Fibroblasts Change Their Character in Response to Chemical Signals

Fibroblasts seem to be the least specialized cells in the connective-tissue family. They are dispersed in connective tissue throughout the body, where they secrete a nonrigid extracellular matrix that is rich in type I and/or type III collagen, as discussed in Chapter 19. When a tissue is injured, the fibroblasts nearby proliferate, migrate into the wound, and produce large amounts of collagenous matrix, which helps to isolate and repair the damaged tissue. Their ability to thrive in the face of injury, together with their solitary lifestyle, may explain why fibroblasts are the easiest of cells to grow in culture—a feature that has made them a favorite subject for cell biological studies (Figure 22-46).

Figure 22-46

The fibroblast. (A) A phase-contrast micrograph of fibroblasts in culture. (B) These drawings of a living fibroblastlike cell in the transparent tail of a tadpole show the changes in its shape and position on successive days. Note that while fibroblasts (more...)

As indicated in Figure 22-45, fibroblasts also seem to be the most versatile of connective-tissue cells, displaying a remarkable capacity to differentiate into other members of the family. There are uncertainties about their interconversions, however. Strong evidence indicates that fibroblasts in different parts of the body are intrinsically different, and there may be differences between them even in a single region. “Mature” fibroblasts with a lesser capacity for transformation may, for example, exist side by side with “immature” fibroblasts (often called mesenchymal cells) that can develop into a variety of mature cell types.

The stromal cells of bone marrow, mentioned earlier, provide a good example of connective-tissue versatility. These cells, which can be regarded as a kind of fibroblast, can be isolated from the bone marrow and propagated in culture. Large clones of progeny can be generated in this way from single ancestral stromal cells. According to the signal proteins that are added to the culture medium, the members of such a clone can either continue proliferating to produce more cells of the same type, or can differentiate as fat cells, cartilage cells, or bone cells. Because of their self-renewing, multipotent character, they are referred to as mesenchymal stem cells.

Fibroblasts from the skin are different. Placed in the same culture conditions, they do not show the same plasticity. Yet they, too, can be induced to change their character. At a healing wound, for example, they change their actin gene expression and take on some of the contractile properties of smooth muscle cells, thereby helping to pull the wound margins together; such cells are called myofibroblasts. More dramatically, if a preparation of bone matrix, made by grinding bone into a fine powder and dissolving away the hard mineral component, is implanted in the dermal layer of the skin, some of the cells there (probably fibroblasts) become transformed into cartilage cells, and a little later, others transform into bone cells, thereby creating a small lump of bone. These experiments suggest that components in the extracellular matrix can dramatically influence the differentiation of connective-tissue cells.

We shall see that similar cell transformations are important in the natural repair of broken bones. In fact, bone matrix contains high concentrations of several signal proteins that can affect the behavior of connective-tissue cells. These include members of the TGFβ superfamily, including BMPs and TGFβ itself. These factors are powerful regulators of growth, differentiation, and matrix synthesis by connective-tissue cells, exerting a variety of actions depending on the target cell type and the combination of other factors and matrix components that are present. When injected into a living animal, they can induce the formation of cartilage, bone, or fibrous matrix, according to the site and circumstances of injection. TGFβ is especially important in wound healing, where it stimulates conversion of fibroblasts into myofibroblasts and promotes formation of the collagen-rich scar tissue that gives a healed wound its strength.

The Extracellular Matrix May Influence Connective-Tissue Cell Differentiation by Affecting Cell Shape and Attachment

The extracellular matrix may influence the differentiated state of connective-tissue cells through physical as well as chemical effects. This has been shown in studies on cultured cartilage cells, or chondrocytes. Under appropriate culture conditions, these cells proliferate and maintain their differentiated character, continuing for many cell generations to synthesize large quantities of highly distinctive cartilage matrix, with which they surround themselves. If, however, the cells are kept at relatively low density and remain as a monolayer on the culture dish, a transformation occurs. They lose their characteristic rounded shape, flatten down on the substratum, and stop making cartilage matrix: they stop producing type II collagen, which is characteristic of cartilage, and start producing type I collagen, which is characteristic of fibroblasts. By the end of a month in culture, almost all the cartilage cells have switched their collagen gene expression and taken on the appearance of fibroblasts. The biochemical change must occur abruptly, since very few cells are observed to make both types of collagen simultaneously.

