Current research on tissue regeneration has evolved from a decades-old study on bone growth. Beginning in 1964, NIDCR (then NIDR) grantee Marshall Urist discovered that bone, which had been demineralized and dried into a powder, could be implanted into the muscle of a rabbit and stimulate the growth of new bone. Urist and collaborators later determined that the active component was proteinaceous and dubbed it bone morphogenetic protein (BMP). This landmark finding started the race to further identify the responsible growth factors that were present in the bone. Dr. Hari Reddi, an NIDCR scientist and grantee, took a closer look at the demineralized bone "matrix", which consists mainly of a network of type 1 collagen. Reddi was able to extract a soluble protein component of the matrix, but neither the soluble component nor the residual collagen matrix could induce new bone growth on their own. The extract had to be recombined with the matrix in order to induce new bone. The finding proved that a soluble osteogenic fraction and a solid carrier were necessary factors for bone regeneration and helped pave the way for working out the structure of bone morphogenetic proteins (BMPs).
Over a ton of bone was needed to provide scientists with a sufficient quantity of the osteogenic fraction for amino acid analysis. The sequencing results revealed striking similarities between components of the osteogenic fraction and transforming growth factor beta (TGF-ß). Initial studies by Reddi and other investigators identified three different BMPs that fit into the TGF-ß superfamily of proteins. Today, over 15 members of the BMP family have been isolated and produced in the laboratory through recombinant DNA technology. It is now also known that BMPs are produced by tissues other than bone and may be instrumental in the formation of the kidney, central and peripheral nervous systems, liver, lung, heart, gonads, skin, and teeth.
Research by NIDCR and other scientists revealed a "bone healing cascade", in which BMPs are believed to play an instrumental role. The natural healing of a broken bone occurs in a series of steps. Undifferentiated stem cells present in the marrow are directed by BMPs to multiply and become specialized cells that produce cartilage (chondrocytes) or bone (osteoblasts). Under the right set of conditions, these stem cells called marrow stromal cells (MSCs) can also be coaxed into forming muscle or tissues that support blood formation or even fat production. In the bone fracture-healing pathway, chondrocytes produce a cartilage framework that is eventually replaced with bone formed by the osteoblasts.
Animal studies have revealed some of the requirements for using BMPs to reproduce the healing cascade at sites that are resistant to normal healing. BMPs must be incorporated into a carrier that holds the molecules in place so that they can react with progenitor cells. Several different carriers have been tested with varying degrees of success. Early attempts used either collagenous bone matrix or hydroxy-apatite, a compound of calcium and phosphate that is similar to the mineral phase of bone. Using natural bone matrix carries the risk of disease and immune rejection, while synthetic hydroxyapatite is not biodegradable and new bone that is formed does not merge well with surrounding bone. An interesting finding from an early NIDCR study was that the pore size and overall shape of the hydroxy-apatite scaffolding was critical in determining the efficiency of bone induction. This work demonstrated the need for a well-defined three-dimensional structure for presenting BMPs, but also pointed out some of the shortcomings of existing materials.
|
Mythology, Evolution, and Tissue Regeneration Although the technology may be just emerging, regeneration of body parts is hardly a new concept. Through the myth of Prometheus, the ancient Greeks are credited with knowledge of the liver's natural regenerative powers. As punishment for stealing the secret of fire from the gods of Olympus, Prometheus was condemned to having a portion of his liver eaten each day by an eagle. His liver would regenerate overnight, providing the eagle with an endless food source and Prometheus with eternal torture. The regenerative capability of a real living creature was recorded as early as 330 BC, when Aristotle observed that a lizard could grow back the lost tip of its tail. In the late 1700s, the scientist Spallanzani reported that a newt could regenerate a complete limb. Since then, the study of regeneration in lower life forms has laid the groundwork for understanding the regenerative capabilities and potential of humans. Bacteria and the single-celled protozoans regenerate complete organisms with each cell division. Many multicellular invertebrates also exhibit extensive regenerative abilities. When cut in half, the at-worm Planaria can grow a new head from one piece and a new tail from the other. However, progression up the evolutionary ladder is generally accompanied by a reduction in regenerative capacity. Limb of Newt, Horn of Stag, Wing of Bat The pinnacle of vertebrate regeneration takes place in the urodele amphibians (salamanders and newts). When a newt's forearm or hindleg is severed, a new one forms in about six weeks. If half of the lower jaw is lost, it grows back complete with new teeth. The process through which this new growth takes place provides insights into some key differences between amphibian regeneration and mammalian wound repair. After a newt limb is amputated, the wound is sealed over by the rapid migration of epithelial cells from surrounding tissue. Mesodermal cells beneath the epidermis lose their differentiated character and reproduce to form a conical mound of cells called the blastema. The