Carl T. Brighton

The pericyte as a possible osteoblast progenitor cell

Bovine capillary and microvessel pericytes were grown in monolayer in standard tissue culture medium supplemented with 10% newborn calf serum at various oxygen tensions for up to ten weeks. The pericytes synthesized alkaline phosphatase and formed colonies that mineralized. Energy dispersive X-ray spectrometry revealed the presence of calcium and phosphate, showed positive staining for collagen and glycosaminoglycan, and, most importantly, demonstrated the synthesis of osteocalcin. Cell proliferation, hydroxyproline production, and alkaline phosphatase synthesis were greatest in 3% oxygen, whereas osteocalcin production was least in 3% oxygen. These findings demonstrate that the capillary or microvessel pericyte exhibits phenotypic expressions in vitro that are similar to that of in vitro bone cells, and these expressions may be somewhat oxygen dependent. It is suggested from these findings that the capillary or microvessel pericyte may be an osteoblast precursor cell.

Early Histological and Ultrastructural Changes in Medullary Fracture Callus

Light and electron microscopic studies of early changes in the medullary callus of a fracture of the rib in rabbits revealed the loss of normal architecture of the marrow and the disappearance of blood vessels in the region of high cellular density adjacent to the fibrin clot; the enlargement and transformation of capillary and venous endothelial cells in the region of low cellular density adjacent to the normal, uninjured marrow; the appearance of polymorphic mesenchymal cells throughout the medullary callus; and the appearance of osteoblasts and new-bone formation by twenty-four hours after the fracture. The meaning of these morphological changes is not clear. However, the spatial relationship between the various cells suggests the possibility that the transformed endothelial cells, reticular cells, and polymorphic mesenchymal cells may be interrelated and may either be osteoblast progenitor cells or may in some way lead to the appearance of osteoblasts in the early callus.

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.

Oxygen Tension of Healing Fractures in the Rabbit

True appreciation of the structure and function of the knee joint meniscus is a relatively recent occurrence. Even decades after studies were published documenting the detrimental effects of meniscectomoy such as osteoarthritis, many physicians continued to advocate total meniscectomy for even minor meniscal pathology. Fortunately, the clinical approach to these problems has undergone dramatic change over the past 20 years. A review of the anatomy and ultrastructure of the meniscus, and its relationship to normal function in terms of load transmission, shock absorption, joint stability, lubrication, and nutrition will enable a better understanding of the rationale for meniscus preservation techniques, including meniscal repair and meniscal transplant.

Oxygen Tension in Zones of the Epiphyseal Plate, the Metaphysis and Diaphysis: An in Vitro And in Viro Study In Rats And Rabbits

1. A technique for measuring oxygen tension in different zones of the epiphyseal plate, the metaphysis and the diaphysis in vitro and in vivo is described. 2. In the in vitro preparations (costochondral junctions of twenty-one-day-old rats), there is a distinct oxygen gradient corresponding to the morphological zones, beginning with a low tension of 19.5 millimeters of mercury in the zone of small size cartilage cells and increasing progressively to a high tension of 95.2 millimeters of mercury in metaphyseal bone. 3. In sharp contrast, the in vivo preparations (proximal tibial epiphyses of six-week-old rabbits) exhibited a very low-oxygen tension in the zone of hypertrophic cells and in the metaphysis. 4. A steep gradient in oxygen tension was present between metaphyseal bone (19.8 millimeters of mercury) and diaphyseal bone (108.7 millimeters of mercury) in the in vivo preparations. 5. The significance of these findings is discussed, and the physiological role of oxygen in epiphyseal plate growth is examined.

Bioelectric Potentials in Bone

Bioelectric phemiomenia have been intensively studied in plamit arid animal tissue. Most. of this research has concentrated on rapidly evoked potentials of short duratiomi, w’hile the study of steady, direct current potentials of lomig duratiomi has proceeded more slowly. Steady, resting potentials, ranging from microvolts to more than one hundred millivolts, have been recorded in biological systems. Single cells, extracellular collagemi and crystallites, membranes, skin, amid whole organs have beeni

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.