Several lines of evidence suggest that the biochemical change is induced at least in part by the change in cell shape and attachment. Cartilage cells that have made the transition to a fibroblastlike character, for example, can be gently detached from the culture dish and transferred to a dish of agarose. By forming a gel around them, the agarose holds the cells suspended without any attachment to a substratum, forcing them to adopt a rounded shape. In these circumstances, the cells promptly revert to the character of chondrocytes and start making type II collagen again. Cell shape and anchorage may control gene expression through intracellular signals generated at focal contacts by integrins acting as matrix receptors, as discussed in Chapter 19.

For most types of cells, and especially for a connective-tissue cell, the opportunities for anchorage and attachment depend on the surrounding matrix, which is usually made by the cell itself. Thus, a cell can create an environment that then acts back on the cell to reinforce its differentiated state. Furthermore, the extracellular matrix that a cell secretes forms part of the environment for its neighbors as well as for the cell itself, and thus tends to make neighboring cells differentiate in the same way (see Figure 19-61). A group of chondrocytes forming a nodule of cartilage, for example, either in the developing body or in a culture dish, can be seen to enlarge by the conversion of neighboring fibroblasts into chondrocytes.

Fat Cells Can Develop From Fibroblasts

Fat cells, or adipocytes, also derive from fibroblastlike cells, both during normal mammalian development and in various pathological circumstances. In muscular dystrophy, for example, where the muscle cells die, they are gradually replaced by fatty connective tissue, probably by conversion of local fibroblasts. Fat cell differentiation (whether normal or pathological) begins with the expression of two families of gene regulatory proteins: the C/EBP (CCAAT/enhancer binding protein) family and the PPAR (peroxisome proliferator-activated receptor) family, especially PPARγ. Like the MyoD and MEF2 families in skeletal muscle development, the C/EBP and PPARγ proteins drive and maintain one another's expression, through various cross-regulatory and autoregulatory control loops. They work together to control the expression of the other genes characteristic of adipocytes.

The production of enzymes for import of fatty acids and glucose and for fat synthesis leads to an accumulation of fat droplets, consisting mainly of triacylglycerol (see Figure 2-77). These then coalesce and enlarge until the cell is hugely distended (up to 120 μm in diameter), with only a thin rim of cytoplasm around the mass of lipid (Figures 22-47 and 22-48). Lipases are also made in the fat cell, giving it the capacity to reverse the process of lipid accumulation, by breaking down the triacylglycerols into fatty acids that can be secreted for consumption by other cells. The fat cell can change its volume by a factor of a thousand as it accumulates and releases lipid.

Figure 22-47

The development of a fat cell. A fibroblastlike precursor cell is converted into a mature fat cell by the accumulation and coalescence of lipid droplets. The process is at least partly reversible, as indicated by the arrows. The cells in the early and (more...)

Figure 22-48

Fat cells. This low-magnification electron micrograph shows parts of two fat cells. A neutrophil cell that happens to be present in the adjacent connective tissue provides a sense of scale; each of the fat cells is more than 10 times larger than the neutrophil (more...)

Leptin Secreted by Fat Cells Provides Negative Feedback to Inhibit Eating

For almost all animals under natural circumstances, food supplies are variable and unpredictable. Fat cells have the vital role of storing reserves of nourishment in times of plenty and releasing them in times of dearth. It is thus essential to the function of adipose tissue that its quantity should be adjustable throughout life, according to the supply of nutrients. For our ancestors, this was a blessing; in the well-fed half of the modern world, it has become also a curse. In the United States, for example, it is estimated that more than 30% of the population suffer from obesity, defined as a body mass index (weight/height²) more than 30 kg/m², equivalent to about 30% above ideal weight.

