas a control monoclonal antibody against osteocalcin did not attenuate the mitogenic action of CMF exposure. The effect of CMF exposure to increase both cell proliferation and IGF-II was cell-density dependent with greater stimulation by CMF observed at lower densities. Together, these data are consistent with the hypothesis that CMF exposure stimulates release/production of IGF-II from bone cells and that increased IGF-II then promotes an increase in cell proliferation.

IGF-II receptor number is increased in TE-85 osteosarcoma cells by combined magnetic fields.

Human osteosarcoma-derived osteoblast-like cells, TE-85, were used to assess the effect of a low frequency alternating magnetic field in combination with a controlled static magnetic field (combined magnetic fields, CMF) on insulin-like growth factor receptor regulation. In our culture system, application of a 15.3 Hz CMF induces a calculated maximum electrical potential in the culture media of 10(-5) V/m. Initial characterization of TE-85 cells demonstrated that (a) TE-85 cells contain both type I insulin-like growth factor (IGF-I) and IGF-II receptors and (b) dose dependence for IGF-stimulated cell proliferation were comparable to the affinities of the IGF's binding to membrane binding sites (i.e., receptors had dissociation constants in the low nanomolar concentration range). The studies with CMF exposure revealed that CMF treatment for 30 minutes increased the number of IGF-II receptors in a frequency-dependent manner without affecting the number of IGF-I receptors. The CMF-dependent increase in IGF-II receptor number was associated with a significant increase in the IGF-II dissociation constant. These results indicate that a membrane receptor levels can be altered by short-term exposure to low-energy, low-frequency electromagnetic fields and suggest a potential biochemical mechanism for electromagnetic effects on bone formation and remodeling.

Overview of effects of electrical stimulation on osteogenesis and alveolar bone.

One endpoint of periodontal therapy is to regenerate structure lost to periodontal disease. Periodontal regeneration requires both formation of a new connective tissue attachment to the tooth and formation of alveolar bone. Several procedural advances may support regeneration of the attachment, however, regeneration of alveolar bone does not occur consistently. Therefore, factors which stimulate bone repair are areas for research in periodontal reconstructive therapy. Effects of cytokines or growth factors on bone repair are examples of such areas. Another one is electrical stimulation which naturally occurs in bone, and as such bone may be particularly susceptible to electrical therapy. This overview describes the potential of electrical stimulation for bone regeneration and applications in alveolar and periodontal research.

Combined magnetic fields increased net calcium flux in bone cells.

Low energy electromagnetic fields (EMF) exhibit a large number of biological effects. A major issue to be determined is "What is the lowest threshold of detection in which cells can respond to an EMF?" In these studies we demonstrate that a low-amplitude combined magnetic field (CMF) which induces a maximum potential gradient of 10(-5) V/m is capable of increasing net calcium flux in human osteoblast-like cells. The increase in net calcium flux was frequency dependent, with a peak in the 15.3-16.3 Hz range with an apparent bandwidth of approximately 1 Hz. A model that characterizes the thermal noise limit indicates that non-spherical cell shape, resonant type dynamics, and signal averaging may all play a role in the transduction of low-amplitude EMF effects in biological systems.

Growth factors and electromagnetic fields in bone.

Stimulation of bone by low-energy electromagnetic fields (EMFs) may prove to be an efficient therapy for skeletal disorders. The study discussed in this article demonstrates that an EMF of 15.3 Hz can increase the proliferation rate of an osteosarcoma cell line, TE-85. The increase in cell proliferation was associated with an increase in binding sites for insulin-like growth factor II and an increase in mitogen activity in culture media.

Low-amplitude, low-frequency electric field-stimulated bone cell proliferation may in part be mediated by increased IGF-II release.

We have developed an in vitro model incorporating a low-amplitude (10(-7) V/cm), low frequency (f less than 100 Hz), capacitively coupled electric field in order to study the mechanism through which an electric field may increase bone cell proliferation. Utilizing this model we have previously shown that electric field-stimulated bone cell proliferation was dependent on release of mitogen activity into the culture medium from exposed cells. The current studies were intended to characterize this mitogen activity. In these studies we found that electric field-stimulated human bone cell proliferation was associated with increased IGF-II mRNA accumulation and IGF-II secretion suggesting that IGF-II may in part mediate the increase in bone cell proliferation following electric field exposure.

