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Organ Systems and Tissues


Treatment of the Injured Knee: Use of Stem Cells to Replace Damaged Cartilage

In the human body the knee, as it supports the entire body weight, is susceptible not only to acute injury but also, in particular, to the development of chronic osteoarthritis. Osteoarthritis can be defined as the degeneration of joint cartilage and the bone beneath it, and it occurs in any joint, especially in middle and older ages, in the hip, knee, and thumb. The most common type of osteoarthritis is in the knee and it leads to the deterioration of the articular cartilage. The articular cartilage covers the bone, and the hyaline cartilage is located within the joints. Arthroscopic surgery has been most widely used to treat this condition but with the advent of new research on stem cells, this condition is being ameliorated without surgery by the injection of stem cells into the site. Normal and osteoarthritic cartilage is shown in Fig. 1.1 .

Figure 1.1, Macroscopic signs of osteoarthritis knee hyaline cartilage: (A) healthy cartilage and (B) osteoarthritis cartilage.

Under the microscope, healthy and osteoarthritic knee hyaline cartilage can be differentiated as shown in Fig. 1.2 .

Figure 1.2, Microscopic signs: (A) microscopic signs of healthy knee hyaline cartilage. The histological (HE staining) analysis of cartilage from normal donor showed a preserved morphological structure with no sign of cartilage degradation. Moreover, the surface of healthy hyaline cartilage appears white, shiny, elastic, and firm. Magnification 20×; scale bars: 100 μm. (B) Microscopic signs of OA knee hyaline cartilage. The histological staining (HE staining) analysis of cartilage from OA donor. The donor demonstrated joint swelling and edema, horizontal cleavage tears or flaps, the surface became dull and irregular and had minimal healing capacity. Magnification 20×; scale bars: 100 μm. Moderate OA cartilage ( black arrow ); the structural alterations included a reduction of cartilage thickness of the superficial and middle zones. The structure of the collagen network is damaged, which leads to reduced thickness of the cartilage. The chondrocytes are unable to maintain their repair activity with subsequent loss of the cartilage tissue. Severe OA cartilage ( blue arrow ) demonstrated deep surface clefts, disappearance of cells from the tangential zone, cloning, and a lack of cells in the intermediate and radial zone, which are not arranged in columns. The tidemark is no longer intact and the subchondral bone shows fibrillation. HE , Hematoxylin-eosin; OA , osteoarthritis.

The zones of the articular cartilage down to the bone, including the tidemark, are shown in Fig. 1.3 .

Figure 1.3, The articular (hyaline) cartilage. The articular cartilage is one of five forms of cartilage: the hyaline or articular cartilage, the fibroblastic cartilage comprising the meniscus, the fibrocartilage located at the tendon and the ligament insertion into bone, the elastic cartilage of the trachea, and the physeal cartilage of the growth plate ( physeal , area of bone separating metaphysis and epiphysis where cartilage grows).

In attempting to repair cartilage of an osteoarthritic knee, chondrocytes are extracted arthroscopically from normal cartilage in the nonload-bearing intercondylar notch or the upper ridge of femoral condyles. These cells are then grown in tissue culture for 4–6 weeks ( Fig. 1.4 ) under conditions where no contamination of any sort can take place (“good practices”). When sufficient numbers of cells have grown up, they are injected into the damaged area, usually in combination with a matrix structure ( Fig. 1.5 ).

Figure 1.4, The development of mesenchymal stem cells: (A) first day of culture; (B) third day of culture; (C) 1 week of culture. Magnification 40×; scale bars: 50 μm.

Figure 1.5, Graphic representation of cartilage tissue engineering. Staring with “X” in upper center, chondrocytes, or mesenchymal stem cells are removed from healthy cartilage; the mesenchymal stem cells are isolated and cultured in tissue culture media for about 7 days under strict conditions so that no impurities can contaminate the culture. The cells are concentrated by centrifugation, combined with a matrix substance and various growth factors and injected back into the damaged knee (“V”). The damaged knee, by itself, cannot produce enough stem cells to repair the damage.

The implanted cells, provided with growth factors, grow in the native environment and form new cartilage tissue. In most cases, there is a definite improvement in which there is a reduction of pain and increased ability to use the affected knee, even in competitive sports. While this sort of success in the treatment of knee cartilage problems has been reported in several clinical trials, there is, as yet, no uniform consensus on the effectiveness of this procedure, indicating the need for further clinical trials. This clinical example serves to highlight some of the features of this chapter, particularly with reference to development in which certain layers of cells are equivalent to stem cells that can, under the appropriate conditions, develop into any tissue in the body.

Development of Organs

Development begins with the fertilization of the ovum by a sperm. The early embryo undergoes a cell division generating the two-cell stage, each daughter cell containing the genome of both parents. The division is of a cleavage type (without cell growth) indicating that the size of the cells becomes progressively smaller with each division. Within 5 days, cell division continues through the 4-cell stage, the 8-cell stage, and the 16-cell stage, the morula (by Day 3) and forming the blastocyst that contains an outer and inner cell mass. At the 16-cell stage, there are 8 large cells outside and 8 smaller cells inside the structure. By the fifth day the trophoblast is distinguishable with a large blastocyst cavity (the blastocoel ) and a distinct inner and outer cell mass. By Day 7 the blastocyst becomes enlarged. This progression is depicted in Fig. 1.6 .

Figure 1.6, The stages of early human development.

