No.7 July 2004

Vol.47, No.7 July 2004

CONTENTS

JMA Policies

Policy Address Haruo UEMATSU ...... 305

Regenerative Medicine

Regenerative Medicine for Cartilage Defects Mitsuo OCHI ...... 307

The Use of Skin Regeneration Technique in the Treatment of Full-Thickness Skin Defects Norio KUMAGAI ...... 311

Regenerative Medicine for the Cornea Shigeru KINOSHITA and Takahiro NAKAMURA ...... 317

Regenerative Medicine for Sclerotic Disorders Toshikazu NAKAMURA ...... 322

Regenerative Medicine for Cardiomyocytes Keiichi FUKUDA ...... 328

Regenerative Medicine for Respiratory Diseases Tatsuo NAKAMURA ...... 333

Centenarians

Implications of Research Findings Obtained from Centenarians Hiroshi SHIBATA ...... 338

Abdominal Aortic Aneurysm

Forefront of Treatment for Abdominal Aortic Aneurysm Hiroshi YASUHARA ...... 344 ⅥJMA Policies

Policy Address

JMAJ 47(7): 305–306, 2004

Haruo UEMATSU

President, Japan Medical Association

The 110th General Assembly of the JMA House of Delegates was held for two days from April 1 to 2 at the JMA Hall. Elections for the offices of the executive board were held on the first day and Dr. Haruo Uematsu (former president of the Osaka Medical Association) was elected as the 16th president of the JMA for the first time. The following is a main part of the policy address of Dr. Uematsu presented on April 2, 2004.

Our country is facing a period of great tional women, to foster their capabilities, and changes as can be seen in the dispatch of Self- to send them to the JMA in future. Defense Forces to Iraq by the government of Since this is such an important time for the Japan amidst an ongoing debate about revising JMA, I believe that making the organization’s the Japanese Constitution. Thus, health care management very transparent while clarifying reforms which are carried out during this its decision-making process will raise the trust period must reflect answers to the question, of our members. Executive members should “What kind of country should Japan strive to address health insurance, long-term care insur- become in the future?” Reforms that address ance, community health care, and other issues only the immediate issues will not contribute to through teamwork, and we are presently pre- national happiness and prosperity. paring to do this. Issues that may be over- looked by one or two individuals can be Striving for Transparency in JMA addressed without fail by a team of three or Affairs and Earning the Trust of four members. JMA Members Firm Support for Social Security and We have been entrusted with the manage- a Universal Health Insurance Program ment of the JMA in times such as these and we are fully aware of the importance and the There are many highly qualified, outstanding significance of this responsibility. The board individuals with accumulated know-how within members that you recently elected into office the Secretariat. I would like to pursue a policy are capable, flexible, and dynamic individuals whereby these talented individuals are utilized who have the confidence and recommendation more often than has been in the past and to of each local medical association. However, allow them to voice their opinions in policy it is regrettable that no women members were decisions as needed. I believe that such a policy elected as executive members. Reflecting on will not only be an added help for the executive this turn of events, we would like each local members, but it will also serve as a means of medical association to actively recruit excep- encouragement for this important pool of out-

JMAJ, July 2004—Vol. 47, No. 7 305 Haruo UEMATSU

standing individuals. Additionally, issues related to women physicians But since the JMA is dedicated to improving and employed physicians must be addressed public health care, I would like JMA’s mainstay as well. policy to be the firm support of social security Improving the membership ratio is an and the national universal health insurance extremely important issue for the future of the program. The foremost important element is JMA. Some of the notable problems that must to provide safe and high quality health care be addressed are how to raise the understand- services efficiently. To accomplish this, some ing of younger and employed physicians about of the existing issues that come to mind are the JMA. Although the merits about joining promoting the CME program for our mem- are discussed, information that cultivates under- bers, enhancing medical ethics, and pursuing standing about the kind of work the JMA is self-reviews. involved in and how it contributes to the world Policies regarding these issues have been at large as well as what the incentives are for adequately debated and compiled into a report physicians to join the association must be dis- by the previous executive board, and what seminated. This information should not simply remains now is their actual implementation. be a discussion about incentives, but measures In implementing these policies, we should must be taken to earn the trust of employed observe the response of not only JMA mem- physicians. bers, but the opinions and response of the general public, while maintaining a very strin- Implementing Responsible Policies gent stance when discussing these policies with and Strategies by the Executive Board members. Among the issues that must be addressed, Raising Incentives to Join the JMA there is the issue regarding the JMARI (JMA and Improving the Membership Ratio Research Institute). I am fully aware of the fact that the JMARI is an effective and competent The most problematic area in health care institute. However, I feel that it is the responsi- services is pediatric care. This is especially bility of the executive board to decide and true in the area of pediatric emergency care. implement JMA’s policies; and the role of the Although pediatric emergency care is available JMARI is to carry out specific tasks, analyze throughout the country, the adequacy of these data, and carry out surveys and studies that services varies according to geographical area. support the decisions made by the executive I hope that a reliable system of pediatric emer- board. I think that it is irresponsible to leave gency care is established uniformly throughout the decision-making process in the hands of the country. To achieve this, the problem of the the institute. maldistribution of physicians must be addressed.

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Regenerative Medicine for Cartilage Defects

JMAJ 47(7): 307–310, 2004

Mitsuo OCHI

Professor, Department of Orthopaedic Surgery, Hiroshima University Graduate School of Biomedical Sciences

Abstract: The treatment of full-thickness defects of articular cartilage remains a problem for orthopedic surgeons. It has been generally accepted that once articular cartilage is injured and forms a defect, the defect cannot be repaired and bordering intact cartilage undergoes degeneration which destroys facing intact cartilage and results in osteoarthritis. Several attempts to repair articular defects with hyaline cartilage have failed. Since the clinical reports of Brittberg et al. in 1994, autologous chondrocyte transplantation has raised the hopes and expectations of orthopedic surgeons that a breakthrough in the repair of damaged articular cartilage is imminent. In Brittberg et al.’s technique, cartilage slices were obtained by arthroscopy from an unloaded area of the femoral condyle; the associated chondrocytes increased in number in a monolayer culture after enzymatic digestion, and the chondrocytes in suspension were then injected into a cartilaginous defect and covered with a ﬂap of the periosteum. According to their report, clinical results were satisfactory and a biopsy of the graft sites showed hyaline-like cartilage repair. However, we had several reservations about their technique in regard to the culture and transplantation procedure in terms of 1) de-differentiation of chondrocytes during a long cultivation, 2) uneven distribution of the grafted chondrocytes throughout the osteochondral defects, and 3) a high risk of leakage of grafted chondrocytes from the defects. We developed a new technique that enables the shift from cell transplantation to tissue transplantation. This technique creates a new cartilage-like tissue by the tissue-engineering technique in which autologous chondrocytes were embedded in atelocollagen gel and cultivated for about 3 weeks. We carefully selected atelocollagen gel as a three-dimensional culture material from the viewpoint of safety and non-immunogenicity, since atelocollagen gel had been used clinically for the treatment of skin wrinkles in plastic surgery and dermatology. Culti- vation results in the proliferation of chondrocytes and the synthesis of an extracellular matrix consisting of chondroitin sulfate and type II collagen at transplantation. By 3 weeks of cultivation, the atelocollagen gel, including chondrocytes had acquired a jelly-like hardness. We have been using our technique since gaining the approval of the ethics committee in 1996. After 3 to 4 weeks’ culture of autologous chondrocytes embedded in atelocollagen gel, a tissue-engineered cartilage was transplanted into a cartilage defect and covered with a periosteal ﬂap, which was sutured with the deep cambium layer facing the subchondral bone plate. We followed up full-thickness cartilage defects of 36 knees from 34 patients treated with our procedure over a minimum period of 2 years. Clinical, arthroscopic and biomechanical results were relatively satisfactory. We conclude that transplanting tissue- engineered cartilage made by the tissue-engineering technique can promote restoration of the cartilage of the knee. Key words: Cartilage injury; Cartilage repair; Three-dimensional culture; Regenerative medicine

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 129, No. 3, 2003, pages 336–338).

JMAJ, July 2004—Vol. 47, No. 7 307 M. OCHI

Introduction chondritis dissecans in young subjects, trau- matic cartilage injury, and osteoarthritis or With the progress in tissue engineering and rheumatoid arthritis in the elderly. molecular biology, basic research in regenera- Currently, there is no other choice than arti- tive medicine aimed at regeneration of defec- ficial joint replacement for reconstruction of tive tissues or organs using autologous cells markedly destroyed articular function due to or tissues and their clinical application have advanced osteoarthritis or rheumatoid arthri- become widespread in recent years. For the tis. For the treatment of localized articular car- regeneration of tissues using tissue engineering tilage defects, a variety of procedures has been techniques, it is generally recognized that cell undertaken, including the drilling method and transplants, biocompatible materials to serve the microfracture technique aimed at inducing as a foothold for growth of transplanted cells marrow cells from a subchondral bone, soft tis- (i.e., scaffold), and growth factors are usually sue grafting such as periosteum-perichondrium required, and studies on regeneration of a wide or meniscus transplantation, and osteochon- variety of tissues have been extensively under- dral transplantation. None of these surgical taken in the field of medical care. Regenerative procedures has proven to provide satisfactory medical care has thus been frequently intro- reconstruction of a natural, smooth cartilage duced by mass media such as newspapers, with hyaline cartilage at the site of defect. periodicals, and television and widely recog- In 1994, Brittberg et al. reported transplanta- nized among the public at large. Knowledge of tion of cultured autologous articular chon- regenerative medicine is now essential to the drocytes utilizing monolayer cell cultures.1) practice of medicine. Cartilage slices obtained by arthroscopy from Repair of articular cartilage, which has little an unloaded region of the knee joint were or no self-repairing capacity, has been taken up trypsinized to isolate chondrocytes, which were as a matter to be pursued in the field of ortho- then incubated to grow into monolayer cul- pedic surgery. Articular cartilage comprises tures. The grown chondrocytes were dispersed chondrocytes and surrounding extracellular in suspension and injected into a cartilaginous matrix (such as type II collagen and proteo- defect covered with a flap of the periosteum. glycans) and is devoid of blood vessels and The technique is currently in widespread use nerve tissues. The chondrocytes are poorly on a commercial basis mainly in Europe and proliferative and produce little extracellular the United States. matrix. Once articular cartilage is damaged, the According to their report, a postoperative usual tissue repair process does not proceed biopsy of the graft sites showed hyaline-like with a consequent lack of restoration of hyaline cartilage repair. The technique thus marked a cartilage. major breakthrough in the repair of damaged When an articular cartilage defect occurs, cartilage. Several problems, however, seem to it gives rise over time to degeneration of the be inherent in this technique, viz. whether surrounding and facing intact joint cartilage, chondrocytes which have de-differentiated which progresses in due course to become during the cultivation in vitro may recover their osteoarthritis. Consequently, pain and limita- function as chondrocytes post-transplantation, tion of excursion occur and functional impair- whether leakage of the grafted chondrocytes ment of the knee joint ensues. This constitutes in suspension may occur from the defect site, a significant clinical problem. Articular carti- and whether the grafted chondrocytes may lage injury may be encountered not only in the become evenly distributed throughout the practice of orthopedic surgery but in other osteochondral defect. clinical settings as well in the form of osteo- To resolve these problems, we have devised a

308 JMAJ, July 2004—Vol. 47, No. 7 TISSUE-ENGINEERING FOR CARTILAGE

technique for tissue-engineered cartilage-like tissue grafting, namely, transplantation of atelocollagen gel-embedded cultured autologous articular chondrocytes. Based on laboratory studies, its clinical application was initiated for the ﬁrst time in the world in 1996.2–7) Gratifying postoperative results have been obtained, and clinical trials are scheduled to be conducted at several university hospitals.

