Defective skin and soft tissue exposing bone or tendon, mostly after injury, infection or tumor surgery, is a major challenge for plastic surgeon. In order to reconstruct the defect, engraftment on the site with tissues that are similar to the injured skin and soft tissue is necessary. Flap operation is commonly available for the reconstruction; however, local flap is limited by the amount, composition and moving range of the flap, while free flap has several other drawbacks, such as the complexity of the operation, longer operation time, and greater loss of donor site. Patients with a chronic wound, such as bedsore and diabetic foot, tend to have poor general condition and tissue flow. For these patients, it is more difficult to perform a flap operation.
Recent development in biotechnology has made it possible to make the tissues to be reconstructed by isolating and culturing cells. Particularly, adipose cells are relatively easily obtainable in considerable amount while minimizing the loss of the donor site; thus, there have been efforts to isolate Adipose Derived Stromal Cells (ADSCs) and, through cell culture and differentiation, to use them for soft tissue reconstruction. The first requisite for tissue regeneration is cells. Of course, there have been reports that wound healing was accelerated and whitening effect could be achieved by using only uncultured ADSCs. However, minimum amount of cells obtained from the donor site can be proliferated ex-vivo and then attached to a 3-dimensional, cell supporting structure, to replace organ or tissue transplantations. Therefore, requisites other than cells would include the cell supporting structure, or scaffold (substrate), blood supply for engraftment after the transplantation, and proteins, such as growth factors, to help differentiation and engraftment.
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1. Cell source
A cell is the most basic component of regeneration. Regeneration medicine approaches can be categorized to 1) cell therapy where the transplanted cells induce the production of materials required for human body to enhance tissue functioning and 2) forming framed tissues or an organ by mixing cells with biomaterials. These cells are mostly allogeneic cells or autogenous cells; allogeneic cells are obtained from the host after immunosuppression or in isolation from the immune system. However, this causes various side effects irrespective of treatment, making autogenous cells without such problems more preferable. A lot of studies using autogenous cells have been performed through animal tests and clinical trials, and the results are good as well. However, the use of autogenous cells sometimes needs an invasive surgery, and cells might be hard to collect when the tissue to be reconstructed is severely injured. Already differentiated somatic cells have limited proliferative activity, making it also difficult to obtain sufficient number of cells, and there is also the possibility of transformation or loss of tissue regenerating ability in the course of proliferation. In order to overcome these limitations, studies have been conducted on methods to secure an alternative source of cells in large amount.
In this context, stem cells than can overcome such limitations of somatic cells are gaining much attention, with a lot of studies on the subject. Stem cells normally have self-renewal ability and are defined as nonspecific cells that can differentiate to various types of mature cells. Stem cells are categorized to embryonic stem cell, fetal stem cell and adult stem cell according to the time of occurrence, or to totipotent cell, pluripotent cell, multipotent cell and unipotent cell according to the differentiation ability.
Embryonic stem cells are recognized as effective for tissue regeneration because they are most potent in proliferation and differentiation abilities. However, cell cloning technology by means of nuclear transfer is required to obtain this cell, and this cell is known to induce teratoma when transplanted directly in the human body and still carries a great risk even when the cell was properly differentiated. The use of embryonic stem cells also carries ethical controversy due to the acquisition of ovum, manipulation of embryo and its artificial destruction. Due to these issues, adult stem cell has been studied a lot in recent years. Adult stem cells have relatively weaker proliferation and differentiation abilities than embryonic stem cells, but are easy to obtain without an ethical issue, and the possibility of easy clinical application makes it an attractive target in many studies. Adult stem cells are distributed around various tissues in the human body from the bone marrow to fat, blood, heart, nerve, muscle and skin, and a lot of efforts are still being made to isolate it from various other tissues. Recent studies are investigating stem cells collected from the fat, amniotic fluid and placenta. These cell groups have similar characteristics to embryonic stem cells but do not induce tumorigenesis in the body and do not stir an ethical issue.
