Part 3 : The Body Electric... The Circuit of Awareness ...The Ticklish Gene

The Body Electric

by Robert O. Becker

Five 

The Circuit of Awareness 

Charlie and I talked all afternoon, beginning fifteen years of fruitful work together. For me, the best part was his friendship and his open mind. He, too, knew there was still a lot to learn. Our relationship also had a side effect of incalculable value: He sent some of his most talented graduate students over to my lab to do their thesis work and later to become my colleagues in research. Andy Marino, Joe Spadaro, and Maria Reichmanis each became an indispensable part of the research group. Like Charlie, they constantly contributed new ideas, and they helped create the atmosphere of intellectual adventurousness that makes a lab creative. 

Closing the Circle 

Charlie's first contribution was to check the equipment and confirm the measurements I'd made on the salamanders. After he'd satisfied himself that everything was real, we discussed what to do next. 

"Well," Charlie said, "to find out more about this current, we'll have to go into the animal—expose a nerve and measure the current there." 

"That's easier said than done," I objected. "Just to cut down into the leg of an animal will damage tissues and produce currents of injury. 

That'll give spurious voltages. Besides, there'd be no stable place to put the reference electrode."  

Charlie gave me a lesson in basic electronics. A voltage entering a wire will decrease as the current travels along, so there'll be a uniform voltage drop in each unit of length. All you need to do is put both electrodes along the conductor, with the reference electrode closer to what you think is the source. If you use a standard distance between the electrodes, you can compare the voltage drop along various wires and measure changes in the whole system from any segment of it. 

All I had to do was the surgery. I decided to work on bullfrogs, whose hind legs were long and contained a nice big sciatic nerve. It was easy to find and could be exposed with just a little careful dissection, going between muscles instead of cutting through them. I was able to isolate over an inch of nerve with no bleeding or tissue damage, slipping a plastic sheet underneath so as not to pick up readings from the surrounding muscles. We measured the voltage gradient over a standard distance of 1 centimeter. It was the same from one frog to the next. In deep anesthesia it was absent or pretty small; as the anesthesia wore off, it became a constant drop of about 4 millivolts per centimeter, always gradually positive toward the spinal cord and negative toward the toes. 

In some frogs we cut the nerve above the measuring site, whereupon the voltages disappeared—another indication that the current was actually in the nerve. Voltages returned a little later, but they weren't the same as before. We figured these secondary voltages were probably an artifact—a spurious measurement produced by extraneous factors— caused by currents of injury from the cut nerve itself or from the other tissues where I'd made the incision to cut the nerve. 

Charlie then suggested that we make measurements on a longer section of nerve, and that was when we ran into a puzzle. The nicely reproducible voltages we'd found before couldn't be duplicated when we extended our measurement distance to 2 centimeters, close to the knee. We expected double the potential we'd found over the 1-centimeter distance, but often it was lower or higher than it should have been. I insisted that my dissection was producing local currents of injury that made our readings unpredictable. However, Charlie pointed out that I was a good frog surgeon and I didn't seem to be doing any more damage than before. He asked, "Could there be any difference in the nerve where you extended the dissection?" 

"Not likely," I said. "The sciatic nerve does split up into two branches, but you only find them below the knee, when one goes to the front of the calf and one to the back." 

"How do you know it doesn't separate before it gets to the knee?" he asked. 

He was right. Not bad for a physicist! The nerve did divide, but the two parts were held together by the nerve sheath until they got past the knee. I was able to remove the sheath and isolate both portions. When we measured these, we found that the two sections were polarized in opposite directions. The voltage drop of the front branch was positive toward the toes. The posterior branch was polarized in the same direction as the sciatic trunk, but it always had a higher voltage gradient. The current in the front branch apparently flowed in the direction opposite to that in the rest of the nerve. The interesting thing was, when we added up the voltage increases from 1-centimeter lengths of the two branches—4 millivolts positive and 8 negative in a typical frog—we got roughly the same voltage gradient that we found in the main nerve, about 4 millivolts negative in every centimeter. At first that didn't make any sense.

On a hunch I took pieces of each nerve and sent them to the pathology department to have microscope slides made. I found that the fibers in the front fork were smaller than those in the other. A light bulb went on! The sciatic nerve is what's called a mixed nerve. It has both  motor and sensory neurons. Sensory fibers are usually narrower than motor fibers, so it looked as though the front branch was all sensory, the back one all motor. Suppose the DC system also had incoming and outgoing divisions. We took readings from other nerves known to be all one type or the other. The femoral nerve along the front of the thigh is almost entirely motor in function, and, sure enough, it had an increasing negative potential away from the spine. The spinal nerves that serve the skin of a frog's back are sensory fibers, and they had increasing negative voltages toward the spine. 

Now we saw that when you put motor and sensory nerves together into a reflex arc, the current flow formed an unbroken loop. This solved the mystery of what completed the circuit: The current returned through nerves, not some other tissue. Just as Gerard had found in the brain, nerves throughout the body were uniformly polarized, positive at the input fiber, or dendrite, and negative at the output fiber, or axon. We realized that this electrical polarization might be what guided the impulses to move in one direction only, giving coherence to the nervous system. 

The Artifact Man and a Friend in Deed 

Charlie had helped develop the electron microscope and as a result knew many of the big names in physiology. Soon after the sciatic nerve experiments, one of these acquaintances visited Syracuse to give a lecture, and we invited him to stop by the lab. After showing him around and talking about the background of the work, we showed him our latest results. We anesthetized four frogs and opened their legs, exposing all eight sciatic nerves and measuring all sixteen branches. The readings were flawless. Every nerve had the voltage and polarity we'd predicted. Proudly, we asked, "What do you think?" 

"Artifact, all artifact," he replied. "Everyone knows there's no current along the nerve." Just then he remembered he had pressing business elsewhere and left in a hurry, apparently afraid some of this might rub off on him. 

Charlie almost never swore, but that day he did. The gist of his remarks was that there sure was a difference between physicists and biologists. The former would at least look at new evidence, while the latter kept their eyes and minds closed. Thereafter we always referred to the "Artifact Man'' when we needed a symbol of dogmatism. 

We continued a few more observations on frog nerves. By now winter  had come. That shouldn't have mattered—the lab temperature was the same all year, and the frogs didn't stop eating and hibernate as they would have in the wild—but there was a difference. The frogs' voltages were much lower, they stayed unconscious longer with the standard dose of anesthetic, and their blood vessels were much more fragile. Did they somehow sense the winter? 

If the DC system was as we theorized, it would be influenced by external magnetic fields. In the Hall-effect experiment I'd already shown that it was, but I'd used a strong field, measuring thousands of gauss. The earth's magnetic field is only about half a gauss, but it does vary in a yearly cycle. At the time there was another scientist who was saying this weak field had major effects on all life. Frank A. Brown, a Northwestern University biologist who was studying the ubiquitous phenomenon of biological cycles—wavelike changes in metabolic functions, such as the alternation of sleep and wakefulness—was claiming that similar rhythms in the earth's magnetic field served as timers for the rhythms of life. Even though his evidence was good, no one paid any attention to him in the early sixties, but it seemed to me that we had something to offer Brown's effort. We had a link by means of which the effect could occur. 

I wrote up the sciatic nerve measurements and added the observation on winter frogs. I sent it to Science but got it back immediately. I guess the editors had second thoughts after running my paper on the Hall effect. Next I tried the even better British equivalent, Nature, which took it. This time I also got some reprint requests. More important, the report led to correspondence with Frank Brown, beginning years of mutual feedback that helped bring about the discoveries described in Chapter 14. 

I thought of one more way we could check whether the current in the nerves was semiconducting. We could freeze a section of nerve between the electrodes. If the current was carried by ions, they would be frozen in place and the voltage would drop to zero. However, if the charge carriers were electrons in some sort of semiconducting lattice, their mobility would be enhanced by freezing and the voltage would rise. 

It worked. Each time I touched the nerve with a small glass tube filled with liquid nitrogen, the voltage shot upward. But perhaps I was damaging the nerve with the glass tube or through the freezing itself. Maybe the increase was merely a current of injury. To check, we simply cut the nerve near the spinal cord; the voltage gradient on the nerve went to zero, and then we applied the liquid nitrogen again. If the cold was really enhancing a semiconducting current, we should find no voltage now even after freezing the nerve—and we didn't. Therefore the increase in current wasn't due to artifact—damage to the nerve by freezing or touching it with the tube. 

That settled it. Test after test had substantiated the direct-current system. Now we had to see where the concept would lead us and try to convince some of the Artifact Men along the way. We had lots of ideas for further work, but now the first priority was to get some reliable system of funding for ourselves. 

I was continuing to have problems with the VA research office. After I'd gotten my second grant from that source, I soon found out that to have it approved and to be able to spend it were two different things. To order supplies—even things as simple as test tubes or electrode wire—I had to fill out a form and give it to the secretary of the research office. She had to fill out another form and get it signed by the research director. This form went to the supply service, where a clerk filled out a third form to actually order the stuff. Well, my orders stopped getting filled. 