Transplantation of Atelocollagen Gel-Embedded Cultured Autologous Fig. 1 Osteochondritis dissecans of the femoral medial Articular Chondrocytes condyle of the right knee in a 27-year-old man Note the cartilage defect extends as deep as the The atelocollagen gel is type I collagen puri- surface of subchondral bone. fied from the bovine corium and causes little or no immune responses to it because the atelocollagen is deprived of the antigenic terminal telopeptide. The gel is a remarkably safe collagen that is already in clinical use in cosmetic surgery for such purposes as eliminating skin wrinkles. The specifics of the procedure are as follows. Cartilage specimens are obtained by arthroscopy from an intra-articular detached cartilage fragment or an unloaded region of knee joint cartilage of the patient. The specimens are sliced and trypsinized/collagenase-digested to isolate chondrocytes. The collected cells are then suspended in an atelocollagen gel and Fig. 2 Two years after transplantation of atelocollagen gelated, followed by incubation with a culture gel-embedded cultured autologous articular medium containing the patient’s serum and chondrocytes antibiotics for 3 weeks. The embedded chon- The defect is repaired with cartilage-like tissue with a smooth surface. drocytes grow in the atelocollagen gel and liberate extracellular matrix to have the gel acquire a jelly-like hardness and turn into a cartilage-like tissue. post-operation and is gradually increased to Three weeks after harvest of the autologous full weight-bearing with muscle training at 6–8 cartilage, the chondral defect lesion is exposed weeks post-operation. via arthrotomy, the chondrocyte-atelocollagen In a laboratory study with human chondro- gel prepared in vitro is transplanted in the cytes collected at an operation of artificial joint defect, and the lesion is covered by a sutured replacement, we confirmed and reported that periosteal flap taken from the proximal medial human chondrocytes cultured in atelocollagen tibia. One to two weeks after the transplanta- gel retained their three-dimensional structure tion, passive movement of the joint is begun. and grew while maintaining their usual round Partial weight-bearing is introduced at 4 weeks shapes without incurring de-differentiation and

JMAJ, July 2004—Vol. 47, No. 7 309 M. OCHI

produced/released extracellular matrix such as opportunities to treat patients suffering from type II collagen and chondroitin sulfate. articular cartilage injury have increased. While Inasmuch as chondrocytes are transplanted it is not easy to repair an articular cartilage as embedded/cultured in the solid gel medium, injury with the original hyaline cartilage, we there is practically no cell leakage from the devised a tissue-engineered technique for periosteal sutured margin, unlike transplanta- transplantation of atelocollagen gel-embedded tion of cells in suspension. Leakage of chondro- cultured autologous articular chondrocytes and cyte transplant is thus quite unlikely even on have been applying it in clinical settings. articular movements, unless gel leakage results Though the postoperative follow-ups have not from significant periosteal damage; this consti- been long enough, satisfactory results have tutes a great advantage. The procedure is also been obtained with this technique. Exploration advantageous in that it provides apparently of the current problems involved and various greater uniformity of chondrocyte transplant approaches to attain more satisfactory out- as compared with the chondrocyte transplanta- comes for cartilage repair are under way. tion in suspension. Treatment with this procedure has been performed on a total of 70 knees, with relatively REFERENCES satisfactory postoperative outcomes (Figs. 1 1) Brittberg, M., Lindahl, A., Nilsson, A. et al.: and 2). The patients were followed by periodic Treatment of deep cartilage defects in the arthroscopic observations postoperatively, and knee with autologous chondrocyte transplan- the graft sites were confirmed to gradually tation. N Engl J Med 1994; 331: 889–895. increase in hardness to become as firm as 2) Ochi, M., Uchio, Y., Kawasaki, K. et al.: Trans- the surrounding normal cartilage. On MRIs, plantation of cartilage-like tissue made by the graft sites were shown over time to gain tissue engineering in the treatment of cartilage defects of the knee. J Bone Joint Surg Br 2002; brightness close to that of surrounding normal 84: 571–578. cartilage. 3) Ochi, M., Uchio, Y., Tobita, M. et al.: Current The method’s drawbacks are: the limited concepts in tissue engineering technique for quantity of chondrocytes obtainable, the long repair of cartilage defect. Artif Organs 2001; period required for the transplanted cartilage 25: 172–179. to mature, and the two-stage operation to com- 4) Matsusaki, M., Ochi, M., Uchio, Y. et al.: plete the procedure. Further spread of cultured Effects of basic fibroblast growth factor on autologous chondrocytes tissue grafting can be proliferation and phenotype expression of expected in the future upon improvement of chondrocytes embedded in collagen gel. Gen Pharmacol 1998; 31: 759–764. the cultivation technique, development of a 5) Kawasaki, K., Ochi, M., Uchio, Y. et al.: better transplant carrier supplanting the atelo- Hyaluronic acid enhances proliferation and collagen, and a technique enabling autologous chondroitin sulfate synthesis in cultured stem cell differentiation into chondrocytes. chondrocytes embedded in collagen gels. J Cooperation and joint research with other Cell Physiol 1999; 179: 142–148. fields such as medicoengineering and mole- 6) Katsube, K., Ochi, M., Uchio, Y. et al.: Repair cular biology are essential for the development of articular cartilage defects with cultured of the method. chondrocytes in Atelocollagen gel. Compari- son with cultured chondrocytes in suspension. Arth Orthop Trauma Surg 2000; 120: 121–127. Conclusion 7) Uchio, Y., Ochi, M., Matsusaki, M. et al.: Human chondrocyte proliferation and matrix With the increases in the elderly population synthesis cultured in Atelocollagen gel. J and the sports population in recent years, Biomed Mater Res 2000; 50: 138–143.

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The Use of Skin Regeneration Technique in the Treatment of Full-Thickness Skin Defects

JMAJ 47(7): 311–316, 2004

Norio KUMAGAI

Professor and Chairman, Department of Plastic and Reconstructive Surgery, St. Marianna University School of Medicine

Abstract: A total of 434 patients have been treated with cultured epithelium transplantation between March 1985 and April 2003, 285 patients by autografting and 149 patients by allografting. Nevertheless, this approach of cultured epithelium is conﬁned only to the epidermis and not the dermis. This results in poor “graft take” and failure of skin regeneration even by autografting in full-thickness skin defects. Regeneration of the dermis is essential for improving the “take” rate of cultured epithelia. Currently, transplantation of cultured autologous epithelium over a previ- ously transplanted dermal allograft is an effective method of treatment for full- thickness skin defects such as third-degree burns and giant nevus resection wounds. However, there are problems with this technique, such as rejection of allograft skin and a short supply of allograft skin. Attention has shifted to the use of cell-free dermis, the development of cultured dermis and cultured skin by tissue engineering techniques. This article describes the present status of skin regeneration techniques for the treatment of full-thickness skin defect via cultured autologous epithelium transplantation and the experience of this department in the treatment of full-thickness skin defects. Key words: Skin regeneration technique; Cell culture; Skin; Tissue engineering

Introduction for harvesting is inadequate. In recent years, many patients have bene- Skin grafting is a widely used operative pro- ﬁted from skin regeneration techniques since cedure in both plastic surgery and general sur- the introduction of “cultured epidermal trans- gery, but it has limitations in the treatment of plantation” for wound coverage in extensive extensive burn wounds in which the skin left full-thickness skin defects. However, the thera-

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 129, No. 3, 2003, pages 339–342).

JMAJ, July 2004—Vol. 47, No. 7 311 N. KUMAGAI

Table 1 Breakdown of Cases Treated with Cultured Autologous or Allogeneic Epidermal Transplantation (April 1985 to April 2001) 2 5 6 8 1122 1111 1133 8

1144 UUlcerlcer 1515 SScarscars ffromrom bburnsurns BBurnurn wwoundsounds Leukoderm 117117 21 6262 GGiantiant nnevievi SScarscars 2323 2828 TTattooattoo BurnBirm 2828 woundswounds TTattoosattoos 3366 2255

Autografting (285 cases) Allografting (149 cases)

Suprabasal layer

Basal layer

Fig. 1 Cultured epithelium and scanning electron micrographs

312 JMAJ, July 2004—Vol. 47, No. 7 CLINICAL APPLICATION OF CULTURED EPITHELIUM

peutic approach for the above technique is very beginning of current skin regeneration confined to the epidermis alone without the techniques. dermal components which has its own problems. This article describes the achievements Effects of Dermal Substance in and the present status of skin regeneration Skin Regeneration techniques in the treatment of full-thickness skin defects. Interaction between the epidermis and the dermis play an important role in skin regener- Results of Clinical Application of ation when autologous cultured epithelia are Cultured Epithelium transplanted onto split-thickness skin wounds retaining the dermis, nearly 100% of the thin The author and colleagues started clinically transplants take and regenerate to become nor- applying autologous cell cultured transplanta- mal skin within one week. The laminin 5, type tion in April 1985. This technique has been IV collagen and type VII collagen which are used for treating various skin disorders such as components of basal layer between wound bed burns, scars, nevi, vitiligo and tattoos. Of the and cultured epithelia are stained. Furthermore, total of 434 patients that were treated, 285 rete ridges are formed. In cases where autolo- patients underwent autografting and 149 patients gous cultured epithelia are transplanted onto received allografting (Table 1). Our labora- full-thickness skin defect wounds devoid of tory’s accomplishments have now made it pos- dermal components, the graft take is extremely sible to yield enough cultured epidermis to low. At 20% one week after transplantation, cover the whole body surface area (1.6 m2) in the graft shows no appreciable change in thick- one month of incubation from a skin graft of ness or formation of the basal layer. The trans- about 10 cm2 (Fig. 1). Such mass epidermal cul- plant gains in thickness and the basal layer tivation from a small quantity of skin graft can finally forms several weeks later. There are no be obtained because the skin contains actively formation rete ridges, the border of the wound proliferative stem cells with high differentiat- bed is even, and the skin shows characteristics ing potential. It has been demonstrated that of cicatricial epithelium, such as hyperkerato- follicular multipotent stem cells capable of dif- sis, hypokeratosis, and dermatoglyphic hypo- ferentiating into internal and external root plasia. As such it takes five months for the sheaths, follicle, sebaceous gland and epidermis basement membrane and continuous fibers to which exist not only in the stratum basal epi- resume their normal morphologic features; the dermis, but also in the follicular bulge.1) region is liable to develop vesicles and to be Clinical application of cultured epithelia began readily detached during this period. The cul- in 1980. In 1984, two children aged 5 and 6 tured epithelial cells do not take on the adipose years old who sustained third-degree burns tissue layer or granulation tissue wounds. over 97% of their total body surface area were saved by autologous cultured human epithe- Reconstruction of the Dermis lium transplantation.2) To raise the graft take rate for cultured epi- Discussion thelia to enable skin regeneration, it is impor- Rheinwald and Green’s3) established a mass tant to generate wound bed formation to serve culture method of epidermal cells by using as a scaffold for cultured epidermal transplants irradiated 3T3 cells as the feeder in 1975, and and to enable regeneration of the dermal com- the subsequent groundbreaking success of its ponents. There have been two different meth- clinical application may be regarded as the ods reported for the generation of the dermis:

