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College London , not the tissue that results. He discusses how emerging concepts and strategies might be blended to revolutionize the engineering of very familiar tissues.

Details of specific techniques or body sites are not his concern here. Among the topics are checking out the tissue groupings and the small print, making support-scaffolds containing living cells, whether three-dimensional complexity and layer engineering are worth the hassle, other ways to grow tissues, and towards four-dimensional fabrication. Figures and tables are available on a companion website. Show more Show less. More information about ebooks. Although numerous investigations have been undertaken to regenerate various kinds of tissue, there are still many critical factors involved in this regenerative program, including cell source, scaffold construction, cell seeding, culture environment, matrix production analysis, mechanical properties of cell—scaffold construct and suitable animal models.

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However, it may be possible someday in the future to isolate patient's cells by means of a small biopsy, expand the cell number in the culture, seed cells onto a three-dimensional scaffold and implant to the same patient. The cell source has an enormous influence on the success of tissue engineering. Based on the living species difference, cells applicable to tissue engineering may be classified into autologous patient's own , allogenic human other than patient and xenogenic animal origin.

Autologous cells are the most appropriate for tissue engineering so far as their activity remains high, whereas allogenic and xenogenic cells are immunogenic and will need an immunosuppressive therapy when a new tissue is engineered from these heterogenous cells. After publishment of reports that have revealed a presence of porcine endogenous retrovirus in pigs Patience et al. A problem associated with autologous cells is the difficulty in harvesting a sufficient amount of cells, especially when a patient is aged or has severely been diseased.

For instance, it is extremely difficult to harvest cardiac cells from a patient suffering from myocardial infarction. If the amount of harvested cells is not sufficient enough for clinical treatment, the cells should be expanded by cell culture. This procedure requires not only a clean cell-processing centre to avoid contamination, but also is time-consuming. In addition, possible viral infection will accompany the fetal calf serum FCS , which is most commonly used in cell culture.

Allogenic cells are useful for skin tissue engineering, because even the allogenic engineered skin tissue serves as better wound cover than non-biological ones, for instance, owing to secretion of powerful growth factors from the engineered tissue. Xenogenic feeder cells have usually been utilized for engineering of epidermal tissue from keratinocyte, because of their high epidermal growth activity, although they have a risk of viral infection.

Cells can be also classified on the basis of the difference in the extent of differentiation. Non-differentiated cells are ES and EG cells that are able to differentiate into all kinds of cells present in the body and have potential to expand without limitation.

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These are the major reasons for why the pluripotent stem cells have attracted so much attention. However, the pluripotent cell involves a number of problems when the cell is used for medical treatments of patients.

Extreme tissue engineering: concepts and strategies for tissue fabrication

If ES cells are obtained from fertilized eggs that have remained not used after the infertile therapy of couples, the ES cells are allogenic to the patient who will receive the cell transplantation. Somatic cell nuclear transfer to an enucleated egg is an alternative approach to circumvent this immune issue because of gene matching, but this technology is controversial as one cannot deny the possible risk of clonal human reproduction through abuse of this novel technology. In the adult body, stem cells exist that can differentiate into many lineages under appropriate conditions.

The haematopoietic stem cell HSC found in bone marrow is most extensively studied, providing eosinophils, erythrocytes, megakaryocytes, osteoclasts and B and T cells. The bone marrow contains also mesenchymal stem cells MSCs that are capable of differentiating into several connective tissue cell types, including osteocytes, chondrocytes, adipocytes, tenocytes, myocytes and bone marrow stromal cells Kotobuki et al.

On the other hand, several tissues in the adult contain progenitor cells that can proliferate and then differentiate to provide organ-specific cell types. Examples include the proliferative keratinocytes found in skin, hepatocytes responding to liver damage, intestinal crypt cells that replenish the absorptive epithelium cells and osteoblasts actively forming new bone and becoming osteocytes.

These progenitor cells appear to have their differentiation limited to a defined lineage. No unique marker has been found that positively identifies the human MSCs. Often they express surface markers associated with distinctive differentiated cell types. Those which are much more attractive and practical for tissue engineering are somatic or adult stem cells that have been believed to exist in tissues of adult, as shown in figure 1.

The most extensively studied stem cell is the HSC that exists in the bone marrow. The stem cell of the epidermal tissue is thought to be present in the basement membrane, but has not yet been identified. The capability of MSC to differentiate into various tissues, including bones, cartilages, adipose, blood vessels, nerves and skin, has attracted much attention of tissue engineers.

An advantage of MSC is the high safety compared with the ES cell that elicits teratoma when transplanted before differentiation into a certain lineage. This suggests that complete differentiation and purification of the cells modified from ES cells are prerequisites for the clinical application of ES cell. By contrast, no tumorigenesis has been reported on bone marrow cells when they have clinically been used.

It is interesting to note that bone marrow regenerates only the tissue specific to the site where the bone marrow cells have been transplanted, although the bone marrow must involve different kinds of stem cells. Figure 1 A long way from undifferentiated to differentiated cells. However, there is strong evidence that much of what has been considered to be transdifferentiation of bone marrow cells is actually attributable to fusion of these cells to host differentiated cells, particularly to the central nervous system, cardiac and liver cells.