In the process of complaining I made friends with the secretaries and found out that the director was holding me up just by not signing my forms. His secretary solved my problem. The director was a procrastinator. A pile of papers would collect on his desk until his secretary told him they had to be taken care of right away. Then he would sign them all at once without looking at each one. His secretary, to whom I owe a tremendous debt, merely slipped my requests back into the middle of the pile, usually late on Friday afternoon. Several times he visited my lab, saw a new piece of equipment, and remarked, "I don't remember ordering that for you." 

"You don't?" I replied sweetly. "We talked about it, and I had plenty of money left in the grant, so you said okay." It was better than arguing over each instrument, and I was careful not to overspend. I don't think he ever caught on. 

Soon I encountered a more serious threat, however. Between the VA and the medical school I had a lot of bosses, and all of them were doing "research." However, the research service's annual report showed that I'd published more than all of my superiors put together on a few thousand bucks a year, while some of them were drawing forty or fifty thousand. I'd broken the old rule that you should never do more than your boss. 

One of these fellows appeared in my lab one day. That was an event in itself, since he'd never been a supporter of mine; in fact, our relations were rather strained That day, however, he evinced great interest in what I was doing and made me an "offer I couldn't refuse." 

"How would you like to have as much money as you need?"  

I said that would be nice, but I wondered how it could come about. 

"No problem. All you have to do is include me in the project. All I would expect in return is that my name would go on all publications." 

It was a few seconds before I could believe I'd heard him right. Then I told him what he could do with his influence. 

A few months later, I found out that the area surgical consultant, practically next to God in the VA hierarchy, was visiting the hospital to act on a report, made by my would-be "benefactor," that I was spending too much time on research and neglecting my patients. Fortunately, there was a lot of infighting among my superiors, and one higher than the guy who'd made the charge was supporting me. His motives were less to save a promising research program and more to embarrass the other man, but I was cleared. 

It was also clear that I was courting disaster by relying on VA money. I needed outside support. I took time off from research to prepare two proposals. One, which I sent to the Department of the Army, emphasized the possibility that direct currents could stimulate healing. Since the Army's business produces quite a few wounds, I thought it would be interested, but it was not. The proposal was turned down promptly, but then a strange thing happened about a month later. I received a long distance call from a prominent orthopedic surgeon, a professor at a medical school in the South. "I have a grant from the Army to study the possibility that direct currents might stimulate wound healing," he purred, "and I wonder if you might have any suggestions as to the best approach to use." 

My God, were they all this sleazy out there? Of course, when I looked up his credentials, I found he had absolutely no background in bioelectricity. He'd just happened to be on the Army review committee, recommended disapproval, and then turned around and submitted the idea in his own name, now getting the go-ahead since he, a man with a reputation and friends on the review board, was going to do it, instead of some unknown upstart. 

I sent the second application to the National Institutes of Health (NIH). I stayed within my specialty and proposed to study the solid state physics of bone, eventually hoping to find out if direct currents could stimulate bone healing. The grant was approved, but only for enough money to do part of what I wanted. And although it was nice to have a cushion, a source not under local control, I nevertheless needed some political clout to stabilize the situation in Syracuse. I went directly to the dean of the medical school. 

Carlyle Jacobson had seemed to be a nice guy, not the type to stand on ceremony or position, and I thought I could talk to him frankly. I gathered up reprints of my papers and went to his office. 

"Sir," I began, "I've been doing research on direct current electrical effects in living things for the past four years. I've gotten some papers published in good journals, and I think this is an important piece of work. Nevertheless, I have great difficulty getting funds from the VA. My requests are blocked by the politicians on the committee. Meanwhile these same guys are spending five times as much as I get, and they don't publish a damn thing." I'm afraid I got carried away, but Dean Jacobsen just sat there listening until I'd finished. 

"Have you done any experiments on the DC activity of the nervous system?" he asked. 

This was an unexpected question, but I told him of our work on salamanders and frog nerves. 

It turned out he'd done some research on nerves years ago—with Ralph Gerard! He was very enthusiastic. "You've gone much further than we ever did," he told me. "We never thought to relate the brain currents to a total-body system. How much do you need?" 

I asked for $25,000 in each of the next two years, but explained that it had to be earmarked for me alone or I would never see it. 

"Don't worry," he said. "Go right back to your lab. I'll get it for you. I wish I could work with you." 

He must have been dialing Washington as the door closed behind me, for the next day the chief of research got a telegram from the VA Central Office authorizing the requested amount for me, and only me. He couldn't understand it, and I professed complete ignorance, too. 

I figured nothing I did now could make the research director like me any less, so I made another move. I went to the hospital director and told him I needed more space. Having heard of my favor from Washington, he was most helpful, and soon I had a suite of rooms on the top floor. 

Suddenly a whole new realm of research was within reach. Charlie and I hardly knew which way to turn. Our first and most important step was to hire Andy Marino as a technician. The salary meant much to him, and his intelligence and dedication meant even more to us. We were on our way. 

The Electromagnetic Brain 

If the current controlled the way nerves worked in the brain as well as in the rest of the body, then it must regulate consciousness to some extent. Certainly the falling voltages in anesthetized salamanders supported this idea. The question was: Did the change in the current produce anesthesia? Apparently it did, for when I passed a minute current front to back through a salamander's head so as to cancel out its internal current, it fell unconscious. How this state compared with normal sleep was impossible to tell, but at least the animal was clinically anesthetized. As long as the current was on, the salamander was motionless and unresponsive to painful stimuli. 

Was this real anesthesia, or was the animal just being continuously shocked? This certainly didn't seem to be the case, but the observation was so important and so basic to neurophysiology that I had to be sure. It was no easy task, however, for there were, and still are, few objective tests known for anesthesia, especially in salamanders. Brain waves had turned out to be useless in gauging depth of anesthesia in humans, because the one good marker—very slow delta waves—only showed up when the patient was dangerously close to death. Lacking any better idea, however, I used my new multi-electrode monitor to make EEG recordings of chemically anesthetized salamanders and found that they showed prominent delta waves even though they all recovered nicely. Delta waves would be my marker. The idea worked beautifully. Very small currents gave me delta waves on the EEG, the waves got bigger as I increased the current, and they all correlated with the animal's periods of unresponsiveness. 

This result naturally led to the question: Did chemical anesthetics work by stopping the brain's electrical current? I couldn't see any way to get direct evidence one way or the other, but I thought maybe chemical anesthesia could be reversed by putting current into the brain in the normal direction. I found this could be done only to a certain extent. I could get a partial return of the higher frequency waves in the EEG, and the anesthesia seemed to become shallower, but I couldn't get a drugged salamander to wake up and walk away.  

In the course of these observations, I found that when the head voltage was dropping as a chemical anesthetic took hold, specific slow waves always appeared briefly in the recordings. They were at the low end of the delta frequencies, 1 cycle per second or even less, and they also showed up when the voltage came back as the drug wore off. To find out if these waves always signaled a major change in the state of consciousness, I decided to use a standard amount of direct current to produce anesthesia, measure the amplitude (size) of the delta waves in the EEG, and then add some one-second waves of my own to the current I was putting into the animal's head. In other words, I would introduce some "change-of-state" waves from outside and see if they produced a shift in the EEG. I couldn't record the EEG simultaneously, because the waves I added would appear on the trace, so I rigged up a switch to cut out the added waves after a minute and turn on the EEG recorder at the same time, without stopping the direct current that would keep the salamander unconscious. 

It seemed to work. The added waves markedly increased the amplitude of the salamander's own deep-sleep delta waves. Was this an artifact? Were the added waves just causing an oscillation in the brain currents that persisted after the external rhythms were removed? It didn't seem likely, because the waves I added were at the change-of-state frequency of 1 cycle per second, while the measured deltas were at the deep-sleep frequency of 3. However, an additional test was possible. I could add waves of other frequencies and see if they worked as well as 1 cycle per second. They didn't; in fact, as the frequency of the added waves increased over that rate, the deep-sleep delta waves got smaller. The one-second waves were a marker of major shifts in consciousness. 

This line of work corroborated one of the main points of my hypothesis. Direct currents within the central nervous system regulated the level of sensitivity of the neurons by several methods by (hanging the amount of current in one direction, by changing the direction of the current (reversing the polarity), and by modulating the current with  slow waves. Moreover, we could exert the same control from outside by putting current of each type into the head. This was exciting. It opened up vast new possibilities for a better understanding of the brain. It was still on the edge of respectability, too, since it was a logical consequence of the work done by Gerard and his co-workers. The next experiment was harder to believe, however. 

I figured the brain currents must be semiconducting, like those in the peripheral nerves. I thought of looking for a Hall voltage from the head but reasoned the brain's complexity would make any results questionable. Then I thought of using the effect backward, so to speak, measuring a magnetic field's action on the brain rather than on the production of the Hall voltage. Since the Hall voltage was produced by diverting some of the charge carriers from the original current direction, a strong enough magnetic field should divert all of them. If so, such a field perpendicular to the brain's midline current should have the same effect as canceling out that normal current with one applied from the outside. The animal should fall asleep. 