JMAJ, July 2004—Vol. 47, No. 7 313 N. KUMAGAI

Fig. 2 Treatment of giant nevi by transplantation of skin allografts and cultured autologous epithelial grafts (Left: preoperative; center: cultured autologous epithelia transplanted over skin allografts; right: 7 years after treatment)

one is to prepare dermal components over the treated according to the procedure for third- wound bed in advance, followed by cultured degree burns. In view of intense allograft re- autologous epithelia grafting and the other is jection of the host with nevi, unlike immuno- to prepare a cultured integrated epidermis- tolerant patients with burn wounds, allografts dermis skin transplant in vitro by the so-called were cryopreserved for more than one month tissue engineering technique. prior to transplantation in order to suppress their antigenicity. Cultured autologous epithe- 1. Utilization of skin allografts lium was transplanted onto the dermal allograft Cuono et al.,4) treated third-degree burns by about ten days after transplantation of the covering the wound surface with allogeneic latter (Fig. 2). As a result, the graft take rate skin grafts after resecting the necrotized wound, for cultured autologous epidermis improved.6) followed by coverage with cultured autologous Therefore, the use of dermal allografts is the epithelium over the dermal allograft. They were most practical because they improve take rates successful in preventing burn wound infection when cultured autologous epithelium is trans- and achieved graft take rates of 80% to more planted over dermal allografts and the treated than 85% owing to the transplantation over the skin presents histological features similar to dermal components. With the same procedure, those of the dermis. the present author and colleagues succeeded in In recent years, cell-free dermal prepara- saving two children from extensive (85% and tions from epidermis-dermis deprived of cellu- 90% body surface area) burn wounds.5) lar components have become commercially In the treatment of giant congenital nevi, the available in various other countries and are lesions are resected in full thickness of the skin used as intra-tissue ﬁlling materials mainly in extending to the underlying adipose tissue plastic surgery. These materials are immuno- layer. Transplantation of cultured autologous logically inert and have been reported to pro- epithelium onto such wounds results in poor mote the take of cultured epithelium.7) Treat- graft take rates. Therefore, the lesions are ment with cultured autologous epithelium used

314 JMAJ, July 2004—Vol. 47, No. 7 CLINICAL APPLICATION OF CULTURED EPITHELIUM

in combination with skin allografts or cell-free Ulcer surfaces covered with cultured dermis dermal preparations seems to be efficacious transplant show accelerated healing as a result as a therapeutic technique for full-thickness of promoted vascularization due to vascular skin defects. However, these materials are not endothelial growth factor (VEGF) secreted by readily available in this country and there may the fibroblasts. However, wound bed formation be a hidden risk of viral infections of human to improve the graft take rate of cultured epi- origin. thelium has not been achieved as yet. Normal dermis contains networks of numerous micro- 2. Dermal reconstruction by vasculatures and has a complicated cytokine tissue engineering technique network. Not only fibrous components such as In attempting skin reconstruction utilizing fibroblasts and collagen fibers, but also the tissue engineering, the type of matrix that vascular system and various cytokines are in- should be used as a scaffold for growing epi- volved in the regeneration of dermis, so that it dermal cells and skin fibroblasts must be deter- is difficult at present to prepare cultured der- mined. Fibrin glue is a preparation employed in mis that can substitute for the cutis vera in vivo. daily clinical practice, and epidermal cells cul- The introduction of multipotent tissue stem tured on this glue retain greater capacity of cells and supplementation with humoral fac- maintaining epidermal stem cells, compared to tors will be needed in the future. the 3T3 feeder layer method. The epidermal cells so cultured are easier to handle and their Conclusion basement membrane structure can be preserved. It has been documented by Ronfard, The use of skin regeneration for the treat- et al.8) that when transplanted over a dermal ment of full-thickness skin defect is still under allograft, epidermal cells cultured on fibrin development and investigation. As mentioned glue were closer in appearance and histological above, remarkable results have been achieved features to normal skin than when transplanted in the clinical setting. Against this background, alone. Ongoing clinical application of cultured the environment surrounding regeneration skin cells containing epidermal keratinocytes and technique has been developing rapidly, not fibroblasts mediated by fibrin glue at this de- only in academic but also in medical industries partment has shown improved graft take rates. as well. Widespread clinical application of cul- Some matrix constituents as well as fibroblasts tured skin prepared with tissue engineering may be contributing to the take rate improve- techniques will be realized in the near future. ment, but tue costliness of fibrin glue poses a Furthermore, value-added cultured skin prepa- practical problem. rations such as those enabling treatment of Meanwhile, studies have been undertaken diseases with gene-transferred epidermal stem to reconstruct the skin in vitro by culturing cell grafting may become clinically available. epidermal keratinocytes and fibroblasts with spongy artificial dermis mainly consisting of REFERENCES atelocollagen as scaffold.9,10) Clinical application of cultured dermis containing cultured 1) Oshima, H., Rochat, A., Kedzia, C. et al.: Mor- allogeneic fibroblasts on spongy sheets com- phogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 2001; 104: prising hyaluronic acid and atelocollagen devel- 233–245. oped by Kuroyanagi at Kitasato University is in 2) Gallico, G.C., O’Connor, N.E., Compton, C.C. 11) progress as part of a skin regeneration study et al.: Permanent coverage of large burn under the governmental millennium project in wounds with autologous cultured human epi- Japan in 2000. thelium. N Engl J Med 1984; 311: 448–451.

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3) Rheinwald, J.G. and Green, H.: Serial cultiva- 1997; 18: 535–544. tion of strains of human epidermal keratino- 8) Ronfard, V., Rives, J.M., Neveux, Y. et al.: cytes: The formation of keratinizing colonies Long-term regeneration of human epidermis from single cells. Cells 1975; 6: 331–343. on third degree burns transplanted with autolo- 4) Cuono, C.B., Langdon, R., Birchall, N. et al.: gous cultured epithelium grown on a fibrin Composite autologous-allogeneic skin replace- matrix. Transplantation 2000; 70: 1588–1598. ment: Development and clinical application. 9) Hansbrough, J.F., Boyce, S.T., Cooper, M.L. et Plast Reconstr Surg 1987; 80: 626–637. al.: Burn wound closure with cultured autolo- 5) Matsuzaki, K., Kumagai, N., Fukushi, N. et al.: gous keratinocytes and fibroblasts attached to Treatment of extensive burn wound patients a collagen-glycosaminoglycan substrate. JAMA with skin allograft/cultured autologous epi- 1989; 262: 2125–2130. thelium; assessments of the grafted sites 4 10) Kuroyanagi, Y., Kenmochi, M., Ishihara, S. et al.: years after the treatment. Keisei-geka (J Plast A cultured skin substitute composed of fibro- Surg) 1995; 38: 193–200. (in Japanese) blasts and keratinocytes with collagen matrix: 6) Kumagai, N., Oshima, H., Tanabe, M. et al.: preliminary results of clinical trials. Ann Plast Treatment of giant congenital nevi with cryo- Surg 1993; 31: 340–349. preserved allogeneic skin and fresh autolo- 11) Kuroyanagi, Y.: Reconstruction of skin by re- gous cultured epithelium. Ann Plast Surg generative tissue engineering; Present status 1997; 39: 483–488. of the development of cultured skin substi- 7) Rennekapff, H.O., Kiessig, V., Griffey, S. et al.: tutes. Seitaizairryo (Biomaterials) 2001; 19: Acellular human dermis promotes cultured 166–176. (in Japanese) keratinocyte engraftment. J Burn Care Rehabil

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Regenerative Medicine for the Cornea

JMAJ 47(7): 317–321, 2004

Shigeru KINOSHITA* and Takahiro NAKAMURA**

*Professor, Department of Ophthalmology, Kyoto Prefectural University of Medicine **Department of Ophthalmology, Kyoto Prefectural University of Medicine

Abstract: Regenerative medicine is a new ﬁeld of medical science that aims to regenerate tissues or organ functions by utilizing such means as stem cells. The cornea is an avascular, transparent structure consisting primarily of three layers, the epithelium, stroma, and endothelium, making it a markedly distinctive body tissue. For the cornea’s regeneration, two approaches are considered feasible: regeneration of the cellular layers from existing tissue speciﬁc stem cells or regeneration by induction of ocular tissues from embryonic stem (ES) cells. This article summarizes current approaches to corneal epithelial regeneration, with particular reference to regenerative medicine as applied to ocular surface reconstruction techniques, such as cultured corneal epithelium transplantation using amniotic membrane as a substrate and transplantation of cultured oral mucosal epithelium. Key words: Amniotic membrane; Corneal epithelium; Oral mucosal epithelium; Ocular surface reconstruction

Introduction cells capable of mitotic division (e.g., stem cells) and restored to their original states”. Following the settlement of gene decoding Ophthalmology study subjects may be roughly studies symbolized by the Human Genome divided into anterior segment domain (corneal Project in the twentieth century, the leading disorders, cataracts) and posterior segment topic has been shifting to post-genome research domain (retinal degeneration, optic neuro- since the beginning of the twenty-first century. pathy). In this paper, we would like to review The field of regenerative medical care and re- our findings and state our thoughts regarding generative medicine is suddenly in the spot- the future of regenerative medicine for the light. Mass media coverage of regenerative cornea (epithelium), with which our study medicine in recent years has been striking. There- group is actively involved. fore, regenerative medicine researchers are responsible for providing accurate information. Structure of the Cornea Regeneration is “a phenomenon in which damaged/lost tissues are repaired with special In vertebrates, including humans, eyes func-

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 129, No. 3, 2003, pages 343–346).

JMAJ, July 2004—Vol. 47, No. 7 317 S. KINOSHITA and T. NAKAMURA

Conjunctiva most aspect of the cornea. It serves to regu- Sclera late hydration of the cornea through an active Ciliary body pumping function and maintains corneal transparency (Fig. 1). Cornea

Vitreous Regeneration of Lens the Corneal Epithelium

Severe ocular diseases refractory to various Anterior chamber treatments among corneal disorders currently Retina Iris ᕇᕆ ᕅ ᕄ ᕃ include Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and chemical burns. It has been documented that corneal epithelial stem Fig. 1 Structures of the eyeball and cornea cells are localized in the radial plicate structure, ᕃ Corneal epithelium, ᕄ Bowman’s membrane, ᕅ Corneal stroma, ᕆ Descemet’s membrane, and called the palisade of Vogt, which is situated in ᕇ Corneal endothelium the pericorneal region (corneal limbus).2,3) In severe ocular surface disorders, corneal epithelial stem cells are extensively damaged with a tion to take charge of the sense of sight, the consequent failure in supply of normal corneal sense that, it is said, provides humans with most epithelial cells, eventually causing coverage of of their information about the outside world. the ocular surface along with inflammation Eyes are important information-perceiving or- of the peripheral conjunctival epithelium and gans for conveying visual information to the marked visual impairment. brain. The cornea of the eye is highly trans- Attempts to regenerate the corneal epithe- parent and serves as a barrier. Its principal lium have been made by various groups of physiological function consists in introducing researchers as a therapeutic strategy against light rays into the eyeball to permit focusing on these disorders. A variety of methods for pre- an image on the retina. The cornea also serves paring transplant sheets has been investigated, as an outer physical and biological layer to pro- e.g., a sheet prepared with corneal epithelial tect the eyeball from extraneous foreign bodies cells alone,4) a sheet comprising corneal epithe- and invasion by microbial pathogens. lial cells cultured on a substrate such as a high The cornea may be divided into three cell polymer, collagen or fibrin,5–7) and a corneal layers from outer to inner: the epithelium, epithelial cell sheet using biomaterial such as stroma, and endothelium. The outermost cor- an amniotic membrane as a substrate.8–10) Our neal epithelium is nonkeratinized, stratified department has conducted studies aimed at and squamous, is of ectodermal origin, and preparing cultured epithelial sheets on the comprises the ocular surface structure together amniotic membrane. with the peripheral limbal epithelium and conjunctival epithelium.1) The corneal stroma is an 1. From in vitro to in vivo —Cultured avascular, transparent tissue, accounting for corneal epithelium transplantation more than 90% of the cornea’s total thickness, Our study team succeeded in preparing sheets and consists mainly of keratocytes and such of differentiated, stratified corneal epithelium extracellular matrix as collagen and proteo- transplantable onto the ocular surface by culti- glycans. The corneal endothelium is a single vating corneal epithelial stem cells from a rab- layer of cells of neural crest origin, like the bit on the amniotic membrane in an animal corneal stroma, and is situated at the inner- model.10) The corneal epithelial stem cells were