Another cell source currently being actively researched is Induced Pluripotent Stem Cells (iPSCs). Shinya Yamanaka at Kyoto University and John Gurdon at the University of Cambridge jointly received 2012 Novel prize for Physiology or Medicine for the study of iPSCs. iPSCs refer to cells induced to have pluripotency by artificial dedifferentiation of differentiated cells without pluripotency, and is also called dedifferentated pluripotent stem cells.
First introduced in 2006, this method transfers Oct4, Sox2, c-Myc and Klf4 genes, which are the main transcription elements of the already differentiated normal cells, by reprogramming cells to overexpress these genes to make pluripotent stem cells. According to a follow-up study, iPSCs were found to have the characteristics of embryonic stem cells, and since first induced in skin fibroblast cells of mice, iPSCs were successfully induced in human somatic cells as well. iPSCs are available for individualized production and can be used for drug screening, disease studies and reconstruction of injured tissues. However, the success rate of current reprogramming is only about 1-2% and requires more time and studies to commercialize them for clinical application.
2. Biomaterials for tissue regeneration
Biomaterials are essential for application of regeneration medicine, along with cells. Biomaterials for regeneration medicine can be used alone or in combination with cells for various purposes. When used alone, biomaterials are used for generally for restoring injured areas by filling or linking defective tissues or for normalizing the function of tissues by inducing their regeneration. On the other hand, when biomaterials are used in combination with cells, the biomaterials work as a medium to deliver cells into the body. Biomaterials for transplantation are composed of ingredients than can facilitate cell adherence and proliferation and has porous structure for smooth cell migration, angiogenesis and supply of nutrition. Such biomaterials should also be helpful for cell culture and be able to control cell phenotype without cell function or gene modification. They also have to provide a supporting role of the mechanical of physical strength required for a particular tissue and should be able to disintegrate spontaneously after a certain period of time, without leaving a foreign substance in the body. The ideal biomaterials for transplantation in the body should have confirmed biological suitability and safety, should be harmonized well with the surrounding tissues once transplanted inside the body, should not induce an inflammatory response in case of synthetic material, and should interact easily with the host. A number of biomaterials have been used so far for regeneration medicine studies, and various materials are currently being synthesized or produced for individual study purposes. Biomaterials can be broadly divided in to synthetic polymers and natural polymers. Studies using absorbable synthetic polymer compounds have been actively performed recently. Synthetic polymer compounds have several merits. They are capable of being produced in various forms, easy to handle, and available for mass production at a low cost. In addition, the raw materials to make them are easy to obtain.
On the other hand, their biosynthesis and tissue affinity are lower than the natural polymers. Along with the use of absorbable polymer compounds, studies on treating in-vivo tissues or organs to eliminate cells and components of the tissue without structural damage has been actively performed to use it as a supporting structure. A supporting structure from natural tissue has similar property to the in-vivo tissues, high affinity to cells and tissues, and contains factors that are helpful for regeneration, which is why it is used for various experiments and clinical studies.
Recent studies on biomaterials are more focused on developing an intelligent supporting structure that plays a more proactive role. Previous studies expected the supporting structure simply to deliver cells and to form a 3-dimentional structure when used in combination with cells. Recently, however, efforts have been made to give the biomaterial and supporting structure the ability to facilitate regeneration through a highly-advanced manipulation.
Such an additional function may be set differently for various purposes of a study, and can induce the production of regulators, which stimulate differentiation and proliferation of cells. Furthermore, there are also studies focused on transplanting a functional supporting structure alone in the body to activate autogenous stem cells of the host and to facilitate tissue regeneration by inducing the activated autogenous stem cells to move into the transplanted supporting structure. Another possibility has been suggested that proper manipulation of the supporting structure alone can procure and differentiate cells necessary for tissue regeneration. Such a method would be able to exclude cumbersome tissue collection, ex-vivo culture and proliferation of cells in the process of tissue regeneration and may reduce sources and time required for cell culture.