We tranquilized a salamander lightly, placed it on a plastic shelf between the poles of a strong electromagnet, and attached electrodes to measure the EEC As we gradually increased the magnetic field strength, we saw no change—until delta waves appeared at 2,000 gauss. At 3,000 gauss, the entire BEG was composed of simple delta waves, and the animal was motionless and unresponsive to all stimuli. Moreover, as we decreased the strength of the magnetic field, normal EEG patterns returned suddenly, and the salamander regained consciousness within seconds, This was in sharp contrast to other forms of anesthesia. 

With direct currents, the EEG continued to show delta waves for as long as a half hour after the current was turned off, and the animals remained groggy and unresponsive just as after chemical anesthesia. 

It seemed to us that we'd discovered the best possible anesthetic, allowing prompt recovery with no side effects. We proposed getting a bigger electromagnet to try this method on larger animals and eventually humans, but we never even got a reply. Our data on direct currents in the nerves weren't quite acceptable, but reactions by living things to magnetic fields were absolutely out of the question in America at that time. 

I was flabbergasted, therefore, to receive a phone call from one of the most prominent biologists in the Harvard-MIT orbit. He told me, "We're in the process of setting up an international conference on high energy magnetic fields at MIT, and we've received a number of questions from respectable scientists in other countries asking why there is to be no session on biological effects of magnetic fields. This is a totally new idea to us, and we really don't believe there are such effects, but some of these fellows are persistent. We've searched the scientific literature and found your paper on the Hall effect. Since you seem to be the only person in this country doing any work along these lines, let me ask if you think there's anything to it." 

I allowed as humbly as I could that there just might be something there, and told him about the latest experiments. There was a long pause filled with disdain. Then I added that Professor Bachman had been working with me. That changed the tone dramatically; this man also knew Charlie through work on the electron microscope during the war. He asked if I would organize the session and arrange for additional speakers. 

There weren't too many investigators to choose from, and some of them were doing very slipshod work. I invited Frank Brown and selected a few others on the basis of published work. I'd just about finished when I got another call. A man with a thick German accent introduced himself as Dietrich Beischer. 

"I have read your paper on the Hall effect," he began, "and I think we have much common interest." He explained that he was studying magnetic field bioeffects for the Navy and had done much work that wasn't published openly. At the time he was conducting a large experiment on human volunteers to check for effects from a null field, a complete absence of magnetism. When I wondered how he produced such a state, he invited me to have a look and perhaps offer suggestions. So off I went to Maryland. 

Beischer was using the compass calibration building in the Naval Surface Weapons Center at Silver Spring. The building was huge. Electrical cables in all the walls, floor, and roof were "servo-connected" (directly cued) to the three axes of the earth's magnetic field, so that the field was canceled out in a sphere about 20 feet across in the center of the structure. Several men were living and being tested in this area. I was impressed by the resources at Beischer's command, and I had a good time, but I wondered what use any discoveries made there might ultimately be put to. My only contribution was to point out that the enclosure had been built before anyone knew about the earth field's low-frequency components, micropulsations ranging from less than 1 to about 25 cycles per second, that were far weaker than the planet's electromagnetic field as a whole. Consequently Beischer's subjects were still exposed to a very weak magnetic field pulsing at these frequencies, and I suspected that that component might be one of the most important for life, because all brain waves were in exactly the same range. Perhaps as a result of this factor, the null-field experiment was turning out to be inconclusive, but I asked Beischer to attend the MIT meeting and present some previous data suggesting that electromagnetic fields could affect embryonic development. 

For my own presentation I decided against trying to compress all the  work I'd done so far into a few minutes before a skeptical audience. Instead I offered some evidence that Charlie and I had gathered with the help of psychiatrist Howard Friedman, which showed a possible relationship between mental disturbances and solar magnetic storms. I'll discuss this study further in Chapter 14. 

The MIT meeting went well. The field of bioelectromagnetism was still young, and the researchers in it didn't make many converts among the mainstream biologists. As usual, we found the physicists more inclined to listen. However, we drew inspiration from each other. I returned to the lab more determined than ever to elucidate the links that I knew existed between electromagnetic energy and life. 

Charlie, Howard, and I decided to find out how the brain's DC potentials behaved in humans. The electrodes we'd been using on salamanders couldn't be scaled up for people, but within a week Charlie invented some that would give us equally precise readings from the human head. We immediately found that the back-to-front current varied with changes in consciousness just as in salamanders. It was strongest during heightened physical or mental activity, it declined during rest, and it reversed direction in both normal sleep and anesthesia. This knowledge led directly to the experiments, described in Chapter 13, that taught us much about how hypnosis and pain perception work. 

At this point I received an invitation from Meryl Rose to speak at the big event in the world of animal science, the International Congress of Zoology. This is not just a yearly convention; it is convened only when its directors agree that there has been enough progress to warrant a meeting. This session, in August 1963, was only the sixteenth since the first one had been set up in 1889. It was an honor to be there, and the conference itself was especially important, since it was one of the first times science formally addressed such ecological emergencies as pesticide pollution, the protection of vanishing species, overpopulation, and urban sprawl. The high point for me came when I gave my paper and saw in the audience Dr. Ralph Bowen, my college biology professor, a kind but exacting teacher who'd inspired me with his unique combination of scientific discipline and respect for life. Afterward, with characteristic caution, he said something like "That's not too bad, Becker. I'd like to see you keep going in this research." 

When I assured him that, despite my M.D. degree, I was still committed to basic biology, he said, "I hope so, but remember, it won't be easy. To change things is never popular." His encouragement meant a lot to me, and I was happy to be able to show him that I'd amounted to something. 

A lot had happened in the four work-filled years since I'd begun studying the current of injury. That first experiment had opened a door into a great hall with passageways leading off in all sorts of fascinating directions. This was really the life! Without leaving the laboratory I'd gone on a journey of exploration as exciting as trekking through the uncharted wilds of New Guinea. Our work on nerves and the brain was leading toward a whole new concept of life whose implications only gradually became apparent. Meanwhile, my colleagues and I were continuing to investigate the processes of healing, leading to insights and practical applications that more than justified my enthusiasm. 

Six 

The Ticklish Gene 

Despite my fascination with fundamental questions about the nature of life, I was, after all, an orthopedic surgeon, and I was eager to find things that would help my patients. In addition, to convince the Artifact Man and all his brothers, Charlie and I were looking for some direct test of semi-conduction in living tissues. The Hall effect and the freezing of frog nerves each demonstrated a characteristic of semi-conduction but didn't confirm it in standard engineering terms. Unfortunately, all the direct tests then known worked only with crystals. You needed a material you could carve into blocks, something that didn't squish when you put an electrode on it. The only possibility was bone. 

To many biologists and physicians, bones are pretty dull. They seem like a bunch of scarecrow sticks in which nothing much happens, plain props for a subtler architecture. Many of my patients were in sad shape because doctors had failed to realize that bone is a living tissue that has to be treated with respect. It's a common misconception that orthopedic surgery is like carpentry. All you have to do is put a recalcitrant fracture together with screws, plates, or nails; if the pieces are firmly fixed after surgery, you're done. Nothing could be further from the truth. No matter how firmly you hold them together, the pieces will come loose and the limb can't be used if the bone doesn't heal. 

The Pillars of the Temple 

The skeleton doesn't deserve this cavalier treatment. The development of bones by the first true fishes of the Devonian era nearly 400 million years The Ticklish Gene 119 ago was a remarkable achievement. It enabled animals to advance, in both senses of the word, quickly and efficiently. Since bone is inside the body, it can live and grow with the animal, instead of leaving it defenseless as an external skeleton does when cast off during a molt. It's also the most efficient system for attaching muscles and increasing the size of animals. 

Bone is extraordinary in structure, too. It's stronger than cast iron in resisting compression but, if killed by X rays or by cutting off its blood supply (barely adequate to start with), it collapses into mush. The part that's actually alive, the bone cells, comprises less than 20 percent of the whole. The rest, the matrix, isn't just homogeneous concrete, either. It's composed of two dissimilar materials—collagen, a long-chain, fibrous protein that's the main structural material of the entire body, and apatite, a crystalline mineral that's mainly calcium phosphate. The electron microscope shows that the association between collagen and apatite is highly ordered, right down to the molecular level. The collagen fibers have raised transverse bands that divide them into regular segments. The apatite crystals, just the right size to fit snugly between these bands, are deposited like scales around the fibers. 

This intricacy continues at higher levels of organization. The collagen fibers lie side by side in layer upon layer wound in opposed spirals (a double helix) around a central axis. The bone cells, or osteocytes, are embedded in these layers, which form units a few millimeters long, called osteons. The center of each osteon has a small canal in which runs a blood vessel and a nerve. The osteons in turn are organized so as to lie along the lines of maximum mechanical stress, producing a bone of the precise shape best able to withstand the forces applied to it. 

Bone has an amazing capacity to grow, which it does in three different ways. In childhood each long bone of the limbs has one or two growth centers, called epiphyseal plates. Each is a body of cartilage whose leading edge grows continuously while its trailing edge transforms into bone. When the bone is the right length, the process stops, and the remaining cartilage forms the bony knob, or epiphysis, at the end of the bone. The "closure" of the epiphyses is an index of the body's maturation. 