318 JMAJ, July 2004—Vol. 47, No. 7 CULTURED EPITHELIAL SHEET TRANSPLANTATION

A B

A: Before operation. There are persistent corneal epithe- B: After operation. Anterior segment 8 months after trans- lial defects covered with inﬂammatory, pathologic con- plantation of cultured corneal epithelium. The ocular sur- junctival tissue, resulting in a marked loss of vision. face is covered with cultured corneal epithelial graft, with retained transparency and recovery of visual acuity. Fig. 2 Cultured corneal epithelium transplantation in the treatment of ocular cicatricial pemphigoid

co-cultured with 3T3 ﬁbroblasts that were pre- tation, graft survival was conﬁrmed in all the treated with mitomycin C to suppress prolifera- patients who underwent cultured allocorneal tive activity in the process of preparation. The epithelium transplantation (Fig. 2).11) cultured corneal epithelial sheet on the amni- Corneal surfaces were cured and retained otic membrane was autotransplanted into the transparency in most cases, but because the same rabbit, was taken over the ocular surface, transplants are allogeneic, problems such as and proved to maintain its transparency. postoperative rejection and infections have Based on the results of the basic data ob- affected therapeutic outcomes. The fate of the tained from laboratory animals, we started clini- transplanted cultured corneal epithelium re- cal application of the allografting of cultured mains uncertain, stressing the need for long- corneal epithelium in patients with severe ocu- term follow-up of the cases. lar surface disorders at this department in 1999 after scrupulously obtaining informed consent 2. From allo- to auto-— Cultured oral in accordance with the approval of the institu- mucosal epithelium transplantation tional ethics committee of the university. The For the treatment of severe ocular surface surgical procedure was as follows. We removed disorders, surgical reconstructive procedures the conjunctival tissue from the cornea and have been developed, such as keratoepithelio- also removed the subconjunctival tissue to plasty, limbal transplantation, and cultured expose the corneal stroma. We then secured corneal epithelium transplantation using allo- the cultured allocorneal epithelium on amni- geneic cells. Because severe ocular surface otic membrane onto the corneal surface with disorders are frequently bilateral, it is in- sutures placed on the limbus. The allografting feasible in such cases to attain reconstruction was performed on 34 eyes with severe ocular with autologous corneal epithelium, which surface disorders, including acute-phase cases eventually requires transplantation of allo- contraindicated for conventional surgical inter- geneic tissue grafts. vention. Forty-eight hours after the transplan- Therefore, taking note of oral mucosal epi-

JMAJ, July 2004—Vol. 47, No. 7 319 S. KINOSHITA and T. NAKAMURA

A B

A: Before operation. The ocular surface structure is B: Anterior segment at 10 days after operation. The covered with conjunctival tissue, presenting a fea- ocular surface is covered with a cultured oral mucosal ture of severe ocular surface disorder. epithelial sheet with retained transparency and stability. Fig. 3 A rabbit cultured oral mucosal epithelial transplantation model

thelium among many other potential sources of bilateral ocular surface disorders, though still cells in view of cytobiological characteristics at the animal experiment level, represents a and ease of tissue collection, we prepared novel surgical procedure.12) This procedure is cultured rabbit oral mucosal epithelial sheets now in the stage of clinical application to on amniotic membrane and autotransplanted humans in collaboration with the Department them onto rabbit ocular surfaces to assess their of Dentistry, and attempts at epithelial repair usefulness. Rabbit oral mucosal epithelium cul- in acute phase corneal alkali burn have been tures on amniotic membrane demonstrated successful in a clinical trial.13) survival and growth of the epithelium. In addition, the cultured epithelium formed a strat- Conclusion ified epithelium consisting of 5 to 6 layers of cells, resembling the normal corneal epithelium. The success in cultured cell transplantation In rabbits that received transplantation of an from in vitro to in vivo has brought about a autologous oral mucosal epithelial sheet onto paradigm shift that operative treatment is now the cornea, the reconstructed ocular surface feasible even in acute-phase severe ocular sur- was practically as transparent as the cultured face disorders that had been contraindicated corneal epithelial sheet. The autografted mucosal for surgical intervention. The idea of expanding epithelial sheet retained its transparency and the source of cells, furthermore, is leading to was confirmed to be surviving on the ocular the possibility of a major breakthrough in surface 48 hours after the transplantation. On surgical reconstruction of the ocular surface Day 10 post-transplantation, the autografted structure. This paper has presented our studies mucosal epithelium was demonstrated to have on regeneration of the corneal epithelium. As spread outward. It was thus confirmed that the the structure of the cornea comprises three oral mucosal epithelial sheet cultured in vitro cellular layers, the epithelium, stroma, and was taken and spread on the ocular surface in endothelium, studies on regeneration of the vivo and its transparency restored (Fig. 3). corneal stroma and corneal endothelium are in This therapeutic concept of ocular surface progress at this department in tandem with reconstruction with cultured oral mucosal epi- corneal epithelial regeneration studies. thelium sheet in the treatment of intractable,

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REFERENCES 8) Tsai, R.J., Li, L.M. and Chen, J.K.: Recon- struction of damaged corneas by transplan- 1) Thoft, R.A.: Conjunctival transplantation. tation of autologous limbal epithelial cells. N Arch Ophthalmol 1977; 95(8): 1425–1427. Engl J Med 2000; 343: 86—93. 2) Schermer, A., Galvin, S. and Sun, T.T.: Differ- 9) Shimazaki, J., Aiba, M., Goto, E. et al.: Trans- entiation-related expression of a major 64K plantation of human limbal epithelium culti- corneal keratin in vivo and in culture suggests vated on amniotic membrane for the treat- limbal location of corneal epithelial stem cells. ment of severe ocular surface disorders. J Cell Biol 1986; 103: 49–62. Ophthalmology 2002; 109: 1285–1290. 3) Cotsarelis, G., Cheng, S.Z., Dong, G. et al.: Ex- 10) Koizumi, N., Inatomi, T., Quantock, A.J. et al.: istence of slow-cycling limbal epithelial basal Amniotic membrane as a substrate for culti- cells that can be preferentially stimulated to vating limbal corneal epithelial cells for au- proliferate: implications on epithelial stem tologous transplantation in rabbits. Cornea cells. Cell 1989; 57(2): 201–209. 2000; 19(1): 65–71. 4) Gipson, I.K. and Grill, S.M.: A technique for 11) Koizumi, N., Inatomi, T., Suzuki, T. et al.: Cul- obtaining sheets of intact rabbit corneal epi- tivated corneal epithelial stem cell transplan- thelium. Invest Ophthalmol Vis Sci 1982; 23: tation in ocular surface disorders. Ophthal- 269–273. mology 2001; 108: 1569–1574. 5) Ohji, M., Kinoshita, S., Watanabe, K. et al.: 12) Nakamura, T., Endo, K., Cooper, L.J. et al.: Corneal epithelium culturing on type IV col- The successful culture and autologous trans- lagen sheet. Folia Ophthalmol Jpn 1991; 42: plantation of rabbit oral mucosal epithelial 784–788. (in Japanese) cells on amniotic membrane. Invest Ophthal- 6) Friend, J., Kinoshita, S., Thoft, R.A. et al.: Cor- mol Vis Sci 2003; 44(1): 106–116. neal epithelial cell cultures on stromal carri- 13) Nakamura, T., Inatomi, T., Sotozono, C., ers. Invest Ophthalmol Vis Sci 1982; 23: 41–49. Amemiya, T., Kanamura, N. and Kinoshita, S.: 7) Rama, P., Bonini, S., Lambiase, A. et al.: Transplantation of cultivated autologous oral Autologous ﬁbrincultured limbal stem cells mucosal epithelial cells in patients with severe permanently restore the corneal surface of ocular surface disorders. Br J Ophthalmol in patients with total limbal stem cell deﬁciency. press. Transplantation 2001; 72: 1478–1485.

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Regenerative Medicine for Sclerotic Disorders

JMAJ 47(7): 322–327, 2004

Toshikazu NAKAMURA

Professor, Division of Molecular Regenerative Medicine, Course of Advanced Medicine, Osaka University Graduate School of Medicine

Abstract: Hepatocyte growth factor (HGF) was discovered in 1984 as a molecular entity of hepatic regeneration factor, which had long been sought after. In ensuing studies, HGF has been proven to enhance the growth and motility of hepatocytes and other functional cells and to induce morphogenesis represented by tubular structures. Furthermore, HGF is endowed with potent anti-apoptotic activity, thereby preventing various tissue injuries. HGF has been demonstrated in a wide variety of animal disease models to be remarkably effective in the preven- tion and treatment of acute and chronic organ disorders. It has marked curative/ ameliorative effects in chronic diseases, especially such sclerotic disorders as liver cirrhosis, renal failure, nephrosclerosis, lung fibrosis and cardiomyopathy for which there is no definitive treatment to date. The remarkable effectiveness of HGF stems from its ability as a tissue-organizing molecule to provide functional improvement by inhibiting fibrosis and reconstructing normal tissues, as well as from its potent anti-apoptotic activity. These show that HGF is an intrinsic tissue repair factor. Key words: Hepatocyte growth factor (HGF); Intrinsic regeneration factor; Lung fibrosis; Liver cirrhosis; Chronic renal failure

Introduction of bone marrow, nerve tissues and muscles. The other, termed the simple duplication sys- Ingenious tissue regeneration mechanisms in tem, is the regeneration system for tissues mammals are roughly divided into two distinct whose cellular components are mature and systems. One is a system in which undifferenti- differentiated, yet vitally capable of prolifera- ated, vigorously proliferative stem cells assume tion as seen in the regeneration of parenchymal the principal role in tissue regeneration. It oper- organs such as the liver, kidneys, and lungs. ates to regenerate and repair tissues comprising Therefore, for organs with complicated multi- differentiated cells that are no longer capable cellular architecture such as the liver, kidneys, of proliferation, such as the hemopoietic tissue and lungs, treatment of an injury by activation

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 129, No. 3, 2003, pages 360–364).