It is not easy to determine to what extent the changes in the quantity of adipose tissue depend on changes in the numbers of fat cells, as opposed to changes in fat-cell size. Changes in cell size are probably the main factor in normal nonobese adults, but in severe obesity, at least, the number of fat cells also increases. The factors that drive the recruitment of new fat cells are not well understood, although they are thought to include growth hormone and IGF-1 (insulinlike growth factor-1). It is clear, however, that the increase or decrease of fat cell size is regulated directly by levels of circulating nutrients and by hormones, such as insulin, that reflect nutrient levels. The surplus of food intake over energy expenditure thus directly governs the accumulation of adipose tissue.

But how are food intake and energy expenditure themselves regulated? A human adult eats about a million kilocalories per year, equivalent to about 200 kg of pure fat. Clearly, if we are not to get hopelessly fat or hopelessly thin over the course of a lifetime, there must be control mechanisms to adjust our eating and energy expenditure over the long term according to the quantity of our fat reserves. The key signal is a protein hormone called leptin, which circulates in the bloodstream. Mutant mice that lack leptin or the appropriate leptin receptor are extremely fat (Figure 22-49). Mutations in the same genes sometimes occur in humans, although very rarely. The consequences are similar: constant hunger, overeating, and crippling obesity.

Figure 22-49

Effects of leptin deficiency. Normal mice are here compared with a mouse that has a mutation in the obese gene, which codes for leptin. The leptin-deficient mutant fails to limit its eating and becomes grotesquely fat (three times the weight of a normal (more...)

Leptin is normally made by fat cells; the bigger they are, the more they make. Leptin acts on many tissues, and in particular in the brain, on cells in those regions of the hypothalamus that regulate eating behavior. The effect in the brain is to lessen hunger and discourage eating, which results in a decreased amount of fat tissue. Thus, leptin, like myostatin released from muscle cells, provides a negative-feedback mechanism to regulate the growth of the tissue that secretes it.

In most obese people, leptin levels in the blood stream are persistently high. Although leptin receptors are present and functional, the effect of leptin on food intake is overwhelmed by other influences, which are poorly understood.

Bone Is Continually Remodeled by the Cells Within It

Bone is a very dense, specialized form of connective tissue, as different as could be from adipose tissue, even though closely related in origin. Like reinforced concrete, bone matrix is predominantly a mixture of tough fibers (type I collagen fibrils), which resist pulling forces, and solid particles (calcium phosphate as hydroxyapatite crystals), which resist compression. The volume occupied by the collagen is nearly equal to that occupied by the calcium phosphate. The collagen fibrils in adult bone are arranged in regular plywoodlike layers, with the fibrils in each layer lying parallel to one another but at right angles to the fibrils in the layers on either side.

For all its rigidity, bone is by no means a permanent and immutable tissue. Running through the hard extracellular matrix are channels and cavities occupied by living cells, which account for about 15% of the weight of compact bone. These cells are engaged in an unceasing process of remodeling: one class of cells (osteoclasts, related to macrophages) demolishes old bone matrix while another (osteoblasts, related to fibroblasts) deposits new bone matrix. This mechanism provides for continuous turnover and replacement of the matrix in the interior of the bone.

Unlike soft tissues, which can grow by internal expansion, bone can grow only by apposition—that is, by the laying down of additional matrix and cells on the free surfaces of existing bone. During development, this process must occur in coordination with the growth of other tissues, in such a way that the pattern of the body can be scaled up without its proportions being radically disturbed. For most of the skeleton, and in particular for the long bones of the limbs and trunk, the coordinated growth is achieved by a complex strategy. A set of minute “scale models” of these bones are first formed out of cartilage. Each scale model then grows, and as new cartilage is formed, the older cartilage is replaced by bone. Cartilage growth and erosion and bone deposition are so ingeniously coordinated during development that the adult bone, though it may be half a meter long, is almost the same shape as the initial cartilaginous model, which was no more than a few millimeters long.