Bone cannot heal. That sounds like a conundrum but it's literally true. Fractures knit because new bone made from other tissues unites the fracture ends. Although we sometimes speak of bone growth as part of fracture healing, the old, preexisting bone doesn't have the capacity to grow. As mentioned in Chapter 1, there are two tissues that produce new bone at a fracture site. One is the periosteum, the bone's fibrous covering. It's the cells of the periosteum innermost layer that have the power of osteogenesis, or bone formation. After a fracture, these cells are somehow turned on. They begin to divide and some of the daughter cells turn into osteoblasts, cells that make the collagen fibers of bone. Apatite crystals then condense out of the blood serum onto the fibers. 

The other tissue that forms new bone to heal a fracture is the marrow. Its cells dedifferentiate and form a blastema, filling the central part of the fracture. The blastema cells then turn into cartilage cells and later into more osteoblasts. This process is true regeneration, following the same sequence of cell changes as the regrowing salamander limb. Whatever a physician does to repair a fracture, he or she must protect the periosteum and marrow cavity from harm. Unfortunately, too often the application of plates, screws, and nails does just the opposite and, rather than helping nature, the treatment impedes healing. 

From a researcher's point of view, the question here is: What activates the cells of the periosteum and marrow? In the case of the marrow, we can expect it to be the same factor that switches on the cells in a salamander's amputated leg. 

There's a third process of growth that's unique to bone. It follows Wolff's law, which is named after the orthopedic surgeon J. Wolff, who discovered it at the end of the nineteenth century. Basically, Wolff's law states that a bone responds to stress by growing into whatever shape best meets the demands its owner makes of it. When a bone is bent, one side is compressed and the other is stretched. When it's bent consistently in one direction, extra bone grows to shore up the compressed side, and some is absorbed from the stretched side. It's as though a bridge could sense that most of its traffic was in one lane and could put up extra beams and cables on that side while dismantling them from the other. As a result, a tennis player or baseball pitcher has heavier and differently contoured bones in the racket arm or pitching arm than in the other one. This ability is greatest in youth, so in childhood fractures it's often best to put the bone ends together gently by manipulation without surgery, settling for a less than perfect fit. Sometimes the hardest part is convincing the parents that a modest bend will straighten itself out in a few months in accordance with Wolff's law. 

Wolff's-law reorganization occurs because something stimulates the periosteum to grow new bone at a surface where there's compressional stress, while dissolving bone where there's tensional stress. Again, the question for researchers is: What turns on the periosteal cells? 

After I'd finished giving my paper on the salamander's current of injury at the orthopedic meeting early in 1961, several people came up to the stage to ask questions. Among them was Andy Bassett, a young orthopedic surgeon who was doing research at Columbia. In our conversation we came up with an angle for investigating Wolff's law—piezoelectricity. Simply put, this is the ability of some materials to transform mechanical stress into electrical energy. For example, if you bend a piezoelectric crystal hard enough to deform it slightly, there'll be a pulse of current through it. In effect, the squeeze pops electrons out of their places in the crystal lattice. They migrate toward the compression, so the charge on the inside curve of a bent crystal is negative. The potential quickly disappears if you sustain the stress, bur when you release it, an equal and opposite positive pulse appears as the electrons rebound before settling back into place. 

Since I'd shown that a stronger-than-normal negative current preceded regeneration, Bassett suggested that maybe bone was piezoelectric and  the negative charge from bending stimulated the adaptive growth of Wolff's law. To find out, we tested both living and dead bones from a variety of animals, and found that bending produced an immediate potential, as expected. The compressed side became negative; the stretched side positive * 

*After writing up this experiment, we found that it had been done before. Iwao Yasuda, a Japanese orthopedist, had shown that bone was piezoelectric back in 1954; he and Eiichi Fukada, a physicist, had confirmed the fact in 1957. We made note of their prior observations but published our paper anyway, since our techniques were different and ours was the first report in English.  

Furthermore, the reversed potentials that appeared when we released the stress weren't nearly as large as the first ones. This was just as it should be. If a negative voltage was the growth stimulus, there had to be some way to cancel out the positive rebound voltage; otherwise it would have negated the growth message. In electronic terms, there had to be a solid-state rectifier, or PN junction diode. 

Despite the intimidating names, this device is fairly simple. It's a filter that screens out either the positive (P) or the negative (N) part of a signal. As mentioned in Chapter 4, current can flow through a crystal lattice either as free electrons or as "holes" that can shift their positions much as the holes migrate when you move the marbles in a game of Chinese checkers. Since current can flow from a P-type to an N-type semiconductor but not the other way, a junction of the two will filter, or rectify, a current. 

The phonograph would be impossible without this device. As the diamond or sapphire crystal of the stylus rides a record's groove, the groove's changing shape deforms it ever so slightly. The crystal, of course, transforms the stresses into a varying electrical signal, which is amplified until we can hear it. It would be an unintelligible hum, however, if we heard both the deformation pulse and the release pulse. Therefore we place a rectifier in the circuit. It passes current in one direction only, so the impulses don't cancel each other out. The signal is rectified, and when we feed it to a loudspeaker we hear music. Bassett and I felt sure we were seeing evidence of rectification in the fact that bone's release pulse was much smaller than the one from stress. 

If the negative piezoelectric signal stimulated growth, maybe we could induce bone growth ourselves with negative current.* We tried out the idea on eighteen dogs. In the thighbone of one hind leg we implanted a battery pack. The electrodes were made of platinum, a nonreactive metal, to minimize any possible electrochemical irritation, and we inserted them through drill holes directly into the marrow cavity. As controls, some of the devices didn't have a battery. After three weeks we found that the active units had produced large amounts of new bone around the negative electrode but none around the positive. In the controls, there was no growth around either electrode

*Again Dr. Yasuda and his colleagues had already done so, but their results seem to have been due to bone's ability to grow in response to irritation from the electrodes. They used alternating current, which is now known to have no direct growth-stimulating effect.  

The results were exciting, but in retrospect I believe we made a serious error when we published this study. In our own minds and in print as well, we confused the negative potentials of the salamander's current of injury, the negative potentials that stimulated bone growth, and the negative potentials from the piezoelectric study. We proceeded as though they were equivalent, but they were not. The piezoelectric potentials were measured on the outside of the bone and appeared only when mechanical stress was applied. They were transitory, and most likely the periosteum was their target tissue. In the implant study, we used continuous direct current applied to the inside of the bone, the marrow cavity. 

What we were stimulating was the DC control system of regenerative fracture healing, not the piezoelectric control system of Wolff's law. We didn't clearly indicate the difference to the scientific reader, and this led to much confusion, some of which still persists twenty years later. As a result, many scientists think electricity stimulates bone growth because bone is piezoelectric. Most of these people don't realize that bone itself doesn't grow when a fracture heals. Moreover, everyone who has proceeded from our technique—and it's being used today to heal nonunions (see Chapter 8)—has done basically the same thing, continuously stimulating the bone marrow. No one has tried to stimulate the periosteum, as the pulsed piezoelectric signal does. 

Our confusion also helped the scientific establishment accept the "trivial" electrical stimulation of bone by considering it something unique to bone. The relationship between our experiment and regeneration proper was lost. 

The Inner Electronics of Bone 

Charlie Bachman and I decided to investigate the electrical properties of bone in more detail and try to figure out how Wolff's law worked. We put together a hypothesis based on my experiments with Bassett. We postulated that the bone matrix was a biphasic (two-part) semiconductor. That is, either apatite or collagen was an N-type semiconductor; the other, a P type. Their connected surfaces would thus form a natural PN junction diode that would rectify any current in the bone. We further theorized that only one of the materials was piezoelectric. On the compressed side of a stressed bone, we expected the positive pulses to be filtered out, leaving a negative signal to stimulate periosteal cells to grow new bone. 

We made several pairs of sample blocks, cut side by side from pieces of bone removed from patients for medical reasons. From one member of each pair we chemically removed the apatite. The other we treated with a compound that dissolved the collagen. The resulting pure collagen was yellowish and slightly rubbery, and the apatite pure white and brittle, but otherwise both blocks still looked like bone. Our first step was to test bone's component materials separately for semi-conduction and piezoelectricity. Collagen turned out to be an N-type semiconductor and apatite a P type. 

Then we tested our samples for piezoelectricity in the same way that Bassett and I had previously tested whole bone. We expected that apatite would be the only one to show an effect, since it was a crystal. However, collagen turned out to be a piezoelectric generator, while apatite was not. We now had the makings of a PN junction—two semiconductors, one an N type, the other a P type, joined together in a highly organized fashion. 

Now came the crucial part of our hypothesis. We had to figure out a way to test for rectification at the PN junction. It was an important crossroads. 

