Solomon R. Pollack

Bone tissue engineering in a rotating bioreactor using a microcarrier matrix system

A novel approach was utilized to grow in vitro mineralized bone tissue using lighter-than-water, polymeric scaffolds in a high aspect ratio rotating bioreactor. We have adapted polymer microencapsulation methods for the formation of hollow, lighter-than-water microcarriers of degradable poly(lactic-co-glycolic acid). Scaffolds were fabricated by sintering together lighter-than-water microcarriers from 500 to 860 microm in diameter to create a fully interconnected, three-dimensional network with an average pore size of 187 microm and aggregate density of 0.65 g/mL. Motion in the rotating bioreactor was characterized by numerical simulation and by direct measurement using an in situ particle tracking system. Scaffold constructs established a near circular trajectory in the fluid medium with a terminal velocity of 98 mm/s while avoiding collision with the bioreactor wall. Preliminary cell culture studies on these scaffolds show that osteoblast-like cells readily attached to microcarrier scaffolds using controlled seeding conditions with an average cell density of 6.5 x 10(4) cells/cm(2). The maximum shear stress imparted to attached cells was estimated to be 3.9 dynes/cm(2). In addition, cells cultured in vitro on these lighter-than-water scaffolds retained their osteoblastic phenotype and showed significant increases in alkaline phosphatase expression and alizarin red staining by day 7 as compared with statically cultured controls.

Signal Transduction in Electrically Stimulated Bone Cells

Background: Electrical stimulation is used to treat nonunions and to augment spinal fusions. We studied the biochemical pathways that are activated in signal transduction when various types of electrical stimulation are applied to bone cells. Methods: Cultured MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields at appropriate field strengths for thirty minutes and for two, six, and twenty-four hours. The DNA content of each dish was determined. Other cultures of MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields for two hours in the presence of various inhibitors of signal transduction, with or without electrical stimulation, and the DNA content of each dish was determined. Results: All three signals produced a significant increase in DNA content per dish compared with that in the controls at all time-points (p < 0.05), but only exposure to capacitive coupling resulted in a significant, ever-increasing DNA production at each time-period beyond thirty minutes. The use of specific metabolic inhibitors indicated that, with capacitive coupling, signal transduction was by means of influx of Ca2+ through voltage-gated calcium channels leading to an increase in cytosolic Ca2+ (blocked by verapamil), cytoskeletal calmodulin (blocked by W-7), and prostaglandin E2 (blocked by indomethacin). With inductive coupling and combined electromagnetic fields, signal transduction was by means of intracellular release of Ca2+ leading to an increase in cytosolic Ca2+ (blocked by TMB-8) and an increase in activated cytoskeletal calmodulin (blocked by W-7). Conclusions: The initial events in signal transduction were found to be different when capacitive coupling was compared with inductive coupling and with combined electromagnetic fields; the initial event with capacitive coupling is Ca2+ ion translocation through cell-membrane voltage-gated calcium channels, whereas the initial event with inductive coupling and with combined electromagnetic fields is the release of Ca2+ from intracellular stores. The final pathway, however, is the same for all three signals—that is, there is an increase in cytosolic Ca2+ and an increase in activated cytoskeletal calmodulin. Clinical Relevance: Electrical stimulation in various forms is currently being used to treat fracture nonunions and to augment spinal fusions. Understanding the mechanisms of how bone cells respond to electrical signals—that is, understanding signal transduction and the metabolic pathways utilized in electrically induced osteogenesis—will allow optimization of the effects of the various bone-growth-stimulation signals.

The proliferative and synthetic response of isolated calvarial bone cells of rats to cyclic biaxial mechanical strain.

Isolated bone cells from the calvaria of newborn rats were grown in monolayer on polyurethane membranes in specially constructed culture chambers. These were subjected to cyclic biaxial mechanical strains of 0.02 per cent (200 microstrain), 0.04 per cent (400 microstrain), and 0.1 per cent (1000 microstrain) at a frequency of one hertz for periods ranging from fifteen minutes to seventy-two hours. DNA content, an index of proliferation, was significantly increased at a strain of 0.04 per cent applied for fifteen minutes and for twenty-four and forty-eight hours. DNA content was not increased at the other amplitudes of strain that were evaluated, nor was it increased after prolonged mechanical stimulation for forty-eight hours or longer. Synthesis of collagen, non-collagenous protein, and proteoglycan, as well as activity of alkaline phosphatase, all indicators of macromolecular synthesis, were significantly decreased at a strain of 0.04 per cent applied for fifteen minutes and for twenty-four, forty-eight, and seventy-two hours. Macromolecular synthesis was not affected by the other amplitudes of strain that were evaluated in this study. At a strain of 0.04 per cent, prostaglandin E2 content was significantly increased after five, fifteen, and thirty minutes of mechanical stimulation, whereas net cAMP content did not change significantly. This suggests that the described cellular events (increased proliferation and decreased macromolecular synthesis) that occur secondary to mechanical strain are mediated, at least in part, by prostaglandin E2.

The Influence of Functional Use of Endosseous Dental Implants on the Tissue-implant Interface. I. Histological Aspects

Functional and non-functional endosseous dental implants were clinically compared in beagle mandibles for up to one year post-operatively. Differing biomechanical conditions led to clinical differences between functional and non-functional implants. Typical clinical tests, however, did not always reveal detailed histological differences between implant-tissue interfaces of functional and non-functional implants.