Defective growth of cartilage during the development of long bones, as a result of a dominant mutation in the gene that codes for an FGF receptor (FGFR3), is responsible for the commonest form of dwarfism, known as achondroplasia (Figure 22-50). Conversely, osteoblasts are lacking in individuals with mutations that disrupt production of a gene regulatory protein (called CBFA1) specifically required for osteoblast differentiation: mice homozygous for this genetic defect are born with a skeleton consisting solely of cartilage and die soon after birth as a result.

Figure 22-50

Achondroplasia. This type of dwarfism has a frequency of one in 10,000–100,000 births; in more than 99% of cases it is caused by a mutation at an identical site in the genome, corresponding to amino-acid 380 in FGF-receptor-3 (a glycine in the (more...)

Osteoblasts Secrete Bone Matrix, While Osteoclasts Erode It

Cartilage is a simple tissue, consisting of cells of a single type—chondrocytes—embedded in a more or less uniform matrix. The cartilage matrix is deformable, and the tissue grows by expanding as the chondrocytes divide and secrete more matrix (Figure 22-51). Bone is more complex. The bone matrix is secreted by osteoblasts that lie at the surface of the existing matrix and deposit fresh layers of bone onto it. Some of the osteoblasts remain free at the surface, while others gradually become embedded in their own secretion. This freshly formed material (consisting chiefly of type I collagen) is called osteoid. It is rapidly converted into hard bone matrix by the deposition of calcium phosphate crystals in it. Once imprisoned in hard matrix, the original bone-forming cell, now called an osteocyte, has no opportunity to divide, although it continues to secrete further matrix in small quantities around itself. The osteocyte, like the chondrocyte, occupies a small cavity, or lacuna, in the matrix, but unlike the chondrocyte it is not isolated from its fellows. Tiny channels, or canaliculi, radiate from each lacuna and contain cell processes from the resident osteocyte, enabling it to form gap junctions with adjacent osteocytes (Figure 22-52). Although the networks of osteocytes do not themselves secrete or erode substantial quantities of matrix, they probably play a part in controlling the activities of the cells that do.

Figure 22-51

The growth of cartilage. The tissue expands as the chondrocytes divide and make more matrix. The freshly synthesized matrix with which each cell surrounds itself is shaded dark green. Cartilage may also grow by recruiting fibroblasts from the surrounding (more...)

Figure 22-52

Deposition of bone matrix by osteoblasts. Osteoblasts lining the surface of bone secrete the organic matrix of bone (osteoid) and are converted into osteocytes as they become embedded in this matrix. The matrix calcifies soon after it has been deposited. (more...)

While bone matrix is deposited by osteoblasts, it is eroded by osteoclasts (Figure 22-53). These large multinucleated cells originate, like macrophages, from hemopoietic stem cells in the bone marrow. The precursor cells are released as monocytes into the bloodstream and collect at sites of bone resorption, where they fuse to form the multinucleated osteoclasts, which cling to surfaces of the bone matrix and eat it away. Osteoclasts are capable of tunneling deep into the substance of compact bone, forming cavities that are then invaded by other cells. A blood capillary grows down the center of such a tunnel, and the walls of the tunnel become lined with a layer of osteoblasts (Figure 22-54). To produce the plywoodlike structure of compact bone, these osteoblasts lay down concentric layers of new matrix, which gradually fill the cavity, leaving only a narrow canal surrounding the new blood vessel. Many of the osteoblasts become trapped in the bone matrix and survive as concentric rings of osteocytes. At the same time as some tunnels are filling up with bone, others are being bored by osteoclasts, cutting through older concentric systems. The consequences of this perpetual remodeling are beautifully displayed in the layered patterns of matrix observed in compact bone (Figure 22-55).

Figure 22-53

An osteoclast shown in cross section. This giant, multinucleated cell erodes bone matrix. The “ruffled border” is a site of secretion of acids (to dissolve the bone minerals) and hydrolases (to digest the organic components of the matrix). (more...)