322 JMAJ, July 2004—Vol. 47, No. 7 HGF THERAPY FOR FABROTIC DISORDERS

B ) ) l 10 l A Acute hepatitis 20 8 Acute renal failure 6 10 Ͱ-chain 4 2 HGF 0 0 Arg Plasma HGF (ng/m 01020304050 Plasma HGF (ng/m 01020304050

s ) )

Val l l s 8 6 Pulmonary ischemia/ Myocardial infarction reperfusion injury ͱ 6 -chain 4 4 2 2 0 0 Plasma HGF (ng/m 01020304050 Plasma HGF (ng/m 0 24 48 72 96 120144168 Time after induction (hr) Time after induction (hr)

c-Met C 50 Normal IgG 80 CCl4-induced 40 Anti-HGF IgG hepatitis Cell membrane 60 (48 hrs 30 post-injection) Tyrosine kinase Y P 40 PLC-Ͳ 20 Y P p85 PI3K PI3K P 20 p85 P hepatocytes 10 hepatocytes Y P Grb2

Ras % PCNA-positive % PCNA-positive SOS Grb2 Y P Gab1 P Crk2 0 0 P Shp2 061224 Normal Anti-HGF Hrs after partial (70%) hepatectmoy IgG IgG

Mitogenic 12 Crush syndrome 2 Angiogenic 8 (48 hrs post-injection) 4 1 injury score renal tubule Renal tubule Motogenic 0 0 No. of PCNA-positive No. Anti-apoptotic epithelial cells (/(200) Normal Anti-HGF Normal Anti-HGF Morphogenic IgG IgG IgG IgG 10 20 Pulmonary ischemia/ 8 15 reperfusion injury A: HGF and diverse biologic activities mediated by (48 hrs 6 HGF receptor 10 post-injection) 4 alveolar

B: Plasma HGF levels associated with 5 injury score 2 epithelial cells

various organ injuries % PCNA-positive 0 0 Normal Anti-HGF Normal Anti-HGF C: Inhibition of regeneration and disease exacerbation Pulmonary parenchymal following neutralization of endogenous HGF IgG IgG IgG IgG

Fig. 1 Biologic activities and roles as an intrinsic regeneration factor of HGF

of the simple duplication system will be a means covered in 1984 as a long-sought-after hepato- of regeneration/repair (therapy) in accordance trophic factor for vigorous regeneration of the with nature. liver, and its structure was clariﬁed by molecu- This paper discusses regenerative therapy for lar cloning in 1989.1) HGF stimulates prolifera- sclerotic disorders that arise from failure of the tion of hepatocytes and practically all types of simple duplication system’s self-repair mecha- epithelial cells, as well as some of mesenchymal nism due to chronic damage. lineage such as vascular endothelial cells. In 1991, the receptor for HGF was identiﬁed to be HGF as an Intrinsic a tyrosine kinase type c-Met gene product. Regeneration Factor Ensuing studies have revealed that HGF is endowed not only with the ability to enhance Hepatocyte growth factor (HGF) was dis- growth of the said target cells (mitogen) but also

JMAJ, July 2004—Vol. 47, No. 7 323 T. NAKAMURA

has other biologic activities such as enhance- organ functions give rise to proliferation of ment of cellular motility (motogen), induction fibroblasts and other interstitial cells and accu- of morphogenesis represented by tubular struc- mulation of interstitial fibrous components tures (morphogen), and anti-apoptotic and which those cells produce, eventually resulting angiogenetic potential2) (Fig. 1A). in sclerosis and dysfunction of the organ. These From the rapid increase in HGF level of studies have indicated that decrease in expres- damaged tissue or circulating plasma occurring sion of the intrinsic regeneration factor HGF in response to visceral injury, it has even been occurs with the progression of chronic disease, suggested that HGF acts as an intrinsic regen- coupled reciprocally with overexpression of eration factor upon various organs (Fig. 1B). the transforming growth factor (TGF)-␤. Blockade of the injury-associated HGF eleva- TGF-␤ acts as an inhibitor of growth of epi- tion by an anti-HGF neutralizing antibody thelial cells and vascular endothelial cells and, results in strong suppression of organ regener- at the same time, as a promoter for apoptosis. ation/repair and expansion of tissue damage Toward interstitial cells, on the other hand, (i.e., aggravation of disease state) (Fig. 1C). It is TGF-␤ functions as a growth-promoting factor obvious also from these events that HGF acts and stimulates production of interstitial fibrous as an intrinsic regeneration factor to promote components such as collagen. Overexpression post-injury regeneration/repair. Conversely, of TGF-␤ is thus a major cause of dysre- administration of recombinant HGF to mice generation and parallel stimulation of tissue after onset of induced acute hepatitis, acute fibrosis. renal failure, myocardial infarction, or pneu- First, we would like to introduce findings monia leads to a conspicuous inhibition of demonstrating HGF’s ability to inhibit the tissue damage (anti-apoptotic effect) and en- development of liver cirrhosis and treat cirrho- hanced regeneration with consequent marked sis. In Japan alone, more than 20,000 patients improvement.2,3) develop liver cirrhosis due to chronic hepatitis We then investigated the site(s) of origin of or congenital biliary atresia annually. Liver HGF, which is promptly generated and sup- transplantation is the only life-saving proce- plied in response to tissue injury. Our results dure currently available, however, registered showed that, in case of liver damage, for ex- donors are extremely few and most cirrhosis ample, HGF mRNA expression occurs imme- patients on the waiting list lose their lives. diately in mesenchymal tissues such as vascular Long-term administration of very low doses endothelial cells, Kupffer cells, and Itoh cells of of dimethylnitrosamine (DMN), a hepatotoxic the damaged liver and that HGF expression is substance, to rats led to overt development of induced also in such distant intact viscera as the hepatic fibrosis and in due course to cirrhosis. lung, spleen, and kidney. Thus, HGF elabo- Deaths began to occur among the animals in rated upon recognition of an injury assumes Week 4 of DMN dosing and practically all ani- physiologic roles for regeneration/repair of mals succumbed to hepatic failure by Week 6. the damaged organ via both endocrine and When rats were started on daily recombinant paracrine pathways. HGF dosing at Week 3 of DMN administration, at which period hepatic fibrosis had devel- Regenerative Therapy for oped, subsequent development of cirrhosis was Chronic Fibrotic Disorders almost completely inhibited along with amelio- ration in preexisting hepatic fibrosis and recon- In chronic disorders involving persistent tis- struction of the normal lobular architecture sue damage, destruction of parenchymal cells (Fig. 2A).4) Thus, the survival rate of rats and loss of vascular endothelial cells that serve increased with increasing dosages of HGF;

324 JMAJ, July 2004—Vol. 47, No. 7 HGF THERAPY FOR FABROTIC DISORDERS

A: Cirrhosis (Masson’s trichrome stain)

100

60 HGF 200Ȑg/kg 40 HGF 50Ȑg/kg Survival rate (%) Survival rate 20 Physiological saline

0 03456 Treatment duration (weeks) Physiological saline HGF

B: Chronic renal failure (Immunostain for type I collagen)

HGF Physiological saline 60 ) l

40 UN (mg/d B

20 0102030 Treatment duration (days) Physiological saline HGF

C: Pulmonary fibrosis (elastica-Masson stain)

Hydroxyproline content

BLM

Simultaneous BLM and HGF injections

HGF injection after BLM

100 120 140 160 180 Physiological saline HGF (Percent of content in normal lungs) % Fig. 2 Therapeutic effects of HGF in fibrous sclerotic disorders Source: A: Matsuda, Y. et al.: J Biochem 1995; 118: 643Ð649. B: Mizuno, S. et al.: J Clin Invest 1998; 101: 1827Ð1834. C: Yaekashiwa, M. et al.: Am J Respir Crit Care Med 1997; 156: 1937Ð1944.

all rats receiving HGF at 200␮g/kg b.w. sur- patic failure. vived in normal health without incurring he- In rats of the HGF-treated groups, there was

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evidence of enhanced growth (regeneration) of showed histological features close to normal hepatocytes, increased collagenolysis, inhibi- renal tissues, indicating that HGF effected glo- tion of TGF-␤ expression as well as hepatocytic merular and renal tubule tissue reconstruction, apoptosis, along with suppressed extracellular as seen in Fig. 2B.6) Similar results were repro- matrix production; the treated animals showed duced in such other chronic renal failure mod- nearly normal histological features of liver els as chronic kidney graft rejection, hydro- tissue architecture.4) Gene therapy with HGF- nephrosis, and 5/6-nephrectomized rats.7) If expressed plasmid produced similar therapeu- reconstruction of renal tissues could be in- tic effects in animals with liver cirrhosis.5) duced by administration of HGF, thereby liber- Next, we would like to discuss the thera- ating chronic renal failure patients from hemo- peutic effects of HGF in chronic renal failure/ dialysis therapy, it would be of great medical nephrosclerosis. ICGN mice, being animal mod- and economic significance. els for chronic nephropathies in humans, show The antifibrotic effect has been demon- gradual impairment of renal function begin- strated following treatment with HGF in a ning just after 10 weeks of age, develop marked mouse model for pulmonary fibrosis induced glomerular and tubulointerstitial fibrosis at 18 with bleomycin (BLM) injections (Fig. 2C).8) weeks of age and after, and eventually develop We would like to omit the details of the data end-stage renal failure. The renal tissue TGF-␤ here on account of limited space. The conspicu- level increases remarkably at 10 weeks of age ous efficacy of HGF lies in regenerating normal and thereafter, and the TGF-␤ elevation is ac- tissue architectures as well as in inhibiting companied by accumulation of fibrous compo- apoptosis of parenchymal cells accountable for nents in renal tissues and increased apoptosis functions of tissues and organs and in stimulat- of renal tubule epithelial cells and glomerular ing growth of those cells. vascular endothelial cells. HGF is also an angiogenetic factor with The renal tissue HGF level is decreased, in potent ability to promote angiogenesis essen- contrast to the TGF-␤ elevation, so that regen- tial to tissue regeneration/repair. In chronic erative capacity of renal tubule epithelial cells disorders in particular, depressed HGF expres- is noticeably depressed. Depressed expression sion occurs and eventually leads to elevated of HGF, which is to act as an intrinsic regenera- expression of fibrosis-promoting factors such tion factor and elevated expression of TGF-␤, as TGF-␤. This, in turn, accelerates fibrosis via is considered to be etiologically largely respon- loss and inhibited regeneration of parenchy- sible for the development and progression of mal tissues. Administration of HGF to subjects chronic nephropathies. In ICGN mice receiv- in such conditions enhances regeneration of ing daily doses of recombinant HGF for 4 parenchymal cells and vascular endothelial weeks starting at 14 weeks of age, the renal cells and suppresses TGF-␤ expression, with function indicator BUN level, which had been consequent inhibition of tissue destruction and continuously increasing, was largely restored to fibrosis. normal 7 days after the start of dosing and re- 6) mained at normal levels thereafter (Fig. 2B). Conclusion In the HGF-treated groups of mice, a pro- nounced suppression of TGF-␤ expression in This article has briefly described the regen- kidney tissues, inhibited accumulation of col- erative therapy of chronic fibrotic disorders for lagen and other fibrous components, suppressed which there has been no definitive treatment apoptosis of renal tubule epithelial cells, and a by vigorous regeneration/repair in vivo using marked enhancement of renal tubule epithelial the intrinsic regeneration factor HGF, which is cells were noted. Consequently, their kidneys considered to play a crucial role in the simple

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duplication system for regeneration/repair in- 4) Matsuda, Y., Matsumoto, K., Ichida, T. and herent in mammals. We feel that there is a fair Nakamura, T.: Hepatocyte growth factor sup- prospect that regenerative medical care using a presses the onset of liver cirrhosis and abro- recombinant HGF protein or HGF gene will be gates lethal hepatic dysfunction in rats. J Biochem 1995; 118: 643–649. a concrete therapy that will remove the suffer- 5) Ueki, T., Kaneda, Y., Tsutsui, H. et al.: Hepato- ing and promote the welfare of countless dis- cyte growth factor gene therapy of liver cir- eased individuals. rhosis in rats. Nat Med 1999; 5: 226–230. 6) Mizuno, S., Kurosawa, T., Matsumoto, K. et al.: REFERENCES Hepatocyte growth factor prevents renal ﬁbro- sis and dysfunction in a mouse model of 1) Nakamura, T., Nishizawa, T., Hagiya, M. et al.: chronic renal disease. J Clin Invest 1998; 101: Molecular cloning and expression of human 1827–1834. hepatocyte growth factor. Nature 1989; 342: 7) Matsumoto, K. and Nakamura, T.: Hepatocyte 440–443. growth factor: renotropic role and potential 2) Funakoshi, H., and Nakamura, T.: Hepatocyte therapeutics for renal diseases. Kidney Int growth factor: from diagnosis to clinical appli- 2001; 59: 2023–2038. cations. Clin Chim Acta 2003; 327: 1–23. 8) Yaekashiwa, M., Nakayama, S., Ohnuma, K. 3) Matsumoto, K. and Nakamura, T.: Hepatocyte et al.: Simultaneous or delayed administration growth factor (HGF) as a tissue organizer of hepatocyte growth factor equally represses for organogenesis and regeneration. Biochem the ﬁbrotic changes in murine lung injury Biophys Res Commun 1997; 239: 639–644. induced by bleomycin. Am J Respir Crit Care Med 1997; 156: 1937–1944.

