Recall from an earlier section that new bone is continually being formed to replace old bone. That is, in contrast to the lengthening of bone, the thickness and strength of bone must continually be maintained by the body. This is accomplished as bone is continually deposited by osteoblasts, while at the same time, it is continually being reabsorbed (broken down and digested by the body) by osteoclasts (refer to Figure 5 earlier in the chapter). Osteoblasts are found on the outer surfaces of the bones and in the bone cavities. A small amount of osteoblastic activity occurs continually in all living bones (on about 4 % of all surfaces at any given time) so that at least some new bone is being formed constantly. Normally, in fact, except in growing bones, the rates of bone deposition and absorption are equal to each other so that the total mass of bone remains constant. Bone mineralization refers to the process of rebuilding bones, including the formation of the collagen/crystalline matrix, over time. Aging and certain disease states such as osteoporosis cause bones to mineralize at a reduced rate. This can also occur when bones are not "loaded" normally, which happens when a leg is in a cast or when ill patients are unable to get out of bed to walk around. When mineralization is reduced, the bones become weaker and more prone to fracture. Whole bone strength is largely dependent on the of the bone tissue, including:

• the architecture and distribution of bone mass, and
• the geometry of the bone.

You already know that bone grows in length, but bone also grows around its diameter, similar to the way a tree trunk grows. If you have ever seen a cross section of a tree trunk, you can estimate the age of the tree by counting the rings. In fact, the wayto measure the rate of growth in a bone's diameter is by producing rings along the cross-section of the bone and timing how long it took to for the bone to grow from ring to ring. Fluorescent bone markers are used to produce colored rings along the diameter of the bone. The idea is simple.

First, the rat is injected with a bone marker. This bone marker will cover the outer boundary of the bone around the periosteum. Then, the bone continues to grow in diameter beyond the colored ring that was produced by the marker. After a certain amount of time, the rat is injected with a different marker that will, again, cover the outermost boundary of the bone with a different color. The bone will then continue to grow some more in diameter beyond the second colored ring. By measuring how much the bone grew and by knowing the amount of time it took to grow that much, one can determine the rate of growth of the bone. But, bone does not only grow "outward" along the edge of the periosteum, it also grows "inward" toward the endosteum, which is the inner boundary of the bone that lines the marrow cavity. Therefore, a marker must be used that can selectively mark both the inner and outer boundaries of the bone. In this way, one can determine if the bone grows faster "outwardly" or faster "inwardly" toward the marrow. The rate of growth of the diameter of a bone would reflect how quickly bone mineralization is taking place.

Dr. Holton performed her experiment in order to compare the mineralization rate of flight animals to that of the control animals. In addition, she was able to compare any effect that the housing arrangements had on bone growth and mineralization. In the cross sectional areas of bone depicted in Figure 21, the bone labels are marked. As you can see, the distance between the labeled rings shows the amount of bone growth between the various injections of fluorescent marker.

Tibia Cross Section: Control	Tibia Cross Section: Flight
Figure 21. A pair of digitized fluorescent images of a cross section of bone from the tibia of a ground control and a flight rat. Preflight, inflight, and postflight bone growth distances are indicated.

The preflight label is a calcein dye which was injected about 13 days before launch.

The inflight label is a marker called declomycin, which was injected 1.5 days before launch.

The postflight label was also calcein and was given at the end of the shuttle flight. The postilight distance leading to the bone surface represents nine days of growth. As mentioned, bone mineralizes both at the endosteal, or inner, surface of bone and at the periosteal, or outer, surface of bone. The most rapid bone growth usually occurs on the outer surface where muscle is attached.

Dr. Holton's results suggest that bone mineralization decreased over time. The results can be broken down into four parts:

(1) At the periosteal surface of the tibia (at a location on the bone known as the tibiofibular junction), the data from Figure 22a shows that the control groups experienced a 13% decrease in mineralization during the flight period. The periosteal mineralization for the flight rats, however, decreased significantly more during the flight period compared to the controls. Those that flew in a group housing arrangement, the AEM-F rats, experienced a 28.8% decrease and those that flew individually, the RAHF-F rats, experienced a 37.7% decrease in mineralization. Postflight, however, is another story. Although all rats exhibited reduced mineralization rates, the flight rats showed less of a decrease than the control rats.