Figure 22-54

The remodeling of compact bone. Osteoclasts acting together in a small group excavate a tunnel through the old bone, advancing at a rate of about 50 μm per day. Osteoblasts enter the tunnel behind them, line its walls, and begin to form new bone, (more...)

Figure 22-55

A transverse section through a compact outer portion of a long bone. The micrograph shows the outlines of tunnels formed by osteoclasts and then filled in by osteoblasts during successive rounds of bone remodeling. The section has been prepared by grinding. (more...)

Through remodelling, bones are endowed with a remarkable ability to adjust their structure in response to long-term variations in the load imposed on them. This adaptive behavior implies that the deposition and erosion of the matrix are somehow controlled by local mechanical stresses, but the mechanisms involved are not understood. The bone cells secrete signal proteins that become trapped in the matrix, and it is likely that these are released when the matrix is degraded or suitably stressed. The released proteins, especially members of the BMP subfamily of TGFβ proteins, may help to guide the remodelling process.

Remodelling carries a risk: defects in its control can lead to osteoporosis, where there is excessive erosion of the bone matrix and weakening of the bone, or to the opposite condition, osteopetrosis, where the bone becomes excessively thick and dense.

During Development, Cartilage Is Eroded by Osteoclasts to Make Way for Bone

The replacement of cartilage by bone in the course of development is also thought to depend on the activities of osteoclasts. As the cartilage matures, its cells in certain regions become greatly enlarged at the expense of the surrounding matrix, and the matrix itself becomes mineralized, like bone, by the deposition of calcium phosphate crystals. The swollen chondrocytes die, leaving large empty cavities. Osteoclasts and blood vessels invade the cavities and erode the residual cartilage matrix, while osteoblasts following in their wake begin to deposit bone matrix. The only surviving remnant of cartilage in the adult long bone is a thin layer that forms a smooth covering on the bone surfaces at joints, where one bone articulates with another (Figure 22-56).

Figure 22-56

The development of a long bone. Long bones, such as the femur or the humerus, develop from a miniature cartilage model. Uncalcified cartilage is shown in light green, calcified cartilage in dark green, bone in black, and blood vessels in red. The cartilage (more...)

Some cells capable of forming new cartilage persist, however, in the connective tissue that surrounds a bone. If the bone is broken, the cells in the neighborhood of the fracture repair it by a sort of recapitulation of the original embryonic process: cartilage is first laid down to bridge the gap and is then replaced by bone.

The capacity for self-repair, so strikingly illustrated by the tissues of the skeleton, is a property of living structures that has no parallel among present-day man-made objects.

Summary

The family of connective-tissue cells includes fibroblasts, cartilage cells, bone cells, fat cells, and smooth muscle cells. Some classes of fibroblasts seem to be able to transform into any of the other members of the family. These transformations of connective-tissue cell type are regulated by the composition of the surrounding extracellular matrix, by cell shape, and by hormones and growth factors. While the chief function of most members of the family is to secrete extracellular matrix, fat cells serve as storage sites for fat. The quantity of fat tissue is regulated in part by negative feedback: fat cells release a hormone, leptin, which acts in the brain to reduce appetite, which leads to a decrease in fat tissue.

Cartilage and bone both consist of cells embedded in a solid matrix. The matrix of cartilage is deformable so that the tissue can grow by swelling, whereas bone is rigid and can grow only by apposition. Bone undergoes perpetual remodeling through which it can adapt to the load it bears; the remodelling depends on the combined action of osteoclasts, which erode matrix, and osteoblasts, which secrete it. Some osteoblasts become trapped in the matrix as osteocytes and play a part in regulating the turnover of bone matrix. Most long bones develop from miniature cartilage “models,” which, as they grow, serve as templates for the deposition of bone by the combined action of osteoblasts and osteoclasts. Similarly, in the repair of a bone fracture in the adult, the gap is first bridged by cartilage, which is later replaced by bone.