Use of Physical Forces in Bone Healing

Abstract During the past two decades, a number of physical modalities have been approved for the management of nonunions and delayed unions. Implantable direct current stimulation is effective in managing established nonunions of the extremities and as an adjuvant in achieving spinal fusion. Pulsed electromagnetic fields and capacitive coupling induce fields through the soft tissue, resulting in low‐magnitude voltage and currents at the fracture site. Pulsed electromagnetic fields may be as effective as surgery in managing extremity nonunions. Capacitive coupling appears to be effective both in extremity nonunions and lumbar fusions. Low‐intensity ultrasound has been used to speed normal fracture healing and manage delayed unions. It has recently been approved for the management of nonunions. Despite the different mechanisms for stimulating bone healing, all signals result in increased intracellular calcium, thereby leading to bone formation.

Stimulation of Fracture Healing by Direct Current in the Rabbit Fibula

An undisplaced fracture of each fibula in a rabbit was permitted to heal for eighteen days. The fracture in one fibula of each animal was subjected to a ten microampere galvanic current, the electrodes being placed in various positions relative to the fracture. Leads were placed in the opposite (control) fibula but did not deliver any current. Each fibular fracture was studied by roentgenogram, stressed for rigidity, and evaluated microscopically. The evidence strongly suggests that a cathodal current of this intensity placed within the fracture site stimulates fracture healing.

The biochemical pathway mediating the proliferative response of bone cells to a mechanical stimulus

Calvarial bone cells of rats were subjected to either a cyclic biaxial strain of 0.17 per cent (1700 microstrain) or a hydrostatic pressure of 2.5, five, or ten pounds per square inch (17.2, 34.5, or sixty-nine kilopascals). The frequency was held constant at one hertz for both types of mechanical stimulation. When cultured bone cells that had been subjected to a cyclic biaxial strain for two hours were harvested twenty-two hours later, it was found that the level of prostaglandin E2 had increased significantly (p < 0.01) as had cellular proliferation (p < 0.01), as indicated by the incorporation of [3H]-thymidine. The addition to the medium of indomethacin, an inhibitor of prostaglandin synthesis, at a ten-micromolar concentration significantly inhibited (p < 0.01) the increase in prostaglandin E2 synthesis but had no effect on the strain-induced increase in cellular proliferation, as indicated by the incorporation of [3H]-thymidine. Twenty-four hours after exposure to the same cyclic biaxial strain for thirty seconds, other cultured bone cells showed a significant increase in the level of cytoskeletal calmodulin (p < 0.05) and in the DNA content (p < 0.05). N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist, was added to the medium at a one-micromolar concentration, which had been shown to have no effect on the increase in the DNA content of control cells; W-7 completely blocked the increase in the level of cytoskeletal calmodulin and in the DNA content in the cells that were subjected to a cyclic biaxial strain. The bone cells subjected to a hydrostatic pressure showed a dose-dependent increase in the concentration of cytosolic Ca2+, as measured with Fura 2-AM, a fluorescent indicator of intracellular calcium. With a pressure of ten pounds per square inch (sixty-nine kilopascals), the increase in the concentration of cytosolic Ca2+ was nearly eight times greater than that at 2.5 pounds per square inch (17.2 kilopascals) (126 ± 15.2 compared with 16 ± 8.0 nanomolar, mean and standard deviation). The addition to the medium of neomycin, an inhibitor of the inositol phosphate cascade, at a ten-millimolar concentration completely blocked the increase in the concentration of cytosolic Ca2+ in these cells; this concentration of neomycin had been shown to have no effect on proliferation in control bone cells. There was also a dose-dependent relationship between the duration of the stimulus and the cellular proliferation. Remarkably, one cycle of pressure at ten pounds per square inch (sixty-nine kilopascals) and a frequency of approximately one hertz produced a 57 per cent increase in the incorporation of [3H]-thymidine at twenty-four hours (p < 0.001). From these findings, we hypothesized that the inositol phosphate cascade-cytosolic Ca2+-cytoskeletal calmodulin system plays a dominant role in the signal transduction of a mechanical stimulus into increased proliferation of bone cells, at least under the conditions reported here. CLINICAL RELEVANCE: Understanding the mechanisms by which bone cells convert a mechanical signal into a biological response is the beginning of an understanding of Wolffs law, which states that form follows function. A complete understanding of Wolffs law eventually should lead to an understanding of the cellular and molecular processes governing bone-remodeling and may allow therapeutic manipulation of bone-remodeling in clinical practice.