blastemal cells eventually redifferentiate into the cartilage, connective tissue, and muscle of the new limb. Signals exchanged between the epidermal and mesodermal cells control the reversal of the differentiated state and subsequent regeneration. Much speculation has centered on whether a similar process can be duplicated in mammals. While mammals have retained the ability to regenerate certain tissues, such as liver, they lack the ability to grow new limbs through blastema formation. But has major regenerative capacity in mammals been lost over the course of evolution, or is it lying dormant waiting for its potential to be unlocked? The basic ability may still be present even though the appropriate trigger mechanisms may not be. Assuming that the same genes that form structures in the developing embryo are also responsible for their regeneration in the adult, the task of scientists is to determine the nature of the blockades and to remove or bypass them. There are several tantalizing examples where mammals have not relinquished the ability to regenerate. The most conspicuous example of mammalian regeneration is the annual replacement of antlers in deer. This is the only example of complete regeneration of mammalian appendages. Other notable examples include bats being able to plug holes in their wing membranes; rabbits, domestic cats, and bats filling in ear holes, and regrowth of the tips of mouse toes. The prospects for human regeneration were greatly enhanced by the discovery that amputated fingertips could grow back in young children. Even though humans and other mammals cannot on their own regenerate teeth, limbs, and organs, specific tissues possess considerable recuperative powers. Mammals are continuously replacing cells in tissues derived from the protective layer of surface epithelium. Appendages such as skin, nails, and hair grow almost continuously. Teeth are constantly replaced in lower vertebrates, but in mammals are shed once and then replaced with a permanent set. However, if the roots remain open as in the case of rodents, the teeth may continue to grow throughout the life of the animal. Other tissues with tremendous cell turnover include blood, intestinal epithelium, and liver. In the intestine, the entire epithelium of each villus turns over every 2 3 days. It is estimated that the total daily production of the various blood cells is in the order of 3 x 1,011 cells for a 150 pound person. In most vertebrates, true regeneration is limited to a very few tissues, such as liver, bone, and skeletal muscle. Liver cells come closest to mimicking the urodele regeneration phenomenon, in which differentiated cells revert to an embryonic dividing state and then differentiate once more into specialized cells. Mature liver cells only partially dedifferentiate, however. In response to tissue destruction, liver cells, which normally don't reproduce, undergo rapid cell division. In true Promethean style, these remarkable cells continue to carry out their normal metabolic functions while multiplying to replace lost tissue. A rat can replace two-thirds of its liver in 5 to 7 days. A baboon liver transplanted into a human will double in size to reach that of a human liver within a week. Other tissues rely on stem or progenitor cells undifferentiated embryonic-type cells that are present in most, perhaps all, adult tissues. Stems cells are dormant until called into action by the proper set of chemical signals, whereupon they differentiate to form the required tissues. However, tissues differ greatly in the effectiveness of their stem cells. Of the major tissues that form the craniofacial complex, (nerve, muscle, skin, bone, blood, cartilage) bone is the most versatile in its regenerative capabilities. |
The search for an ideal scaffolding material continues to be a hot research area. Such material should provide a sufficiently rigid framework to fill the defect, yet degrade after the new tissue forms, and leave a product that is fully integrated with surrounding tissue. More recent studies have looked at BMPs incorporated into collagen gels, as well as biodegradable polymers like polylactic acid (PLA) or polyglycolic acid (PGA), the basic suture materials. BMPs, incorporated into various matrices, have been subjected to intensive preclinical and clinical research for both orthopedic and craniofacial applications. The sites investigated have included the calvaria (skullcap), jaw, and palate, where nonhealing defects were created in different animal models, including primates.
Scientists at the University of Michigan have taken the growth factor-biodegradable matrix combination a step further. Instead of incorporating a protein factor into the matrix, they substituted a plasmid containing the gene for the growth factor. The reasoning behind these "gene-activated matrices (GAMS)" was that a protein is quickly degraded, but a gene would get incorporated into a host cell and remain active over a longer period of time. When put to the test, BMP GAMS have been able to repair long bone defects in rats and dogs.
Hydrogels are a new generation of matrix materials currently being tested for cartilage and bone regeneration. The method is based on polymers that can house progenitor cells and growth factors, and then be injected into the repair site, thus avoiding surgical intervention. An external light source is used to trigger "photopolymerization" of the injected liquid so that a solid structure is established to support the repair process. An NIDCR grantee is currently looking at this approach for repairing craniofacial structures and treating dental defects, like poor bonding between teeth and under-lying jawbone.
Next Chapter | Biomimetics and Tissue Engineering -Table of Contents