JMAJ, July 2004—Vol. 47, No. 7 327 ⅥRegenerative Medicine

Regenerative Medicine for Cardiomyocytes

JMAJ 47(7): 328–332, 2004

Keiichi FUKUDA

Assistant Professor, Institute for Advanced Cardiac Therapeutics, Keio University School of Medicine

Abstract: Heart transplantation is the ultimate treatment option for severe cardiac failure, but is available only for a very small fraction of cases due to a serious donor shortage. Increasing attention has become focused upon a novel therapeutic approach, regenerative medicine, to break the present impasse. Attempts to regenerate cardiomyocytes have been made by using pluripotent embryonic stem cells or marrow-derived mesenchymal stem cells (adult stem cells). Cardiomyocytes can be regenerated from embryonic stem cells as well as from adult stem cells, but the regenerated cells differ in characteristics depending on the source stem cells. These two groups of stem cells differ with respect to proliferative potency, pluripotency, method of induction of differentiation into cardiomyocytes, rejection reactions, and tumorigenic potential. Further studies to ascertain which type of stem cell will be more useful and safer for this purpose remain to be carried out. Studies in laboratory animals have reportedly demonstrated improvement of cardiac function through regenerated cardiomyocyte transplantation into the heart, encouraging the hope that a new treatment modality has been found for severe heart failure. Key words: Embryonic stem cell; Adult stem cell; Cardiomyocyte; Regenerative medicine

Introduction treatment option. There is still no increase in brain-dead donors and heart transplantation is In Japan, cases of heart disease have consis- a treatment available only for a very few cases. tently been increasing with the aging of the To break the present impasse, therefore, a population and the Westernization of the diet. method to treat intractable cardiac failure by A wide variety of pharmacotherapies has been regenerating and transplanting cardiomyocytes developed for the treatment of intractable is being sought. Studies of heart muscle cell severe heart disease, with proven efﬁcacy, how- regeneration have been making a steady ever, heart transplantation is the sole radical progress, though at the level of laboratory ani-

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 129, No. 3, 2003, pages 365–368).

328 JMAJ, July 2004—Vol. 47, No. 7 REGENERATION OF CARDIOMYOCYTES

Table 1 Comparison of ES Cells and Adult Stem Cells as Materials for Regenerative Cardiomyocytes

ES Cells Adult Stem Cells Post-fertilization early-stage embryo Marrow stromal cell Origin (inner cell mass of blastocyst) Cell isolation Method of cell establishment is already Sparse among bone marrow cells, and method of technique, etc. established, and is relatively easily performed. cell establishment is yet to be established. Proliferative At present, cells are considered to infinitely Proliferate to some extent but the number of potency proliferate. divisions is unknown. Differentiate into any type of cell in vivo. Recognized to be able to differentiate into The cells differentiate into early developmental mesodermal cells such as osteoblasts, chondroblasts Pluripotency stage cells in vitro, but it is thought to be difficult and adipocytes, but reportedly undergo for them to differentiate into cells that appear late differentiation into nerve cells (ectoblast-derived) in the fetal stage. and cells of the liver (entoblast-derived) as well. Differentiate relatively easily, but a method to Demonstrated to differentiate into cardiomyocytes, Differentiation into have ES cells specifically differentiate into but a method to have the cells specifically cardiomyocytes cardiomyocytes has not been established. differentiate into cardiomyocytes has not been established. Rejection reactions Occur No rejection reactions if the cells are autologous. Tumorigenic There is potential risk of teratomas after No potential transplantation if undifferentiated cells remain.

mal experiments, and will lead to development embryo having reached the stage of blastocyst. of research with human cells and their clinical These cells are known to differentiate into any application. type of cell in vivo and have been shown to Stem cells currently used for regenerative differentiate in vitro into a variety of organic medical therapy for the myocardium are cells such as cardiomyocytes, skeletal muscle broadly divided into two groups: embryonic cells, vascular endothelial cells, smooth muscle stem cells (ES cells) obtained from early stage cells, neurons, and hepatocytes. However, most embryos post in vitro fertilization and marrow types of cells that appear late in the fetal stage adult stem cells obtained from the bone mar- have not been demonstrated from ES cells row of adults. Whether one type is superior in vitro. to the other for cardiomyocyte regeneration It is generally recognized that various cell remains to be seen. Characteristics of the two growth factors, cytokines, and cell adhesion types of stem cells are summarized in Table 1. factors are required for ES cell differentiation This article outlines the current status and into those various cells. Recent studies have future prospects of regenerative medicine for clariﬁed a cascade operating for selective dif- the myocardium. ferentiation of ES cells into motor neurons, however, the cascade for ES cell differentiation Differentiation from ES Cells to into cardiomyocytes has not been fully eluci- Cardiomyocytes dated to date. A method to have ES cells form a cell mass Figure 1 illustrates the outline of cardio- (embryoid) has been introduced as a general myocyte regeneration using ES cells. ES cells means of inducing ES cell differentiation into are those cells constituting the inner cell mass, cells capable of differentiation. The frequency destined to form the fetus, from an early with which differentiation from the embryoid

JMAJ, July 2004—Vol. 47, No. 7 329 K. FUKUDA

Fertilization Morula

Ovary Germ cell Testis

Liver Entoderm Pancreas Nuclear transplantation Thyroid into egg cell Enucleation Myocardium, Skeletal muscle Mesoderm Bone, cartilage Fat, ligament Inner cell mass of blastocyst Tendon Collection of somatic cells Epithelial cell Ectoderm Neuron Pigment cell Established ES cells Nerves

Cell differentiation Blood, visceral organs

Cell transplantation Adult body Skeletal muscles, myocardium

Fig. 1 Myocardial regeneration from ES cells

to cardiomyocytes is regarded as being about predominance of hematopoietic stem cells. In 7–8% at best. Humoral factors known to fact, more than 99% of marrow cells take part induce ES cell differentiation speciﬁcally into in the production and development of blood cardiomyocytes include bone morphogenetic cells. It was discovered recently that, among protein-2 (BMP-2) and Wnt 11, which facilitate bone marrow cells, there are cells termed stro- the differentiation, and Wnt 3 and Wnt 8, which mal cells that essentially do not represent act as inhibitors. Possible involvement of vari- blood cells but secrete cytokines and growth ous other factors is presumed; the process factors to support cells of the hematopoietic seems to be quite intricate. Selective differen- system. The presence of pluripotent stem cells tiation of ES cells into cardiomyocytes may capable of differentiating into various types become feasible from analysis of the pathways of cells among the marrow stromal cells has of the differentiation process. become recognized. These marrow stromal cells with pluripotent capacity are referred to Adult Stem Cell Differentiation into as mesenchymal stem cells on account of their Cardiomyocytes ability to differentiate into such mesenchymal cells as osteoblasts, chondroblasts, and Bone marrow has been universally recog- adipocytes. nized to be the site of hematopoiesis with the In view of the mesenchymal stem cells being

330 JMAJ, July 2004—Vol. 47, No. 7 REGENERATION OF CARDIOMYOCYTES

Adult Induction of stem cell differentiation Hematopoietic stem cell

Bone marrow

Blood corpuscles

Regenerated cardiomyocytes Cell transplantation

Fig. 2 Myocardial regeneration from adult stem cells and cell transplantation

able to differentiate into mesoblast-derived cytes exhibited the sinus node pattern of action organs, we wondered if they could differentiate potentials early after their differentiation from to become cardiomyocytes as well, which are adult stem cells, and the pattern gradually also of mesodermal origin. Our studies demon- changed to the ventricular muscle cell type. strated that cardiomyocytes that beat regularly Catecholamin ␣1 receptor (cardiac hyper- by themselves can be obtained from mesenchy- trophic effect) and ␤1 and ␤2 receptors (positive mal stem cells. Figure 2 shows an outline of the chronotropic and positive inotropic effects) process. Mesenchymal stem cells have recently play important roles in the muscle cells of the been reported to undergo differentiation into heart. In regenerated cardiomyocytes of bone nerve cells (ectoblast-derived) and cells of the marrow origin, ␣1 receptor switched to myocar- liver (entoblast-derived) as well, and are now dial type (mainly ␤1A and ␤1B receptors) as the termed adult stem cells. differentiation proceeded into heart muscle

cells, and simultaneously, ␤1 and ␤2 receptors Characteristics of Regenerated that had not existed early in the course of dif- 2) Cardiomyocytes ferentiation became expressed. Stimulation of these catecholamine receptors led to activation Cardiomyocytes derived from bone marrow of subreceptor signaling to produce cardiac show expression of fetal ventricular muscle hypertrophy, increases in heart rate and in- type genes soon after their differentiation creases in myocardial contractile force. from adult stem cells, and thereafter gradually The above ﬁndings indicate that the regener- express adult type genes.1) Expression of genes ated cardiomyocytes are endowed with practi- for atrial natriuretic polypeptide and cerebral cally normal characteristics of cardiomyocytes.3) natriuretic polypeptide that are considered cardiomyocyte-speciﬁc have also been demon- Treatment of Cardiac Failure by Cell strated. The cells proved to self-beat, and those Transplantation differentiated from murine adult stem cells showed 120–250 beats/min. The cardiomyo- Cardiomyocyte transplantation has been

JMAJ, July 2004—Vol. 47, No. 7 331 K. FUKUDA

extensively investigated since the mid-1990s at plantation to realization requires securing the level of animal experiments. Cells har- regenerated cardiomyocytes and supplying vested in primary cultures of heart muscle cells those cells safely and at moderate expense. obtained from the fetus or the neonate were When adult stem cells and ES cells are com- transplanted into the heart of sexually mature pared, this author’s view is that the latter will animals, and the transplantation was shown to come into use earlier in the future. Supply of improve post-infarction cardiac function. Clini- regenerated cardiomyocytes derived from ES cal experience with the transplantation of fetal cells will become a reality within several years. midbrain obtained through artiﬁcial termina- Major problems are rejection reactions and tion of pregnancy into patients with Parkinson’s method of transplantation. To avoid rejection, disease has yielded some gratifying therapeutic it is essential to transplant the nucleus of a results. The amount of cell transplants required somatic cell into an egg cell, as shown in Fig. 1. is apparently greater in the case of cardiomyo- The nuclear transplantation eventually has a cyte transplantation, so that it is not practical to close bearing upon the matter of human clon- use aborted fetuses as the source. ing, therefore it is important to hold nation- Regenerated cardiomyocytes derived from wide discussion of this ethical problem. ES cells, when transplanted, reportedly proved to electrically bound to recipient myocardium and contract synchronously with surrounding REFERENCES cells, thus fueling hopes for the use of regenerated cardiomyocytes. Our experience with 1) Makino, S., Fukuda, K., Miyoshi, S. et al.: regenerated cardiomyocyte transplantation Cardiomyocytes can be generated from mar- into the hearts of adult patients showed long- row stromal cells in vitro. J Clin Invest 1999; 103: 687–705. term engraftment with gratifying outcomes. 2) Hakuno, D., Fukuda, K., Makino, S. et al.: However, there have been reports demonstrat- Bone marrow-derived regenerated cardio- ing that the number of cardiomyocytes taken as myocytes (CMG Cells) express functional compared to that of cardiomyocytes trans- adrenergic and muscarinic receptors. Circula- planted diminished due to cellular necrosis tion 2002; 106: 380–386. during the course of engraftment. Further 3) Fukuda, K.: Differentiation from bone mar- study is needed, including assessments of trans- row mesenchymal stem cells into cardiomyo- plantation methods. cytes. In: Asajima, M., Iwata, H., Ueda, M. and Nakatsuji, N. (Eds.): “Regenerative medicine and life science—Reproductive engineering, Problems Associated with Myocardial stem cell engineering and tissue engineering”, Regeneration and Future Prospects Protein, Nucleic Acids and Enzymes, (Special Issue), Kyoritsu Shuppan, Tokyo, 2000; 45(13): To bring regenerative cardiomyocyte trans- 2078–2084. (in Japanese)