Fluoride pharmacokinetics in good and poor responders to fluoride therapy.

In this study, the relationship between fluoride pharmacokinetics and the response in spinal bone density to fluoride treatment was studied in 14 patients with primary osteoporosis treated with fluoride for at least 1 year. Serum concentrations and urinary excretion of fluoride were determined after ingestion of 10 mg fluoride as monofluorophosphate. The pharmacokinetic parameters were calculated according to a linear one-compartment open model. The fasting serum fluoride level was 8.8 +/- 0.98 mumol/liter. The peak serum fluoride level was 20.5 +/- 1.4 mumol/liter and was reached within 2 h after ingestion of fluoride. When the patients were divided into good and poor responders, based on whether they did or did not exhibit a change in spinal bone density of 13 mg/cc per year or more, we found that good responders had decreased renal fluoride clearance (-62 +/- 13%, p less than .02), increased maximum change in serum fluoride (+38 +/- 18%, p less than .01), increased extrarenal clearance (+62 +/- 57%, p less than .05) and increased change in serum alkaline phosphatase (ALP) (+241 +/- 169%, p less than 0.02) compared with poor responders. Our data suggest that one factor accounting for a good response is a relatively high serum level of fluoride. However, although the maximum change in serum fluoride was greater in good responders compared with poor responders, variations in fluoride levels could not explain all of the variation in spinal bone density. Therefore, we propose that in addition to differences in serum fluoride, other factors are also responsible for the good response.

Frequency dependence of increased cell proliferation, in vitro, in exposures to a low-amplitude, low-frequency electric field: evidence for dependence on increased mitogen activity released into culture medium.

In order to investigate the mechanism(s) through which an electric field can increase bone cell proliferation, we have developed an in vitro model incorporating a low-amplitude (estimated 10(-7) V/cm in the serum-free culture medium), low-frequency, capacitively coupled electric field. In previous studies with this model, we have shown that electric field exposure can increase bone cell proliferation both in chick tibiae organ cultures and in calvaria-derived monolayer cell cultures. The current in vitro studies demonstrate that skeletal tissue responses to a 30 min electric field exposure are characterized by a) a frequency window for both increased cell proliferation and increased release of mitogen activity into the cell-conditioned medium, with a peak near 16 Hz; b) a dependence on conditioned medium from exposed cells for increased cell proliferation; and c) a correlation between the alkaline phosphatase content of the bone cell cultures and effects of electric field exposure on both cell proliferation and release of mitogen activity into the conditioned medium.

Embryonic bone matrix formation is increased after exposure to a low-amplitude capacitively coupled electric field, in vitro.

In order to investigate the mechanism(s) of electric field-stimulated osteogenesis, we have developed an in vitro model in which embryonic chick tibiae have consistently demonstrated increased bone matrix formation in response to a low amplitude (estimated 10(-5) V/m in the serum-free culture medium), capacitively coupled, 10 Hz sinusoidal electric field. Initial applications of this model revealed that 72 h of continuous exposure to the electric field increased tibial collagen production by 29% compared to untreated controls, P less than 0.01. Additional studies further revealed: (a) that when electric field exposure was limited to 30 min/day during the 72 h in vitro incubation, embryonic bone matrix formation was increased by 83%, compared to non-treated controls (P less than 0.001), suggesting an inductive mechanism; (b) that the osteogenic response to electric field exposure in vitro was not unique to embryonic chick tibiae, since a similar response was also seen with newborn mouse calvaria (+133%, P less than 0.02); (c) that electric field-exposure-stimulated chick bone matrix formation was associated with increased bone cell proliferation; and (d) that this mitogenic response to in vitro electric field exposure could also be observed with embryonic chick calvarial cells in monolayer, serum-free cultures.

Free-standing emergency clinics: community development guidelines.

When a free-standing emergency clinic (FEC) unexpectedly opened in Montgomery County, Maryland, the local emergency medical services council thought the FEC's operational plan was in conflict with the community concept of emergency medical services (EMS). Because there were no agreed upon factual guidelines with which to judge the FEC, the council established a task force to study the problem and to develop standards and guidelines. This article is based on that report. We recommend that if a facility physically separate from a hospital uses the words "emergency" in offering medical services, it means at least 24 hours of operation with standards at least equal to those offered by the local EMS system.