Here we ran up against what's known in the trade as a technical problem. To test for rectification we had to put one electrode on the collagen and one on the apatite as they appeared in whole bone. Unfortunately, the apatite crystals are each only 500 angstroms long. Now, the angstrom (named after the Swedish pioneer of spectroscopy Anders Jonas Angstrom) was invented for measuring atoms and molecules, and it is not large. Five hundred of them are only one tenth as long as a single wave of green light. Even today's thinnest microelectrodes are 1 micron (10,000 angstroms) wide, and at the time the thinnest ones available to us were much larger. It would have been like trying to measure a grain of rice with a telephone pole. 

We would have to do it in a sort of statistical way. Because of the way bone is built—millions of little scales glued onto larger fibers arranged in more or less lengthwise spirals along the osteones—I reasoned thus: If we put an electrode on the lengthwise cut, we'd be contacting mostly apatite, while an electrode on a face cut across the grain should connect to a greater proportion of collagen. If that method of electrode hookup worked and if we had a rectifier in our bones, then we'd be able to pass current through our samples only in one direction. That was exactly what happened. Our bone samples weren't as efficient as a commercial rectifier, but the amount of current we could put through them from a battery of constant voltage was much greater in one direction than in the other. 

Current flowing "uphill," against the normal flow from P to N semiconductors, is called a reverse bias current, and we used it to look for photoelectric effects. Many semiconductors absorb energy from light, and any current flowing through the material gets a boost. We arranged our apparatus so only a small spot of light shone on the bone, because our silver electrodes were slightly sensitive to light and could produce a real artifact. With the voltage constant, the light produced an unmistakable increase in the current. Now, if bone really contained a rectifier, the photoelectric effect should be sensitive to the current's direction. The current in reverse bias should rise more with the same light intensify than the current in forward bias The experiment was simple. We reversed the battery and turned on the light. The amperage rose higher than before. The rectifier was real. 

We could now follow the entire control system of Wolff's law. Mechanical stress on the bone produced a piezoelectric signal from the collagen. The signal was biphasic, switching polarity with each stress and-release. The signal was rectified by the PN junction between apatite and collagen. This coherent signal did more than merely indicate that stress had occurred. Its strength told the cells how strong the stress was, and its polarity told them what direction it came from. Osteogenic cells where the potential was negative would be stimulated to grow more bone, while those in the positive area would close up shop and dismantle their matrix. If growth and resorption were considered as two aspects of one process, the electrical signal acted as an analog code to transfer information about stress to the cells and trigger the appropriate response. 

Now we knew how stress was converted into an electrical signal. We had discovered a transducer, a device that converts other forces into electricity or vice versa. There was another transducer in the Wolff's-law system - the mechanism that transformed the electrical signal into appropriate cell responses. Our next experiment showed us something about how this one worked. 

Collagen fibers are formed from long sticks, like uncooked spaghetti, of a precursor molecule called tropocollagen. This compound, much used in biological research, is extracted from formed collagen—often from rat tails—and made into a solution. A slight change in the pH of the solution then precipitates collagen fibers. But the fibers thus formed are a jumbled, feltlike mass, nothing like the layered parallel strands of bone. However, when we passed a weak direct current through the solution, the fibers formed in rows perpendicular to the lines of force around the negative electrode. This fit our new discoveries perfectly, because the lines of force on the negative (compressed) side of a bent bone would be in precisely the same alignment as the collagen fibers of the new bone that formed there.

This was the first time that a complete circuit diagram of a growth process could be made. To us, this seemed an achievement of some note, but, although we got published, no one seemed to pay any attention. 

The scientific community wasn't ready for biological semi-conduction,and the notion of diodes in living tissue seemed ridiculously farfetched to most of the people I told about it. For that reason, I never even bothered to try publishing one of our follow-up experiments. It was just too weird. 

In the mid-1960s, solid-state devices were only beginning to hit the market, and one of the PN junction's most interesting properties hadn't yet been exploited. When you run a current through it in forward bias, some of its energy gets turned into light and emitted from the surface. In other words, electricity makes it glow. Nowadays various kinds of these PN junctions, called light-emitting diodes (LEDs), are everywhere as digital readouts in watches and calculators, but then they were laboratory curios. 

We found that bone was an LED. Like many such materials, it required an outside source of light before an electric current would make it release its own light, and the light it emitted was at an infrared frequency invisible to us, but the effect was consistent and undeniable. 

Even though we'd already proved our hypothesis, Charlie and I did a few more experiments on bone semi-conduction, partly for additional confirmation and partly for the fun of it. It was known that some semiconductors fluoresced—that is, they absorbed ultraviolet light and emitted part of it at a lower frequency, as visible light. We checked, and whole bone fluoresced a bluish ivory, while collagen yielded an intense blue and apatite a dull brick-red. Here we found a puzzling discrepancy, however, which eventually led to a discovery that could benefit many people. When we combined the fluoresced light from collagen with that from apatite, we should have gotten the fluoresced light from whole bone. We didn't. That indicated there was some other material in the bone matrix, something we'd been washing out in the chemical separation. 

Charlie and I were stumped on that line of research for a couple of years, until our attention was caught by a new development in solid state technology called doping. Tiny amounts of certain minerals mixed into the semiconductor material could change its characteristics enormously. The making of semiconductors to order by selective doping would become a science in itself; to us it suggested trace elements in bone. We already knew that certain trace metals - such as copper, lead, silver, and beryllium - bonded readily to bone. Beryllium miners had a high rate of osteogenic sarcoma—bone cancer—because beryllium somehow removed normal controls from the osteocytes' growth potential. Radioactive strontium 90 worked its harm by bonding to bone, then bombarding the cells with ionizing radiation. Perhaps some trace element normally found in bone changed its electrical properties by doping it.

To find out, Charlie and I used a very complex device called an electron paramagnetic resonance (EPR) spectrometer on our bone samples. There's no easy way to explain just how this instrument works, but basically it measures the number of free electrons in a material by sensing a resonance produced in the electrons' vibrations by in applied magnetic field. We used it to measure the free electrons in collagen and apatite, and we found the same kind of discrepancy as in our fluorescence experiment: When we added together the free electrons of collagen and apatite, we fell short of the number we found in whole bone. That made us certain that we were washing out some trace mineral. 

We decided to work backward. We prepared a solution containing small amounts of a wide variety of metals. Then we soaked our collagen and apatite cubes in this broth to see what they'd pick up. 

We knew we were on the way to solving this mystery when we examined the results. Only a few of the metals had bonded to the bone materials: beryllium, copper, iron, zinc, lead, and silver. The diameters of all the absorbed atoms were exact fractions of one another. The results showed that the bonding sites were little recesses into which would fit one atom of silver or lead, two of iron or copper or zinc, or six of beryllium. 

Only one of these metals gave us an electron resonance of its own, indicating that it had a large number of free electrons that could affect the electrical nature of bone. That metal was copper. We made a batch of broth containing only copper. We expected that copper's EPR signal would change to one value as it bonded with collagen, and to another as it bonded with apatite. Since the molecular structure of each was quite different, we figured that each would bind copper in a different way. 

We could hardly believe the results. Bonding had indeed changed copper's resonance, but the change was the same in both materials. By analyzing it we deduced that each atom of copper fit into a little pit, surrounded by a particular pattern of electric charges, on the surface of apatite crystals and collagen fibers. Because the pattern of charges was the same in both materials, we knew that the bonding sites were the same on both surfaces and that they lined up to form one elongated cavity connecting the crystal and fiber. In other words, the two bonding sites matched, forming an enclosed space into which two atoms of copper nestled. The electrical forces of this copper bond held the crystals and fibers together much as wooden pegs fastened the pieces of antique furniture to each other. Furthermore, the electrical nature of peg and hole suggested that we had found, on the atomic level, the exact location of the PN junction. 

This discovery may have some medical importance. The question of how the innermost apatite crystals fasten onto collagen had eluded orthopedists until then, and the finding may have opened a way to understand osteoporosis,a condition in which the apatite crystals fall off and the bone degenerates. The process is often called decalcification, although more than calcium is lost. It's a common feature of aging. I surmise that osteoporosis comes about when copper is somehow removed  from the bones. This might occur not only through chemical/metabolic processes, but by a change in the electromagnetic binding forces, allowing the pegs to "fall out." It's possible that this could result from a change in the overall electrical fields throughout the body or from a change in those surrounding the body in the environment. 

Osteoporosis has been a major worry of the American and Russian space programs. As flights got longer, doctors found that more and more apatite was lost from the bones, until decalcification reached 8 percent in the early Soviet Salyut space-station tests. Serious problems were known to occur only when apatite loss reached about 20 percent, but the trend was alarming, especially since depletion of the calcium reservoir might affect the nervous system and muscular efficiency before the 20-percent level was reached. Although the immediate cause was their inability to get a malfunctioning air valve closed before all the cabin air escaped, weakness from muscle tone loss might have contributed to the death of the three cosmonauts who succumbed while returning from their twenty-four-day flight aboard Soyuz 11 at the end of June 1971. 

