Robert J. Fitzsimmons

tissue Engineered Bone Repair of Calvarial Defects Using Cultured Periosteal Cells

&NA; 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 I 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 &mgr;g/mg dry weight versus 0.08 ± 0.11 &mgr;g/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.

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 nonspherical cell shape, resonant type dynamics, and signal averaging may all play a role in the transduction of lowamplitude EMF effects in biological systems.

A pulsing electric field (PEF) increases human chondrocyte proliferation through a transduction pathway involving nitric oxide signaling

A potential treatment modality for joint pain due to cartilage degradation is electromagnetic fields (EMF) that can be delivered, noninvasively, to chondrocytes buried within cartilage. A pulsed EMF in clinical use for recalcitrant bone fracture healing has been modified to be delivered as a pulsed electric field (PEF) through capacitive coupling. It was the objective of this study to determine whether the PEF signal could have a direct effect on chondrocytes in vitro. This study shows that a 30‐min PEF treatment can increase DNA content of chondrocyte monolayer by approximately 150% at 72 h poststimulus. Studies intended to explore the biological mechanism showed this PEF signal increased nitric oxide measured in culture medium and cGMP measured in cell extract within the 30‐min exposure period. Increasing calcium in the culture media or adding the calcium ionophore A23187, without PEF treatment, also significantly increased short‐term nitric oxide production. The inhibitor W7, which blocks calcium/calmodulin, prevented the PEF‐stimulated increase in both nitric oxide and cGMP. The inhibitor L‐NAME, which blocks nitric oxide synthase, prevented the PEF‐stimulated increase in nitric oxide, cGMP, and DNA content. An inhibitor of guanylate cyclase (LY83583) blocked the PEF‐stimulated increase in cGMP and DNA content. A nitric oxide donor, when present for only 30 min, increased DNA content 72 h later. Taken together, these results suggest the transduction pathway for PEF‐stimulated chondrocyte proliferation involves nitric oxide and the production of nitric oxide may be the result of a cascade that involves calcium, calmodulin, and cGMP production.

Electromagnetic fields as first messenger in biological signaling: Application to calmodulin-dependent signaling in tissue repair

BACKGROUND The transduction mechanism for non-thermal electromagnetic field (EMF) bioeffects has not been fully elucidated. This study proposes that an EMF can act as a first messenger in the calmodulin-dependent signaling pathways that orchestrate the release of cytokines and growth factors in normal cellular responses to physical and/or chemical insults. METHODS Given knowledge of Ca(2+) binding kinetics to calmodulin (CaM), an EMF signal having pulse duration or carrier period shorter than bound Ca(2+) lifetime may be configured to accelerate binding, and be detectable above thermal noise. New EMF signals were configured to modulate calmodulin-dependent signaling and assessed for efficacy in cellular studies. RESULTS Configured EMF signals modulated CaM-dependent enzyme kinetics, produced several-fold increases in key second messengers to include nitric oxide and cyclic guanosine monophosphate in chondrocyte and endothelial cultures and cyclic adenosine monophosphate in neuronal cultures. Calmodulin antagonists and downstream blockers annihilated these effects, providing strong support for the proposed mechanism. CONCLUSIONS Knowledge of the kinetics of Ca(2+) binding to CaM, or for any ion binding specific to any signaling cascade, allows the use of an electrochemical model by which the ability of any EMF signal to modulate CaM-dependent signaling can be assessed a priori or a posteriori. Results are consistent with the proposed mechanism, and strongly support the Ca/CaM/NO pathway as a primary EMF transduction pathway. GENERAL SIGNIFICANCE The predictions of the proposed model open a host of significant possibilities for configuration of non-thermal EMF signals for clinical and wellness applications that can reach far beyond fracture repair and wound healing.

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.

The role of insulin-like growth factor II in magnetic field regulation of bone formation

Abstract Musculoskeletal tissue is uniquely sensitive to biophysical input, as demonstrated by both mechanical and electrical stimulation experiments. However, the mechanism by which biophysical input couples to cellular processes is not well understood. The results presented in these studies suggest that these stimuli are bioactive due to stimulation of growth factor biosynthesis by musculoskeletal target cells. We chose insulin-like growth factors (IGFs) as the model growth factor, as the IGFs are capable of stimulating chemotaxis, proliferation, and differentiation of osteoprogenitor cells. These specific studies addressed whether short-term exposure to combined a.c. and d.c. magnetic fields (CMF) would increase production of IGF-II by both osteoblast-like cell cultures as well as rat fracture callus cultures. In vitro studies on human osteoblast-like cell cultures demonstrated statistically significant increases in IGF-II levels and DNA synthesis after only 30 min CMF exposure. In rat fracture callus explant cultures, IGF-II levels were increased at least two-fold dependent on the callus differentiation stage, and these results were comparable with the effect of the osteotropic agent, parathyroid hormone. In summary, these results suggest that the mechanism by which CMF, and other biophysical stimuli, regulate musculoskeletal repair is by modulation of endogenous growth factor (IGF-II) synthesis and secretion.

Coupling of Bone Formation and Bone Resorption: A Model

Publisher Summary This chapter presents a model to explain the coupling of formation to resorption and the factors that could influence the amount of resorption cavity fill-in and thereby determine whether there is again or a loss of bone. One of the major functions of bone is to serve the mechanical needs of the body. The control mechanism that determines the extent to which bone accomplishes this function is the coupling of bone formation to bone resorption; this, in turn, regulates bone mass. Under normal steady-state conditions during the bone remodeling process, the level of resorption cavity fill-in by osteoblasts is identical to the size of the resorption cavity and, as such, bone formation is said to be coupled to bone formation. There are several local sources of cytokines and growth factors that are relevant to the proliferation and differentiation of osteoblasts, cells whose synthetic activity is required for resorption cavity fill-in. Resorbing cytokines not only regulate bone resorption, but also either increase or decrease osteoblast proliferation. Osteoclasts can produce the IGFs and TGFβ and probably other growth factors. The bone tissue itself is a storage depot for many growth factors. Osteocytes and osteoblasts produce growth factors in response to several conditions, including mechanical forces. The coupling of formation to resorption is an important mechanism in health and disease and is also the target of therapeutic agents to replete an osteoporotic skeleton.

Biophysical stimulation of tissue healing mediated by IGF-II

Cells and tissues respond to a large variety of extracellular signals, including electromagnetic fields (EMF). Recent studies have demonstrated that combined AC and DC magnetic fields may couple specifically to ion dependent cellular processes. This coupling suggests an extraordinary potential for use of these combined magnetic fields for tissue healing applications in clinical situations. To this end, we have perfomed studies on in vitro osteoblast and in vivo rat osteoporosis model systems. Since osteoporosis is a result of impaired bone formation/bone resoprtion, we proposed to test whether direct osteoblast activation via calcium-dependent pathways would prevent bone loss in a model of hormonally induced osteoporosis. The cellular studies addressed the question of whether combined magnetic fields could induce cell proliferation, and whether this effect was based on autocrine growth factor (insulin-like growth factor; IGF-II) stimulation by osteoblasts.