332 JMAJ, July 2004—Vol. 47, No. 7 ⅥRegenerative Medicine

Regenerative Medicine for Respiratory Diseases

JMAJ 47(7): 333–337, 2004

Tatsuo NAKAMURA

Associate Professor, Institute for Frontier Medical Sciences, Kyoto University

Abstract: Tissue engineering is an important technology for supporting regenerative medicine, producing tissues in incubation bottles in a tissue culture laboratory. In addition, a newer approach to regeneration of tissues in the human body, called in situ tissue engineering, has been devised in Japan and is attracting attention as a new means of bringing out our body’s potential. New therapeutic methods for chronic respiratory disorders such as pulmonary emphysema and pulmonary ﬁbrosis, as well as for primary pulmonary hypertension for which there have been no curative treatment measures available, are currently under investigation. This paper provides a full account of the developments. In situ tissue engineering causes regeneration of the lung parenchyma itself within the lungs using various growth factors and collagen, which serves as a scaffold for tissue regeneration. A new type of tracheal prosthesis has been in clinical use since 2002 in which new technique was applied to induce autologous tissue regeneration. Key words: Tissue engineering; Artiﬁcial trachea; In situ tissue engineering; Primary pulmonary hypertension

Introduction state of regenerative medical care for these respiratory disorders, with particular reference The number of patients suffering from to the progress of research aimed at application dyspnea after surgical resection of carcinoma in clinical settings. of the lungs and patients with chronic respiratory disorders such as pulmonary emphysema Artificial Trachea (Bronchus) or pulmonary fibrosis is expected to increase continuously in the future. Primary pulmonary Obstructive disorders and stenosis of the hypertension is also a progressive disease, central airway are mainly caused by infiltration though lower in incidence, with an extremely of lung cancer or thyroid carcinoma. These dis- poor prognosis. This paper reviews the present orders are rarely indicated for surgery because

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 129, No. 3, 2003, pages 369–372).

JMAJ, July 2004—Vol. 47, No. 7 333 T. NAKAMURA

ﬁrmed over a 5 years observation.3) This tissue regeneration-type tracheal prosthesis is now clinically used as a trial upon an ongoing ethical committee review.

Regeneration of Pulmonary Parenchyma and In Situ Tissue Engineering

Regeneration of the lungs, unlike the liver, Fig. 1 Autologous tissue regeneration type has not been recognized. However, the recent tracheal prosthesis A structural equivalent of tracheal cartilage comprising a advances in regenerative medicine have shown polypropylene stent spirally wrapped around a Marlex mesh that the lungs can also be regenerated. Regen- (left) and coated with collagen sponge. Autologous tissue regenerates in this collagen scaffold layer. eration of destroyed pulmonary parenchyma, if feasible, will enable treatment of disorders for which there has long been no curative treatment, such as pulmonary emphysema, pulmo- of lack of reconstructive procedures, despite an nary fibrosis, and pulmonary hypertension. extensive resection of the malignancies. Devel- A group of investigators at Harvard Medical opment of an artificial trachea that can be used School seeded the lung cells obtained from a safely even in such patients has already begun sheep that had been trypsinized on biodegrad- in early 1940s. Initially, a variety of attempts able polyglycolic acid (PGA) fiber mesh and were made to replace the affected trachea with incubated with added growth factors such as an artificial tube like a vascular prosthesis, but epithelial cell growth factor (EGF) and basic all proved unsuccessful. Artificial tracheas fibroblast growth factor (bFGF). The investi- were then developed using the new idea of hav- gators implanted the cells on fiber mesh in the ing autologous tissue regenerate on the surface subcutis of nude mice. One week later, the of a tracheal prosthesis. This artificial trachea is implant was noted to have become a tissue composed of a Marlex mesh tube reinforced by resembling lung tissue capable of producing a spiral stent and is conjugated with collagen collagen and surfactants. At 2 weeks after that has a good affinity for tissues (Fig. 1). the implantation, an extracellular matrix with Okumura et al. were the first to succeed in an alveolar structure (i.e., type I and type II the use of this artificial trachea for regen- alveolar epithelium and bronchioid patterns) eration of a 5-cm canine cervical trachea.1) had formed.4) Thus, even the lungs can be The host’s tissues replace the collagen on the formed if pulmonary cells are used under implanted artificial trachea while the mesh certain conditions. and stent integrate as being embedded with Apart from such attempts, studies aimed at the host’s tissues. Further, the epithelium regeneration of new lung tissues with a scaffold spreads from the stumps, and eventually, con- placed in the lungs have been conducted by tinuously covers the luminal surface. The use Itoi et al. in Japan.5) When a collagen sponge, of a Y-shaped collagen-conjugated prosthesis which serves as a scaffold for regeneration, is was demonstrated to be safe and effective in implanted in the lung of a rabbit, the implanted the reconstruction of the carina tracheae, collagen is gradually degraded and absorbed which is the most difficult operation in the and cells infiltrate it. On Day 5 after the opera- surgery of respiratory organs.2) The long-term tion, tubular structures had formed in the safety of carinal reconstruction has been con- border of the surrounding lung tissue and had

334 JMAJ, July 2004—Vol. 47, No. 7 REGENERATION OF THE LUNG USING IN SITU TISSUE ENGINEERING

lular matrix due to inflammatory cells. The strategy of treatment has been mainly suppression of inflammation, but recently, progress has been made in the areas of tissue repair and regeneration. The pathogenesis of pulmonary fibrosis are implicated in cytokines, such as fibroblast growth factor (FGF) and transforming growth factor (TGF)-␤, which are elaborated by inflammatory cells. Meanwhile, hepatocyte growth factor (HGF) inhibits the effects of these cytokines; intrapulmonary collagen levels are maintained via equilibrium between these factors. Degradation of extracellular matrix is Fig. 2 Tissue regenerated in the collagen sponge scaffold effected by matrix metalloproteinase (MMP) implanted in the lung and other proteases, and it has been demon- (Courtesy of Dr. S. Itoi, Institute for Frontier strated that the tissue level of MMP is elevated Medical Sciences, Kyoto University) Note the regenerated duct cells forming tubular structures in patients with pulmonary fibrosis. In view of continuous to adjacent normal regions. Photomicrograph at this, research in regenerative medicine is cur- 5 weeks after implantation. Normal lung tissue at left. Lung tissue can be regenerated with the new tissue engineering rently in progress to regenerate a normal lung technique. from fibrotic pulmonary structure by effective intrapulmonary administration of HGF and other cytokines and MMP.6) joined with normal lung tissue (Fig. 2). The Pulmonary Emphysema changes move in due course to the central portion of the implant, so that the implanted The number of the patients of pulmonary collagen scaffold becomes replaced by newly emphysema has been rising progressively; the regenerated lung tissue. The cells showing disease is the third or fourth leading cause of tubular structures are considered to be of Clara death in Western countries. Corticosteroids, cell or type II alveolar cell origin from the expectorants and bronchodilators prescribed results of the immunostaining examination. for the treatment of pulmonary emphysema The method of regenerating tissue in situ are aimed at suppressing the progression of the with a scaffold placed in the body is referred to disease. as in situ tissue engineering, and there is grow- Pulmonary emphysema is known to progress ing hope for its clinical application. In addition, due to imbalance between proteases and development of treatment methods to repair antiproteases in lung tissues. Accordingly, and regenerate lung tissues using growth fac- administration of such growth factors as HGF, tors and cells is also in progress as described EGF and TGF-␤, use of protease inhibitors below. to elastase, MMP and cathepsin, and use of inhibitors to all-trans retinoic acid (ATRA) Pulmonary Fibrosis and other neutrophil activators are being assessed as a drastic therapy.7) Medication It is now generally recognized that pul- with these substances represents an attempt monary fibrosis arises from progressive lung to regenerate pulmonary tissues by stimulat- remodeling caused by proliferating extracel- ing local stem cells that may remain in the

JMAJ, July 2004—Vol. 47, No. 7 335 T. NAKAMURA

ᕃᕄ

Alveoli Alveoli

Primary pulmonary Blood EPC injection hypertension vessel

ᕅᕆ

Blood vessel regeneration Regenerated blood vessels

Fig. 3 Shema showing the new treatment method for primary pulmonary hypertension Endothelial progenitor cells (EPC) collected from autologous peripheral blood and grown in vitro are injected directly into the lung to allow neoangiogenesis for improvement of pulmonary hypertension.

damaged lung. an invariably fatal outcome in 2–10 years (aver- Studies in this ﬁeld had been conﬁned to age: 5 years). While lung transplantation is laboratory investigations using small rodents, used to treat this disease mainly in Western therefore, there is a wide gap from the situation countries, a novel treatment based on regen- in clinical settings. Toba et al. demonstrated erative medicine has been devised and is regeneration from emphysematous lung tissues attracting attention. Endothelial progenitor with an improvement in pulmonary function by cells (EPC) concerning angiogenesis have been trans-bronchoscopic administration of TGF-␤ demonstrated to be present in a peripheral in a canine (beagle) model of pulmonary blood cell subpopulation called peripheral emphysema induced with intra-airway elastase blood mononuclear cells (PBMCs). Autolo- spraying, thus taking a distinct step toward gous EPC isolated from the peripheral blood of clinical application.8) the patient and grown in culture are injected into the lung to regenerate new blood vessels in Primary Pulmonary Hypertension (PPH) the lesion for treatment of PPH (Fig. 3). Takahashi et al. trans-bronchoscopically in- Primary pulmonary hypertension is a disease jected autologous EPC that had been isolated with an extremely poor prognosis that is char- from the peripheral blood and grown in vitro acterized by incipient symptoms of shortness of into the lung tissue of a canine PPH model. This breath on exertion and right cardiac failure and resulted in regeneration of new blood vessels

336 JMAJ, July 2004—Vol. 47, No. 7 REGENERATION OF THE LUNG USING IN SITU TISSUE ENGINEERING

in the lung of the dog, which then showed Organs 2000; 23: 718–724. improvement of cardiac function, which had 4) Cortiella, J., Vacanti, C.A. and Vacanti, M.P.: been depressed due to pulmonary hyperten- Tissue engineered lung. Tissue Eng 2000; 16: sion.9,10) The pioneering results from Japan have 661. 5) Itoi, S., Nakamura, T. and Shimizu, Y.: In situ shed a great ray of hope for the treatment of tissue engineering for lung regeneration using PPH, for which lung transplantation has been a collagen sponge scaffold. ASAIO J 2001; 47: the only curative treatment available. 173. 6) Yaekashiwa, M., Nakayama, S., Nukiwa, T. et Conclusion al.: Simultaneous or delayed administration of hepatocyte growth factor equally represses With the advent of the twenty-first century, the fibrotic changes in murine lung injury regenerative medical care has been steadily induced by bleomycin. A morphologic study. applied in clinical settings to respiratory dis- Am J Respir Crit Care Med 1997; 156: 1937– 1944. eases as well. A new treatment modality is thus 7) Barnes, P.J.: Novel approaches and targets for being developed even for disorders for which treatment of chronic obstructive pulmonary no radical treatment has been available. disease. Am J Respir Crit Care Med 1999; 160: S72–S79. REFERENCES 8) Toba, T., Takahashi, M., Fukuda, M. et al.: A study on bronchoscopic preparation of dog 1) Okumura, N., Nakamura, T., Natsume, T. et al.: models for interstitial pneumonia and emphy- Experimental study on a new tracheal pros- sematous lungs. Jpn J Chest Surg 2002; 16: 473. thesis made from collagen-conjugated mesh. (in Japanese) J Thorac Cardiovasc Surg 1994; 108: 337–345. 9) Takahashi, M., Nakamura, T., Toba, T. et al.: 2) Sekine, T., Nakamura, T., Matsumoto, K. et New therapeutic option for primary pul- al.: Carinal reconstruction with a Y-shaped monary hypertension utilizing endotherial collagen-conjugated prosthesis. J Thorac progenitor cells. ASAIO J 2002; 48: 188. Cardiovasc Surg 2000; 119: 1162–1168. 10) Takahashi, M., Nakamura, T. and Katou, H.: 3) Nakamura, T., Teramachi, M., Sekine, T. et al.: Transplantation of endothelial progenitor cells Artificial trachea and long term follow-up in into the lung to alleviate pulmonary hyperten- carinal reconstruction in dogs. Int J Artif sion in dogs. Tissue Engineering in press.