Space osteoporosis may result from unnatural currents induced in bone by a spacecraft's rapid motion through the earth's magnetic field, with a polarity reversal every half orbit, or it may be a direct effect of the field reversal. This abnormality, which may change the activity of bone cells directly, would be superimposed on abnormal responses of bone's natural electrical system, which is almost certainly affected by weightlessness. The unfamiliar external field reversals could also weaken the copper pegs, at the same time that the bones are in a constant state of "rebound" from their earthly weight-induced potentials, producing a signal that says, "No weight, no bones needed." 

We know that the more even distribution of blood caused by weightlessness registers in the heart as an excess; as a result, fluid and ions, including calcium, are withdrawn from tht blood. However, the effect probably isn't due to weightlessness alone, for the Skylab astronauts did rigorous exercise, which would have  supplied plentiful stresses to their bones. They worked out so hard that their muscles grew, but decalcification still reached 6.8 percent on the twelve-week mission. 

The Soviets at first claimed to have solved the problem before or during the Soyuz 26 mission of 1977—78, in which two cosmonauts orbited in the Salyut 6 space lab for over three months. Subsequent Soviet space persons, who have remained weightless as long as 211 days, reportedly have showed no ill effects from osteoporosis, and chief Soviet space doctor Oleg Gazenko said it simply leveled off after three months. However, this claim was later officially withdrawn, and I have a hunch that the Soviets are working on a way to prevent the condition by simulating earth-surface fields inside their space stations, a method that perhaps hasn't yet worked as well as they'd hoped. 

Andy Bassett has suggested giving our astronauts strap-on electromagnetic coils designed to approximate their limb bones' normal gravity stress signals, but so far NASA has shown no interest. 

Unfortunately for earthbound victims of osteoporosis, the copper peg discovery still hasn't been followed up, even though I published it over fifteen years ago. Charlie and I wanted to continue in that direction, but we knew that we couldn't sustain more than one major research effort at a time. We decided that regenerative growth control was our primary target, so we reluctantly dropped osteoporosis. Fortified by our new knowledge that electricity controlled growth in bone, we returned instead to the nerves, taking a closer look at how their currents stimulated regrowth. 

A Surprise in the Blood 

I felt as though the temple curtain had been drawn aside without warning and I, a goggle-eyed stranger somehow mistaken for an initiate, had been ushered into the sanctuary to witness the mystery of mysteries. I saw a phantasmagoria, a living tapestry of forms jeweled in minute detail. They danced together like guests at a rowdy wedding. They changed their shapes. Within themselves they juggled geometrical shards like the fragments in a kaleidoscope. They sent forth extensions of themselves like the flares of suns. Yet all their activity was obviously interrelated; each being's actions were in step with its neighbors'. They were like bees swarming: They obviously recognised each other and were communicating avidly, but it was impossible to know what they were saying. They enacted a pageant whose beauty awed me. 

As the lights came back on, the auditorium seemed dull and unreal. I'd been watching various kinds of ordinary cells going about their daily business, as seen through a microscope and recorded by the latest time lapse movie techniques. The filmmaker frankly admitted that neither he nor anyone else knew just what the cells were doing, or how and why they were doing it. We biologists, especially during our formative years in school, spent most of our time dissecting dead animals and studying preparations of dead cells stained to make their structures more easily visible—"painted tombstones," as someone once called them. Of course, we all knew that life was more a process than a structure, but we tended to forget this, because a structure was so much easier to study. This film reminded me how far our static concepts still were from the actual business of living. As I thought how any one of those scintillating cells potentially could become a whole speckled frog or a person, I grew surer than ever that my work so far had disclosed only a few aspects of a process-control system as varied and widespread as life itself, of which we'd been ignorant until then. 

The film was shown at a workshop on fracture healing sponsored by the National Academy of Sciences in 1965. It was one of a series of meetings organized for the heads of clinical departments to educate them as to the most promising directions for research. A dynamic organizer named Jim Wray had recently become chairman of the Upstate Medical Center's department of orthopedic surgery, but Jim's superb skills were political rather than scientific. Since I was an active researcher and had just been promoted to associate professor, Jim asked me to go in his place. I tried to get out of it, because I knew my electrical bones would get a frosty reception from the big shots if I opened my mouth, but Jim prevailed. The meeting was mostly what I'd expected, but there were three bright spots. One was that micro cinematic vision. Another was the chance to get acquainted with the other delegate from my department, a sharp young orthopedic surgeon named Dave Murray. The third was the presence of Dr. John J. Pritchard. 

A renowned British anatomist who'd added much to our knowledge of fracture healing, Dr. Pritchard was the meeting's keynote speaker—the beneficent father-figure who was to evaluate all the papers and summarize everything at the end. Dave and I almost skipped his talk. We hadn't been impressed with the presentations, and we figured there had been so few new ideas that Pritchard would have nothing to say. However, our bus to the Washington airport didn't leave until after Pritchard's luncheon address, so we stayed. With a tact that seemed peculiarly English, he reached the same assessment we had, but phrased  it so as to offend no one. He stressed that fracture healing should be considered a vestige of regeneration. Most past work on fractures had described what happened when a bone knit, as opposed to the how and why. As Pritchard pointed out, "Not a great deal of thought has been given to the factors that initiate, guide, and control the various processes of bone repair." Just as in regeneration research, this was the most important problem about fractures, he concluded. 

Dave and I had to wait several hours before our flight back to Syracuse. We sat in the airport lounge and talked excitedly about broken bones. Dave agreed that, since I'd found electrical currents in salamander limb regeneration, it was at least plausible that similar factors controlled the mending of fractures. Having just deciphered the control system for stress adaptation (Wolff's-law growth) in bone, I felt prepared to get back to the more complex problems of regeneration via its remnant in bone healing. Dave and I decided to collaborate, and we planned our experiment on the plane. We would break the same bone in a standardized way in each of a series of experimental animals. I would study the electrical forces in and around the fractures as they healed. We would kill a few of the animals at each stage of healing, and Dave, an expert histologist (cell specialist), would make microscope slides of the healing tissues and study the cellular changes. Along the way we would fit our findings together to see whether electricity was guiding the cells. 

Our first task was to choose the experimental animals. We wanted to use dogs or rabbits, since ultimately we were trying to understand human bones and wanted to work with animals as closely related to us as possible. But we would need scores of them to study each phase of healing adequately, and we had neither the funds nor the facilities to house so many large mammals. We thought of rats, but their longest bones were too short to study clearly and were curved as well. We were looking for nice, long, straight bones, in which we could produce uniform breaks. 

We settled on bullfrogs. They were cheap to buy and care for; we could even collect some ourselves from nearby ponds. I already had a lot of experience in working with them. Best of all, the adult frog's lower leg had one long bone—the tibia and fibula found in most vertebrates had merged into a tibiofibularis. It was about two inches long with a fine, straight shaft. 

Our misgivings about the evolutionary distance between frogs and humans were allayed when we went to the library to read up on what was then known about fracture healing in frogs. Dr. Pritchard himself, along with two of his students, J. Bowden and A. J. Ruzicka, had determined that frogs mended their bones the same way people did. Our question was: What stimulated the periosteal and marrow cells to change into new bone-forming cells? 

We began by anesthetizing the animals and resolutely breaking all those little green legs by hand, bending them only to a certain angle so as not to rupture the periosteum around the fracture. I found I had to put little plaster casts on them—not because the frogs seemed in great pain but because their movements kept shifting the broken bones and making systematic observations impossible. They would have healed anyway; in our first sixty frogs we found two that had broken their legs in the wild and mended them, but I'm sure ours were the first that ever had casts. 

The electrical changes were complex but were almost the same in every fracture. There were two distinct patterns, one on the periosteum and one on the bone. Before fracture the ankle end of both the bone and the periosteum had a small negative potential of less than 1 millivolt as compared to the knee end. At the moment of fracture, the negative potential on the intact periosteum over the break shot up to 6 or 7 millivolts, while areas of positive charge formed above and below the break. After a week, the periosteum normal progression of negative charge toward the ankle was restored. When a fracture ruptured the periosteum, its negative potential went even higher than 7 millivolts, but amputation of the dangling lower leg immediately reversed the polarity, producing a positive current of injury from the stump, as in the frogs of my first regeneration experiment. The bone itself underwent a short-term electrical change opposite to that in the periosteum. A small positive charge appeared on each of the broken ends during the first hours, then fell to near zero after three hours. 

The electricity had two different sources. When cut the leg nerves, the periosteum readings dropped dramatically, indicating that these potentials were coming from currents in the nerves to the periosteum and the surrounding wound area. Measurements on the bone, which has almost no nerves, were unaffected. Many piezoelectric materials emit a continuous current for several hours after their charge-producing structure has been left in a state of unresolved stress by fracture; I surmised that this was true of bone and soon found that another research team had recently proven it. Two separate currents, then, one from the nerves and one from the bone matrix, were producing potentials of opposite polarity, which acted like the electrodes of a battery. These living electrodes were creating a complex field whose exact shape and strength reflected the position of the bone pieces. The limb was, in effect, taking its own X ray. 

While I was busy with my probes and meters, Dave was taking samples of the bones and blood clots and preparing them for the microscope. We killed a few of the frogs every fifteen minutes during the first two hours, then every day for two weeks, every other day for the third week, and weekly for the last three weeks. Preparing the slides took several days. 