Stem Cells

Cells of the blastocyst can form all the tissues of all the organs in the body. The only cells that are totipotent (can develop into every cell type in the body, including placental cells) are the embryonic (stem) cells existing after the first and second cell divisions following fertilization. Similar in potency are pluripotent stem cells (embryonic stem cells capable of generating all cell types in the body) derived from the inner cell mass of the blastocyst. These cells, in the appropriate environment, can differentiate into every cell type present in the organism. What is more, this differentiation can take place either in vivo or in vitro. Stem cells also can be obtained from adults (generally multipotent stem cells that can develop into more than one cell type) as most tissues in the body have stores of stem cells (caches) that are used to replace the cells in the tissue that have died. Some caches are more plentiful than others, and a great deal of research has centered on the stem cells in caches stored in joints (mesenchymal stem cells), for example. Adult stem cells, in addition to embryonic stem cells, can be transformed into many types of tissue cells under the appropriate environmental conditions. The various stem cells and tissue progenitor cells have different properties reflected in differences in their metabolism as listed in Table 1.1 .

Table 1.1
Summary of Metabolism in Respective Stem and Progenitor Cells.
Source: Reproduced from Shyh-Chang, N., Daley, G.Q., Cantley, L.C., 2013. Stem cell metabolism in tissue development and aging. Development 140, 2535–2547.
Mammalian Cell Type Active Metabolic Pathways
Totipotent stem cells/blastomeres Pyruvate oxidation, bicarbonate fixation
Pluripotent stem cells/embryonic stem cells Anabolic glycolysis, PPP, Thr-Gly metabolism
Differentiating embryonic stem cells OxPhos, ROS, eicosanoids
Long-term hematopoietic stem cells Catabolic glycolysis, fatty acid oxidation
Hematopoietic progenitors Anabolic glycolysis, OxPhos, ROS, eicosanoids
Neural stem cells Low glycolysis
Neural progenitors High glycolysis, OxPhos, ROS, eicosanoids, fatty acid synthesis
Mesenchymal stem cells Low glycolysis
Chondroblasts High glycolysis
Osteoblasts OxPhos
Preadipocytes OxPhos, ROS
Myoblasts Anabolic glycolysis, PPP
Myotubes Glycolysis, OxPhos
Cardiomyocyte progenitors Lactate oxidation
Intestinal stem cells ( Drosophila ) OxPhos
OxPhos , Oxidative phosphorylation; PPP , pentose phosphate pathway; ROS , reactive oxygen species.

Mesenchymal stem cells have been proliferated in vitro and subsequently used to regenerate cartilage in knee joints that have suffered degeneration, for example. Many such clinical interventions, however, may not be permanent, and the process has to be repeated after a time. There is some concern that stem cells injected into patients could generate into cancer cells, although the evidence that this is an inherent danger in using them for clinical treatments, so far, has not borne out. Since each tissue in the body may have a cache of stem cells, the aging process could be the result of insufficient stem cells to replace tissue cells that become damaged and die out. Consequently the generation of such stem cells in vitro constitutes the practice and hope for further clinical treatments to replace tissue cells and organs. Stem cells generated in vitro must be safe from any contaminating substances or organisms and all are generated under good manufacturing practices promulgated by the FDA (Food and Drug Administration). A newer development under the heading of “therapeutic cloning” involves the use of pluripotent stem cells (without destruction of a human embryo) that are genetically identical to the donor to produce pluripotent stem cells that can be used to treat disease. In this case, adult skin cells from a donor can be used to extract his/her DNA, and this DNA can be fused electrically with donated adult human eggs, DNA of which has been removed. The resulting embryo at the level of the blastocyst is the source of pluripotent stem cells. The resulting pluripotent cells should be useful in treating many human diseases, including Parkinson’s disease, type 1 diabetes, heart disease, and others.

The next stage from the blastula is the gastrula developed by the invasion ( invagination ) of the bottom layer of cells of the blastula up into the blastocoel forming two layers with a pore ( blastopore ) on the bottom where the invasion began as shown in Fig. 1.7 . Now, two germ layers are determined, the outer layer of ectoderm and the inner layer of endoderm .

Figure 1.7, The formation of the gastrula from the blastula.

Further development reveals the internal mesoderm as shown in Fig. 1.8 .

Figure 1.8, Further development of the gastrula: (A) early development showing layers of ectoderm and mesoderm; (B) development of intermediate mesoderm and paraxial mesoderm; (C) further development of structures and parietal mesoderm, visceral mesoderm and endoderm; (D) rise of the intermediate mesoderm, the intraembryonic body cavity of the somite.

These three layers of cells the ectoderm, endoderm, and mesoderm lead to the development of specific tissues and organs. From the endoderm is derived the primitive gut that, in turn, generates the lungs, liver, pancreas, and digestive tubes. The ectoderm leads to the development of the epidermis, the forerunner of the skin, hair, and mammary glands. It also leads to the epidermal placodes and then to the lens of the eye and the inner ear. Placodes are thickenings of the ectoderm. Apparently, there are a number of different groups of placodes but they all have a similar developmental history and they lead to different aspects of the nervous system. Importantly, the ectoderm is the precursor of the neural tubes and neural tissue leading to the development of the brain and spinal cord (central nervous system, CNS) and the peripheral nervous system (PNS). The mesoderm gives rise to the axial skeleton (bones of the head and trunk), skeletal muscle, as well as the connective tissue of the skin. It also gives rise to other organs: the oviducts and uterus, kidney, ovary, and testes, the connective tissue of the body wall and limbs, the mesenteries, heart, and blood vessels. The endoderm is the precursor for the remaining bodily systems. The summary of the further development of these three embryonic layers of cells is shown in Fig. 1.9 .

Figure 1.9, Summary of the three embryonic layers of cells and their fates in the human body.

Gross Structures and Functions of Organ Systems

Most of this information is already obvious to you. However, the organ systems will be described briefly. Organs usually contain more than one type of tissues. Tissues will be discussed in the next section.

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