(2) Like the periosteal surface of the tibia, the endosteal surface of the tibia showed decreased formation. Unlike the periosteal surface, however, the mineralization rate of the endosteal surface was very similar in all groups. Figure 22b shows this decrease averaged about 45%.

Figure 22 (a&b).

(3) In the humerus, no difference between flight and control animals occurred during the preflight period, but during the flight and postflight periods, the flight rats showed significantly reduced periosteal and endosteal mineralization. Figures 22c and 22d show the results of the periosteal and endosteal mineralization rate measurements, respectively.

Figure 22 (c&d).
Figure 22 (a,b,c,d). Bone mineralization rates at the periosteal (outer) and endosteal (inner) surface of the tibia and the humerus of the rat.

Notice that the units of measurement for the bone mineralization rate is mm/day. That means that the measurement was based on how many square millimeters of bone were formed per day. What does all of this mean? Well, essentially, there was no real difference in mineralization rate due to flight at the endosteal surface of either the tibia or humerus. The main differences existed in the mineralization of the periosteal surface, with the largest difference being between the singly housed flight (RAHF-F) and control animals (VIV-C) for both the tibia and the humerus. This study was also designed to determine whether space flight affected the strength characteristics, or stiffness, of certain bones. In order to measure stiffness, the humerus and the tibia bones were placed onto a three-point bending chamber that was equipped to bend the bones while keeping them in a special "physiological fluid." This fluid was important to keep the bones in a state that resembles living bone. The bones were then bent until "failure," or until they fractured. All bone samples failed at a midpoint on the diaphysis. Figure 23 shows the results of the humerus stiffness test for animals that flew on both the RAHF (housed individually) and the AEM (group-housed).

Figure 23. The stiffness levels of the rat humerus for both the individually houses animals (RAHF) and the group-housed animals (AEM).

In both cases, values for the flight and control animals are shown. From Figure 23, the data suggests the following:

• The RAHF flight animals that were housed individually seemed to maintain the strength of their humerus bones compared to the control animals that stayed on Earth. However, their humerus bones were found to be significantly weaker than those of their matching controls after being home for nine days. That is, their bones did not seem to develop normally, even when they returned from space flight. From Figure 23, the data also suggests the following:
• The AEM flight animals that were housed in groups of five rats per cage seemed to have somewhat stronger humerus bones than their controls, both at landing and after nine days of recovery back on Earth. In addition, they seemed to have stronger bones overall than the animals that were housed individually in the RAHF. Does this mean that the animals that were housed in more crowded conditions get more exercise, thereby being able to "load" their bones more often You can imagine that they do have to constantly bounce off one another,but it is unclear if their stiffer bones can be fully attributed to their housing arrangement.

In space or during restricted activity or exercise, we've seen evidence of changes in the bone structure of rats, and in a previous chapter we learned of the decrease in the mass of the gravity-dependent muscles. These space flight effects certainly compound one another, rendering the animals weaker overall. Because of this weaker condition, the animals may be prone to bone fractures when exercise or structural loading is increased (i.e., return to Earth from space). The question that we are faced with is how all of this applies to the human system.We need to find out what components of the bone structure are changed, the extent to which they change, the impact of the changes on bone strength, and the reversibility of any changes. Only then can we develop countermeasures for astronauts to inhibit potentially damaging changes in bone structure during space flight This research can also help to design appropriate therapy for patients here on Earth who suffer from various forms of bone disease.

Now it is time to return to Dr. Holton's hypotheses:

Hypothesis 1

Gravity is necessary for normal development of bone structure on Earth, and decreased gravity or skeletal unloading causes changes in skeletal growth patterns.

Hypothesis 2

The response of bone to space flight will be localized and will differ not only from bone to bone, but also at different sites within the same bone.

Hypothesis 3

The type of housing (group vs. individual) will influence the bones response to space flight and the recovery from space flight.