In the normal sequence of bone healing in frogs, a blood clot forms after about two hours and develops into a blastema during the first week. It turns into the rubbery, fibrous callus during the second and third weeks, and ossifies in three to six weeks. In this last period, islands of bone first emerge near the broken ends. Next, bony bridges appear, connecting the islands. Then the whole area is gradually filled in and organized with the proper marrow space and blood canals to join the segments of old bone. 

Dave began his work with specimens taken nearly a week after the fractures, when we expected to see the first signs of the callus forming. "This is mighty damn funny," he said as he walked in with the first box of slides. "I can't see any mitoses in the periosteum. There's no evidence that the cells there are multiplying or migrating." 

We agreed that we must have done something wrong. Pritchard's work had been quite conclusive on this point. He'd even published photographs of the periosteal cells dividing and moving into the gap. We thought maybe we were looking at specimens from the wrong time period, but we could see with our own eyes that the callus was starting to form. Dave went back to study specimens from the first few days, even though we didn't expect to see much then except clotting blood. Soon he called me from his lab and asked, "What would you say if I told you that the red corpuscles change and become the new bone-forming cells?" 

I groaned. "Nonsense. That can't be right." But it was right. We went over the whole slide series together. Beginning in the second hour, the erythrocytes (red cells) began to change. 

All vertebrates except mammals have nuclei in their red blood cells. In mammals, these cells go through an extra stage of development in which the nucleus is discarded. The resulting cells are smaller, can flow through smaller capillaries, can be packed with more hemoglobin, and thus can carry oxygen and carbon dioxide more efficiently. Nucleated erythrocytes are considered more primitive, but even in these the nucleus is pyknotic—shriveled up and inactive. The DNA in pyknotic nuclei is dormant, and such cells have almost no metabolic activity; that is, they burn no glucose for energy and synthesize no proteins. If you had to choose a likely candidate for dedifferentiation and increased activity, this would be the worst possible choice. 

In our series of slides the red cells went through all their developmental stages in reverse. First they lost their characteristic flattened, elliptical shape and became round. Their membranes acquired a scalloped outline. By the third day the cells had become ameboid and moved by means of pseudopods. Concurrently, their nuclei swelled up and, judging by changes in their reactions to staining and light, the DNA became reactivated. We began using an electron microscope to get a clearer view of these changes. At the end of the first week, the former erythrocytes had acquired a full complement of mitochondria and also ribosomes (the organelles where proteins are assembled), and they'd gotten rid of all of their hemoglobin. By the third week they'd turned into cartilage-forming cells, which soon developed further into bone-forming cells. 

I wasn't happy with this turn of events. How could we reconcile what we saw with the well-documented findings of Pritchard, Bowden, and Ruzicka? I'd expected evidence for the semiconducting electrical system I'd been investigating, a concept that was already strange enough to keep me out of the scientific mainstream. I would have been happy if the electrical measurements had fit in with straightforward changes in the periosteal cells. The difference between them and erythrocytes was crucial. Periosteal cells were closely related precursors of bone cells; blood cells couldn't have been further removed. They couldn't possibly have built bone without extensive job retraining on the genetic level. These bullfrogs were bringing us up hard against a wall of dogma by showing us metaplasia—dedifferentiation followed by redifferentiation into a totally unrelated cell type. The process took place in some of the most specialized cells in the frog's entire body, and it looked as though the electric field set the changes in motion while at its strongest, about an hour after the fracture. 

Our next move was a respectful letter to Dr. Pritchard asking if there  was any way he could make sense of the contradictory observations. He replied in the negative but sent our inquiry to Dr. Bowden, who had done the actual work on frogs as his doctoral thesis. Bowden had a possible explanation. He'd done the experimental work under a time limit, and to finish before the deadline he'd kept his frogs at high temperatures—only a few degrees short of killing them, in fact—in order to speed up their metabolism. 

Bowden also mentioned that two researchers cited in his bibliography had seen fracture healing in frogs much the same way we had. In the 1920s, a German named H. Wurmbach, also working on his doctorate, noted some strange cellular transformations in the blood clot and worried over his inability to explain them. However, Wurmbach also found mitoses in the periosteum and ascribed healing to the latter process, since it didn't involve dedifferentiation. A decade later, another German scientist, A. Ide-Rozas, saw the same changes in the blood cells, but he was more daring. He proposed that this transformation was the major force behind fracture healing in frogs and further suggested that regenerating salamanders formed their limb blastemas from nucleated red blood cells. Other experiments seemed to contradict Ide-Rozas' idea about limb blastemas, so his work was discredited and ignored, but Bowden wished us better luck. 

Bowden's letter gave us a framework for understanding our results. We already knew that mammals did not heal bones by dedifferentiation of their red corpuscles, because their red cells had no nuclei and thus no mechanism for change. Mammals also had a thicker periosteum than other vertebrates, so we reasoned that periosteal cell division played a larger healing role in mammals. Frogs, it seemed, had both methods available but activated the periosteal cells only at high temperatures. 

Do-lt-Yourself Dedifferentiation 

Now we were sure that our results were real. We repeated the same fracture studies, but this time we also observed the cells while they were alive. We took tissue samples from the fractures and made time-lapse film sequences using techniques like those in the movie that had impressed me so much at the NAS workshop. We confirmed that the changes began in the first lew hours, just after the electrical forces reached their peak. 

Now we decided to try a crucial test. If the electricity really triggered healing, we should be able to reproduce the same field artificially and start  the same changes in normal blood cells outside the frog. If this didn't work, then I probably had spent the last seven years "collecting stamps"— accumulating facts that were interesting but, in the end, trivial. 

I calculated the amount of current that would produce the fields I'd found. I came up with an incredibly small amount, somewhere between a trillionth and a billionth of an ampere (a picoamp and a nanoamp, respectively). Again I thought there must be some mistake. I didn't see how such a tiny current could produce the dramatic effects we'd seen, so, figuring that even if my numbers were right, more juice would simply hasten the process, I decided to start with 50 microamps, a current level that would be just shy of producing a little electrolysis—the breakdown of water into hydrogen and oxygen. 

I designed plastic and glass chambers of various shapes, fitted with electrodes of several types. In these chambers we would place healthy red blood cells in saline solution and observe them by microscope while the current was on. 

I set up the experiment in a lab across the street from the medical center, where there was available one of the inverted microscopes we would need to observe the cells through the bottoms of the chambers, where most of them would settle. I put a young technician named Frederick Brown in charge of the long grind of watching the cells hour after hour at different current levels and field shapes in the various chambers. We began in the summer of 1966. Fred was to enter medical school that fall, and I figured two months would be more than enough time! He was to run one test batch of frog blood each day and report to me the next morning as to what he'd found. 

It didn't start well. Nothing had happened after six hours of current. We couldn't increase the amperage without electrocuting the cells, so we ran it longer. Still nothing happened. In fact, the cells started dying when we left them in the chambers overnight. We decided to lower the current, but I still didn't believe in the absurdly low values I'd calculated, so I told Fred to drop the amperage only a little bit day by day. He and I stared at a lot of blood cells over those two months, all stubbornly refusing to do anything. Finally, two days before Fred had to leave, we'd gotten the current down as far as our first apparatus could go, and well within the range I'd calculated—about half a billionth of an ampere. At eleven that morning he called me excitedly and I rushed across the street. 

With the room darkened and the microscope light on, we saw the same cell changes as in the blood clot, first at the negative electrode, then at the positive electrode, and finally spreading across the rest of the  chamber. In four hours all the blood cells in the chamber had reactivated their nuclei, lost their hemoglobin, and become completely unspecialized in form.

We repeated the experiment many times, working out the upper and lower limits of the effective current. The best "window" was somewhere between 200 and 700 picoamps. I say "somewhere" because the susceptibility of the cells varied, depending on their age, the hormonal state of the frog, and possibly other factors.* This was an infinitesimal tickle of electricity, far less than anything a human could feel even on the most sensitive tissue, such as the tongue, but it was enough to goose the cell into unlocking all its genes for potential use. 

* Rather than continually renewing a small part of their red-cell stock, frogs generate a whole year's supply in late winter as they emerge from hibernation. Thus, all their erythrocytes age uniformly as the year progresses. The cells become less sensitive to electricity as they get older, and that may be why frogs, even when warm and not hibernating, heal fractures more slowly in winter. The red blood cells dedifferentiate most readily in the spring, and the female's become even more sensitive than the male's when she ovulates early in that season. At that time her red corpuscles will despecialize in response to less than one picoamp. In fact, we saw red cells from ovulating females dedifferentiate completely in chambers to which we supplied no current whatever. Apparently an unmeasurably small current created by the charge difference between the plastic chamber and glass cover slip was enough. 

The effect depended on having the proper cells as well as the proper current—white blood cells, skin cells, and other types didn't work. Only erythrocytes seemed to serve as target cells in frogs. We found the same response in the blood cells of goldfish, salamanders, snakes, and turtles. The only variation was that the fish cells despecialized faster and the reptilian cells more slowly than frog blood cells. In all erythrocytes the shift in the transparency and staining characteristics of the nucleus was a point of no return. These changes seemed to indicate reactivation of the DNA, for afterward the rest of the process continued even if we switched off the current. 