Intracellular Ca2+ stores and extracellular Ca2+ are required in the real-time Ca2+ response of bone cells experiencing fluid flow

In this study, we sought to determine if there is a requirement for calcium entry from the extracellular space as well as calcium from intracellular stores to produce real-time intracellular calcium responses in cultured bone cells subjected to fluid flow. Understanding calcium cell signaling may help to elucidate the biophysical transduction mechanism(s) mediating the conversion of fluid flow to a cellular signal. An experimental design which utilized a scheme of pharmacological blockers was employed to distinguish between the biochemical pathways involved in this cell signaling. A parallel-plate flow chamber served as the cell stimulating apparatus and a fluorescence microscopy system using the calcium-sensitive dye fura-2 measured the intracellular calcium changes. In the present study, evidence for a role by the inositol-phospholipid biochemical pathway, specifically inositol trisphosphate (IP3) was obtained using neomycin which completely inhibited the calcium response to flow. Additionally, a concomitant role of extracellular calcium was demonstrated through experiments performed in calcium-free medium which also eliminated the flow response. Experiments conducted with gadolinium, a stretch-activated channel blocker, partially inhibited (approximately 30%) the flow response while verapamil, a type-L voltage sensitive channel blocker, had no effect on the flow response. These results suggest a requirement of extracellular calcium (or calcium influx) as well as IP3-induced calcium release from intracellular stores for generating the intracellular calcium response to flow in bone cells.

Dielectric Permittivity and Electrical Conductivity of Fluid Saturated Bone

The dielectric permittivity and electrical conductivity of freshly excised and formalin fixed samples of rat femoral bone were determined over a frequency range of 10 Hz-100 MHz. Impedance measurements were performed in the frequency domain using a vector impedance meter and an impedance analyzer. The results of these measurements show that the conductivity of fixed and fresh bone is nearly independent of frequency below 100 kHz, with the conductivity of fresh bone being two to three times greater than that of the fixed sample. At higher frequencies, the conductivity increases as a power function of frequency. The permittivity of bone reaches very high values at low frequencies, but decreases rapidly with increasing frequencies and approaches a limiting value of about ten. This high-frequency limit is consistent with the water content of the tissue, and with the permittivity of the anhydrous matrix. It is suggested that the olarizability observed at audio and radiowave frequencies is in part sssociated with the collagen phase, although other interfacial polarization effects can also be present.

Electromechanical potentials in cortical bone--II. Experimental analysis.

The electrokinetic model developed in Part 1 of this paper is used to characterize the electromechanical effect in cortical bone. Low frequency characteristics of stress-generated potentials are measured to provide insight into the origin and generation of these potentials induced in fluid-filled cortical bone. The results support the proposed model and indicate that fluid movement within the microporosity of bone is responsible for observed potentials whose origin is electrokinetic. The microporosity in bone, composed of the fluid spaces in and around mineral crystals encrusting collagen fibrils, constitutes an enormous surface area and appears to dominate surface-related phenomena at low frequencies. Previous experimental results, reported by many researchers, are also supported by this mechanism.

Electromechanical potentials in cortical bone—I. A continuum approach

An electrokinetic model to characterize the electromechanical effect in cortical bone has been developed using the basic principles of the biphasic theory of porous materials and a simple model for permeability and charge distribution for cortical bone. The model is developed analytically in Part I of this paper and is shown to account qualitatively for the principal experimental results reported to date. Part II of this paper concerns experimental analysis of this model, reporting results of low frequency testing of the dynamic characteristics of stress-generated potentials. Quantitative analysis of these results indicates that the microporosity of bone, made up of the channels around the hydroxyapatite encrusting the collagen matrix, is the compartment responsible for the electromechanical effects in fluid-saturated cortical bone. This microporous compartment would seem to be the obvious source of the electrokinetic effect, because it has the greatest surface area in bone and constitutes the rate limiting fluid flow compartment in deformation-induced fluid flow at low frequency.

AN ANATOMICAL MODEL FOR STREAMING POTENTIALS IN OSTEONS

An anatomical model for streaming potentials in osteons is developed to characterize the electromechanical effect in bone. The model accounts for the microstructure of the osteon and is based upon first principles of electrochemistry, electrokinetics, continuum mechanics and fluid dynamics. Intra-osteonal potentials and their relaxation times are numerically evaluated. Many of the previously reported observations of potentials in osteons and across macroscopic specimens are explained for the first time in terms of an electrokinetic model. The cusp-like behavior of intra-osteonal potentials is explained, the dependence of the potentials on solution viscosity and conductivity is demonstrated, and insight is gained relative to the time dependence of stress generated potentials.

Biochemical pathway mediating the response of bone cells to capacitive coupling.

Rat calvarial bone cells or mouse MC3T3-E1 bone cells subjected to a capacitively coupled electric field of 20 m V/cm consistently showed significant increases in cellular proliferation as determined by deoxyribonucleic acid content. Verapamil, a membrane calcium channel blocker; W-7, a calmodulin antagonist; indocin, a prostaglandin synthesis inhibitor; or bromophenacyl bromide, a phospholipase A2 inhibitor, each at a concentration that did not interfere with cell proliferation in control cultures, inhibited proliferation in those cultures subjected to the electric field. In contrast, neomycin, an inhibitor of the inositol phosphate cascade, did not inhibit this electrically induced cellular proliferation. Prostaglandin E2 production also was increased significantly with electrical stimulation, and this increase was inhibited by verapamil or indocin but not by neomycin. Thus, the data suggest that the signal transduction mediating the proliferative response of cultured bone cells to a capacitively coupled field involved transmembrane calcium translocation via voltage gated calcium channels, activation of phospholipase A2, and a subsequent increase in prostaglandin E2. Increases in cytosolic calcium and activated calmodulin are implied. The inositol phosphate pathway, unlike its dominant role in signal transduction in mechanically stimulated bone cells, does not appear to play a role in signal transduction in the proliferative response of bone cells to electrical stimulation.