3. Vascularization of regenerated tissues
Transplanted cells and supporting structure in the body can survive by receiving nutrition and oxygen from blood vessels; therefore, rapid vascularization is essential. In order for cells to survive for a long period of time in a sizable supporting structure produced as a 3-dimentional form, oxygen and nutrition should be able to be delivered everywhere throughout the supporting structure by sufficient vascularization. To achieve this purpose, studies often use angiogenesis factor, such as Vascular Endothelial Growth Factor (VEGF). Such factors are known to stimulate neovascularization, and recent studies reported that these factors can form relatively large tissues by synergistic effect when combined with endothelial cells. Another approach is to insert genes that can produce angiogenesis factors for the same consequence, although this method may not be easy for clinical application. However, various combinations of such methods are continuously attempted. Another method to induce neovascularization is to optimize the synthesis of biomaterials and the microstructure of the supporting structure. A supporting structure with pores of proper size helps the provision of nutrition and oxygen, thereby facilitating neovascularization and infiltration.
Recently, a lot of studies are investigating the technology to make a supporting structure with microvascular networks for endothelial cells to grow on and methods to use organs or blood vessels as a supporting structure. However, such methods are still limited for forming large tissues required for actual clinical application, because biological methods using angiogenesis factors may temporarily stimulate angiogenesis but cannot maintain the effect continuously. To overcome such limitations, recently there have been efforts to prolong the lifetime of cells contained in the supporting structure, with the purpose of providing elements required for cell survival continuously until angiogenesis occurs.
One example is to insert a particulate that generates oxygen inside the supporting structure or biomaterial, where it can release oxygen continuously to maintain cell survival. Development and application of various advanced technologies are expected to accelerate the research processes of regeneration medicine and its clinical application.
4. Enhanced function of cells and supporting structures
The objective of regeneration medicine is to restore injured tissues rapidly. Therefore, more attempts are focused on transplanting tissues that have almost fully matured structure and function for more rapid recovery of the injured tissue, rather than transplanting cells or tissues with the expectation of functional recovery during the follow-up. For example, treatments using regulating factors, which mediate cell to cell interactions, and growth factors are under development. Development of new technologies, such as nanotechnology, enables genetic modification to release certain factors continuously or recently more effective delivery system of growth factors.
Among the methods to mature tissue function outside the body as best as possible is bioreactor. Biomechanical factors are known to have great influence on growth, maintenance, regression and recovery of tissues. In the process of producing tissues ex-vivo, such biomechanical stimulations similar to those inside the body are essential for successfultransplantation of the tissues into the body.
Such a bioreactor typically modifies the temperature, pH, oxygen, carbon dioxide, various nutrition, metabolites, and other regulators as required for tissue or cell culture. Recently developed high-end bioreactors can automate such a regulating function, with monitoring ability, and provides more varied biomechanical environments. Biomechanical stimulations are more effective for tissue generation of the cardiovascular system, musculoskeletal system, skin and blood vessels, which require higher strength and durability in dynamic environment. Recent developments in computer engineering and the production technique of biomaterials made more detailed simulation of biomechanical environments available. This data is used for making more functional, biomimetic artificial tissues and organs through the production of a more ideal bioreactor of the environment wanted for each tissue.
The next article will look into the acellular dermal matrix, which plays a role as a template for regeneration.
References
❶ Regenerative Surgery 3rd ed. Yu Ji and Lee Il Woo. Koonja Publishing. June 10, 2010.
❷ Neligan PC, Plastic Surgery, 3rd ed, Elsevier2013,
❸ Clinical Application of Adipose DerivedStromal Cell Autograft for Wound CoverageSeo DL, Han S, Chun KW, Kim WK J KoreanSoc Plast Reconstr Surg 2008 Nov 035(06):653-658.
- To be continued -
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