This was a breakthrough. We'd learned something hitherto unsuspected about fracture healing in frogs, and it was almost certain to benefit human patients a few years down the road. Because we'd used frogs instead of mammals, we'd also stumbled upon the best proof yet for dedifferentiation—a do-it-yourself method. If we'd studied fracture healing in mammals, we almost certainly would not have made the discovery, for periosteal cells don't dedifferentiate and marrow cells are hard to experiment with. Instead, we even had movies of dedifferentiation happening and electron photomicrographs of air its stages, including brand-new ribosomes being made in the nucleus and deployed into the surrounding cytoplasm. Moreover, all the steps in dedifferentiation, including the activities in the nucleus and the assembly of ribosomes and mitochondria, exactly paralleled the changes found by the most recent research on salamander limb blastemas. We'd found the electrical common denominator that started the first phase—the blastema—in all regeneration. 

The Genetic Key 

Soon after we'd finished this experiment, I was invited to a meeting on electromagnetism in biology at the New York Academy of Sciences. This was basically a one-man show. Kenneth MacLean, a prominent surgeon and highly placed member of the academy, had been using magnetic fields on his patients for years and was convinced that they helped. Independently wealthy, he'd set up a lab in his office, with a large electromagnet. The meeting was a testament to his persistence rather than any widespread belief within the academy that he was right. So in February 1967 I presented our recent work. I played down the full role of electricity in fracture healing, with its overtones of vitalism, and concentrated on our method for inducing dedifferentiation in vitro. That was enough to call forth many attacks from the audience, most of which were variations on "I just don't believe it." Some said we were just electrocuting the cells, despite the fact that they survived for ten days in culture.

One audience member responded with some thoughtful and constructive criticism, however. He accepted the fact that we'd seen what we'd seen. Nevertheless, there were, he said, other barely possible explanations. In particular, he stated, we hadn't gone far enough in proving that the cells weren't slowly degenerating from some small but harmful change caused by the current. While the cells in our chambers looked like those we'd photographed in the fracture clot, our idea that these cells were electrically dedifferentiated healing cells was so at variance with current views that we must have more direct proof. For such a radical departure, seeing wasn't quite believing. 

Stimulated by this one honest reaction, Dave and I returned to Syracuse and planned how we could use the latest knowledge about DNA to test our evidence further. A few years before, James Watson and Francis Crick had proposed what became known as the central dogma of genetics. In simplified form, it stated that the active DNA in each specialized cell imprinted its own specific patterns onto transfer RNA, which relayed them to messenger RNA. This second RNA molecule moved outside the nucleus to the ribosomes, where it translated the genetic instructions into the particular proteins that made the cell what it was. 

We reasoned that, since a dedifferentiating cell was not going to divide immediately, it wouldn't duplicate its genes. Therefore there should be no increase in the amount of DNA it contained. However, since the cell changed its type by manufacturing a whole new set of proteins, the amount of RNA—the protein blueprints—should increase dramatically. 

Using radioactive labeling and fluorescent staining techniques, we found there was indeed no new DNA but dramatic increases in RNA. Another test showed that our despecialized cells contained not only different proteins but also twice as many as their precursor red blood cells. The most conclusive experiment was one suggested by Dan Harrington, a student who'd taken Fred Brown's place and who later went on to a Ph.D. in anatomy. Dan proposed that we use certain well-known metabolic inhibitors that disrupt the DNA-RNA-protein system, to see if we could prevent dedifferentiation. One such inhibitor, an antibiotic called puromycin, blocks the transfer of information from messenger RNA to the ribosomes and thus prevents proteins from being built. 

Dan proposed that we set up our plastic chambers in pairs. In one member of each pair we would place blood cells in saline solution and pass current through them as before. The other chamber would contain cells from the same frog and be connected to the same generator, but the solution would contain puromycin. The setup would thus be precisely controlled. If the current was actually unlocking new genes, the puromycin should stop dedifferentiation by intercepting the DNA's protein-making instructions. If the current was merely making the cells degenerate, however, the puromycin should have no effect and the transformations should continue. 

Our conclusion held up: The cells in the puromycin solution didn't change. Next Dan suggested that we bathe these cells with several changes of water to wash out the puromycin. They promptly dedifferentiated with no current flowing! Apparently the current caused the genetic change in spite of the antibiotic, and the genes stayed unlocked, so the system still worked perfectly as soon as the puromycin blockade was lifted.

By early 1970 we had solid proof for nearly every detail of the control system for fracture healing in frogs, and by extension probably in mammals as well. Like all other injuries, a fracture produced a current of injury, in this case derived from the nerves in and around the periosteum.* At the same time, the bone generated its own current piezoelectrically due to residual stress in the mangled apatite-collagen matrix. These signals combined to stimulate the cells that formed new bone. 

*The lack of the periosteal (nerve-derived) current may explain the uncontrolled, deformed growth that often follows fractures in the limbs of paraplegics and lepers. Their bones still generate a positive potential in the gap, but because of nerve damage it isn't balanced by the negative periosteal potential that normally surrounds the break. 

Except for the identity of the target cells, bone repair seemed to be basically the same in all vertebrates, proceeding through the stages of blood clot, blastema, callus, and ossification. In fish, amphibians, reptiles, and birds, the red cells in the clot dedifferentiated in response to the electric field, especially the positive potentials at the broken ends of the bone. They then re-differentiated as cartilage cells and continued on to become bone cells.

In some animals the periosteal cells responded to the current of injury by migrating into the gap and specializing a bit further into bone cells. This process seemed to be available to amphibians only at high temperatures, but it was the dominant method in mammals, whose thick periosteum made up for the lack of nuclei in their red corpuscles. By this time we'd become pretty sure that the marrow component of bone healing in humans involved the dedifferentiation of at least the immature erythrocytes, which still contained a nucleus, and possibly other cell types. 

The electrical forces turned the key that unlocked the repressed genes. The exact nature of that key was the one part still missing from the process. The current couldn't act directly on the nucleus, which was insulated by the cell's membrane and cytoplasm. We knew that the current's primary effect had to be on the membrane. The cell membrane itself was known to be charged. Its charge probably occurred as a specific pattern of charged molecules, different for each type of cell. We postulated that the membrane released derepressors—molecules that migrated inward into the nucleus, where they switched on the genes. Based on recent findings about the structure of RNA, we suggested that the derepressor molecules might be a stable form of messenger RNA that persisted in the mature red cell even after its nucleus shriveled up and turned itself off. RNA molecules can be stable for a long time when they are folded, the strands secured together by electron bonds. If such folded RNA molecules were stored in the cell membrane, the tiny currents could release their bonds and unfold them. This hypothesis has not yet been tested.

Fracture healing was ended by a straightforward negative-feedback system. As the gap was filled in with new matrix, the bone gradually redistributed its material to balance the stresses on it from the action of the surrounding muscles during cautious use, in accordance with Wolff's law. Repair of surrounding tissues lessened and then stopped the periosteal injury current. As a result the electrical field returned to normal, shutting off the cellular activities of healing. 

When we'd finished this series of experiments, I was sure this was the most important piece of work I would ever do, and I was determined to get it published as a major article, not just a short note. Luck was with me. On several speaking engagements during the previous two years I'd been able to talk at length with Dr. Urist. He was enthusiastic about our findings, and since he was the editor of one of the major orthopedic journals—Clinical Orthopedics and Related Research—I submitted our report there. The editorial board published it uncut, and I was pleased to use Dr. Pritchard's statement at the 1965 bone-healing workshop as an epigraph. To me it's still the most satisfying of my publications. 

This was the first time the control system for a healing process had been worked out in such detail. Except for the less conclusive account Dave and I had presented to the New York Academy of Sciences three years before, it was also the first really incontrovertible proof of dedifferentiation and metaplasia. 

These were hardly new ideas, of course. Dedifferentiation had often been proposed during the previous four decades as the simplest explanation of blastema formation, and a great deal of evidence for it had been amassed. Elizabeth Hay had even published an electron microscope photograph of a blastema cell that hadn't despecialized completely and still contained a piece of muscle fiber. Nevertheless, the idea was dismissed by most of the biologists who wielded influence in grant review committees and universities. 

Today, however, dedifferentiation is no longer a dirty word. In part, this is because Dave and I devised a way to produce it artificially, which could be repeated by anyone who cared to. Art Pilla, an electrochemist working with Andy Bassett in New York, was the first to confirm our method. I'm happy to have been able to play a major role in this hardwon advance of knowledge. 

Even more important, this was the first work of mine that led directly to a technique that helped patients—electrical stimulation of bone healing (see Chapter 8). Meanwhile, our results led to another major question: Couldn't the currents we'd found be used artificially to stimulate other types of regeneration? We decided to see if we could bring limb regrowth a step closer to humans by trying to induce it in rats. 

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https://exploringrealhistory.blogspot.com/2022/04/part-4the-body-electric-good-news-for.html

Good News for Mammals 

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