Whether the observed developmental potential of bone marrow cells has a single or multiple explanations matters little, if the end result restores tissue structure and function. That is why the apparent ability of bone marrow cells to tissue differentiation has caused so much excitement.

It has been reported that the fat tissue contains MSCs Zuk et al. This tissue may be easier for harvesting than bone marrow, and hence numerous studies have been undertaken to isolate MSCs from the fat tissue. When combined with knowledge of growth factor and cytokine that promote differentiation, and suitable scaffold or carrier for delivery to a particular tissue site, MSC may appear to be the ideal candidate cell for development of therapeutic tissue regeneration and tissue engineering. Neither unpredictable nor unwanted cell types have been detected among the differentiated human MSCs.

This differs from all reports investigating the differentiation of ES cells which form multiple and unpredictable cell types as they differentiate. If the adult stem cell of each tissue becomes readily available to tissue engineering investigators as a result of great advances in cell biology and biotechnology, clinical application of tissue engineering will be remarkably accelerated.

However, in many cases, the number of harvested stem cells is not sufficient enough for clinical applications. We need to multiply the harvested cells by cell culture. This is a challenge because their proliferative ability is generally not high and de-differentiation will eventually take place during the proliferation of stem cells. When stem cells are cultivated on a two-dimensional cell culture dish, cell proliferation proceeds at a reasonable rate but accompanies de-differentiation.

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On the contrary, de-differentiation does not readily take place when stem cells are cultured on three-dimensional substrates, but the cell proliferation rate is reduced to very low levels. Therefore, it has been attempted to switch the de-differentiated state of cells to their original state by three-dimensional culture, but the switching efficiency is not as high as expected.

The most desirable approach is to multiply stem cells at a high rate keeping the undifferentiated original state. The FCS that has most frequently been used for cell culture contains xenogenic species that might induce some infection Louet Replacement of the FCS with synthetic sera using a range of recombinant growth factors has been explored for a long time, but the proliferative capability is still lower than FCS.

However, it was recently found that the proliferative ability of human serum increased to the level similar to that of FCS, if platelets involved in the human blood had been in advance broken before serum preparation and the platelet contents were joined to the serum Kawaguchi et al. The conventional human serum is prepared after removal of the whole platelets. It would be very convenient to both patients and physicians if devastated tissues or organs of patients can be regenerated by simple cell injection to a target site, but such cases are relatively rare, including haematopoietic diseases, cardiovascular diseases with malfunction of capillary or small blood vessels like arterioles, diseases due to deficiency of physiologically active substances e.

Most of large-sized tissues and organs with distinct three-dimensional form will require support for their formation from cells. The support is called scaffold, template, or artificial extracellular matrix ECM. The major function of scaffold is similar to that of the natural ECM that assists proliferation, differentiation, and biosynthesis of cells.

In addition, a scaffold placed at the site of regeneration will prevent disturbing cells from invasion into the site of action. A few technical terms have been used without clear definition. For convenience, they are classified here depending on the scaffold use, as depicted in figure 2.

According to this definition, regenerative medicine involves two concepts, cell therapy without any use of scaffold and tissue engineering that needs scaffold as a support of tissue regeneration. Figure 2 Classification of regenerative medicine based on the use of scaffolds. To fulfil the functions of a scaffold in tissue engineering, the scaffold should meet a number of requirements. First, it should have interconnected micropores, so that numerous cells can be seeded, migrate into the inside, increase the cell number and should be supplied by sufficient amounts of nutrients.

Micropores make both vascular formation and waste transport possible. This is important for the survival of cells inside the scaffold. Furthermore, scaffold should have an optimal porosity with adequate surface area and mechanical strength. The absorption kinetics of scaffold is also critical and depends on the tissue to be regenerated. If a scaffold is used for the tissue engineering of skeletal system, degradation of the scaffold biomaterial should be relatively slow, as it has to maintain the mechanical strength until tissue regeneration is almost completed.

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For the skin tissue engineering, the scaffold does not need to stay longer than one month. If the scaffold remains for a longer time than desired, the remaining material may retard the tissue regeneration rather than promote it. This indicates that the absorption kinetics of scaffold material will profoundly affect the success rate of tissue engineering.

In general, polyglycolide PGA and its copolymers, such as lactide—glycolide copolymer PLGA , degrade too quickly when used as a scaffold, because their tensile strength reduces to the half within two weeks. In contrast, poly l -lactide PLLA degrades too slowly, requiring 3—6 years for complete resorption.

Figure 3 Schematic for absorption rate of absorbable polyesters.

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This leads to limited applications of these biopolymers. Alginates do not contain any hydrolysable bonds, but are often used as a resorbable biomaterial.

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  6. Other water-soluble biopolymers are mostly rendered water-insoluble through covalent crosslinking with use of glutaraldehyde or carbodiimide.