JMAJ, July 2004—Vol. 47, No. 7 337 ⅥCentenarians

Implications of Research Findings Obtained from Centenarians

JMAJ 47(7): 338–343, 2004

Hiroshi SHIBATA

Professor of Gerontology, Graduate School of Obirin University

Abstract: The number of centenarians in Japan has increased more than a hundred times over the past four decades, reaching nearly 20,000 persons currently. The rate of centenarians in the population is higher in the western part of Japan, where mortality from stroke is low. Centenarians generally have nutritionally well-balanced diets that are rich in animal food and vegetables. They tend to be extroverted, optimistic, hard working, and scrupulous. A recent national survey on centenarians revealed that they have a positive view of their health status, although the degree of independent functioning in activities of daily living is low. Key words: Long life; Lifestyle; Quality of life

Introduction Caucasus, another of the areas famous for its inhabitants’ longevity, can also be ruled out, According to the author’s research, the longest- because the demographic statistics of the lived person in Japan has reached the age of former Soviet Union were problematic. 116 years. Although some records of longer- Thus, according to reliable statistics, the lived individuals have been found, they all human life span appears to be less than 120 tended to be ﬂawed for one reason or another.1) years. Therefore, centenarians are those who Since Japan has an excellent system of cen- have lived nearly to the limit of the human life sus taking, reports of the existence of very span, and their existence provides hope for long-lived individuals in other societies also the longevity of humankind. In Japan, several are unlikely to be trustworthy. In fact, it national surveys on centenarians have been was revealed by a US research group that carried out, in addition to various regional not a single centenarian had been found in surveys. A review of such surveys has been Vilcabamba, an Ecuadoran city in South reported by Shinkai, who indicated the direc- America, which has been considered one of the tion of future research.3) world’s three main areas of longevity.2) The Most of the previous studies of centenarians

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 128, No. 7, 2002, pages 1075–1079). The Japanese text is a transcript of a lecture originally aired on April 15, 2002, by the Nihon Shortwave Broadcasting Co., Ltd., in its regular program “Special Course in Medicine”.

338 JMAJ, July 2004—Vol. 47, No. 7 RESEARCH IN CENTENARIANS

1920 113 1925 187 1930 105 1935 228 1940 185 1947 55 1950 97 1955 158 1960 144 1963 153 1964 191 1965 198 1966 252 1967 253 1968 327 1969 331 1970 310 1971 339 1972 405 1973 495 1974 527 1975 548 1976 666 1977 697 1978 792 1979 937 1980 968 1981 1,072 1982 1,200 1983 1,354 1984 1,563 1985 1,740 1986 1,851 1987 2,271 1988 2,668 1989 3,078 1990 3,298 1991 3,625 1992 4,152 1993 4,802 1994 5,593 1995 6,378 1996 7,373 1997 8,491 1998 10,158 1999 11,346

0 2,000 4,000 6,000 8,000 10,000 12,000 No. of centenarians

Sources: National census (1920–1960) and surveys by the Elderly People’s Welfare Division, Social Affairs Bureau, Ministry of Health and Welfare (since 1963).

Fig. 1 Changes in the number of centenarians7) were case studies that attempted to determine activities, focused on the quality of cente- factors involved in longevity.4) However, the narians’ lives.5,6) In this study, which was project known as the national cross-sectional conducted under the Ministry of Education, study of 1/2 samples of centenarians, which Culture, Sports, Science and Technology, the was part of the study on regional systems to aid author and other researchers attempted to the elderly in maintaining or improving daily address the day-to-day needs of the elderly. living and to promote participation in social

JMAJ, July 2004—Vol. 47, No. 7 339 H. SHIBATA

Centenarian rate Number of centenarians per elderly population of 100,000 81~203 (5) 47~53 (7) 70~81 (4) 43~47 (4) 65~70 (4) 41~43 (3) 59~65 (5) 38~41 (7) 53~59 (3) 31~38 (5) Number of prefectures in parentheses

Fig. 2 Centenarian rate by prefecture (1999)7)

1) Historic Changes and Geographic 110 years, with little change over the years. Distribution of Centenarians This underscores the author’s view that the outer margin of longevity for humans is about Figure 1 shows changes in the number of this level. centenarians in Japan.7) Statistical data on cen- The geographic distribution of centenarians tenarians became more accurate after 1963, in Japan shows that the western part of the when the Old-Age Persons’ Welfare Law was country fares better in producing them.7) The enacted on their behalf. The latest data show distribution of centenarians shows an inverse that centenarians total nearly 20,000 individu- correlation with mortality from stroke, and, in als, representing more than a 100-fold increase relation to longevity, there is a closer correla- from 1963. Interestingly, despite this dramatic tion with mean life expectancy at age 65 than increase in centenarians, there has been no that at age 0. When comparing the distribution tendency for the upper age limits to increase, of centenarians according to prefecture, the and no one has exceeded 116 years of age. The rate of centenarians to the total prefectural highest age of centenarians has remained about population is often used, but, for fair compari-

340 JMAJ, July 2004—Vol. 47, No. 7 RESEARCH IN CENTENARIANS

(%) 20 16.9 16.0 (%) 15 14.6 75

59.6 57.6 48.7 10 50

5 25

0 0 Overall average in Japan Male Female Overall average in Japan Male Female Average in centenarians Average in centenarians Fig. 3 Comparison of the proportion of protein calories Fig. 4 Comparison of the proportion of animal protein to total calorie intake4) to total protein intake4)

son, this measure requires adjustment by the associate calorie restriction with long life, con- rate of individuals over the age of 65. There- sidering the fact that average height is greater fore, use of the rate of centenarians to the in eastern Japan. The proportion of animal pro- prefectural population of those 65 years old tein to total protein intake in persons in their or older is appropriate. Figure 2 shows the rate 60s showed a signiﬁcant positive correlation of centenarians to the population of those with the rate of centenarians. However, this 65 years old or older by prefecture in 1999, index has a geographical distribution similar to according to our calculations. The number of that of temperature, so that careful multivari- centenarians per population of 100,000 aged ate analysis may be necessary before causality 65 or older was highest in Okinawa (202.8), can be discussed. followed by Kochi (113.4), Kumamoto (92.7), Shimane (91.4), and Kagoshima (82.7). At the Dietary and Lifestyle Habits of other end of the spectrum, the corresponding Centenarians rate was lowest in Saitama (31.9), Aomori (32.1), Ibaraki (34.7), Shiga (35.9), and Akita Our national survey carried out in 1972 (36.5). and 1973 provided a detailed inquiry into the An analysis of factors determining the geo- eating habits of centenarians for the purpose of graphical distribution of centenarians yielded nutritional analysis.4) Probably because cente- interesting results.6) There was a strong corre- narians had a smaller physique and were less lation between average temperature and the active, total daily calorie consumption was ,pϽ0.001). How- 1,073 kcal for males and 939 kcal for females ,0.740סrate of centenarians (r ever, the rate of centenarians was higher in about half the corresponding totals for the gen- Hokkaido, where the average temperature was eral Japanese population. Data on the overall lowest, than in the entire Tohoku area, indicat- Japanese population were derived from the ing that the outside temperature is not neces- 1972 National Dietary Survey. Interestingly, as sarily a decisive factor. Although there was an shown in Fig. 3, the rate of calories derived inverse correlation with total calorie intake at from protein to total calories was higher in 60–69 years of age, it may be premature to centenarians than in the general population.

JMAJ, July 2004—Vol. 47, No. 7 341 H. SHIBATA

Namely, they consumed a low-calorie, high- With regard to alcohol consumption, 45% protein diet. More surprisingly, the proportion of males and 77.6% of females were never of animal protein to total protein intake in drinkers, whereas 31.1% of males and 14.9% of centenarians was much higher than that in the females were past drinkers, and 21.9% of males general population (Fig. 4). These data indi- and 5.8% of females were current drinkers. cated a diet almost opposite to a vegetarian Thus, habitual drinking was more common diet. When data collection was limited to cente- than habitual smoking, consistent with data narians with good memory function, we found from previous studies on long life in various that their eating habits before the war were parts of the world. similar to those of the general population, indicating that these centenarians altered their Characteristics and Personalities of eating habits after reaching their middle 70s, Centenarians almost as if they took the lead in the westernization of Japan’s eating habits. In our survey, data on the characteristics of The tendency of centenarians to adopt 80 centenarians in their younger days were positive, well-balanced eating habits has also obtained mainly from their families. It was been demonstrated by survey research con- found that they tended to be extroverted ducted by the Japan Health Promotion & Fit- (81.2%), cheerful and friendly, and optimistic, ness Foundation.8) while they were also hard workers and scrupu- The 1993 survey by this foundation included lous.10) A national survey carried out later in the exercise habits of centenarians.9) The cente- 1975 also revealed similar features.11) However, narians lived through the era dominated by the it is unclear to what extent these character and nation’s policy of increasing wealth and mili- personality traits are peculiar to centenarians. tary power, and their occupational life chiefly consisted of physical labor. Therefore, it is pre- Quality of Centenarians’ Lives sumed that they began to exercise for pleasure only after retirement. In the 1993 survey, 52% Our recent study of centenarians provided a of centenarians replied that they exercised, and better understanding of the quality of their 43% of them replied that they exercised daily. lives.6) Many of them had a decreased current Specifically, “walking” was predominant for level of functioning. In fact, 22.2% of men and both men and women, followed by “field 41.1% of women fell under the category of labor/weeding/caring for plants”, “gymnastic “bedridden”. This, however, is understandable: exercise”, and “work”. Compared with cente- nearly one-half of the centenarian population narians who had no regular exercise, cente- is replaced every year, indicating that those in narians who engaged in regular exercise were the terminal stage of life accounted for a con- better in activities of daily living (ADL), siderable proportion at the time of investiga- showed more interest in hobbies, associated tion. However, it is apparent that they have led more actively with friends, and had eating an independent life for most of their existence, habits that were improved from those in with only a few people bedridden for a pro- middle age. longed period. Therefore, they have a generally According to our most recent survey on good view of their health status. As shown in centenarians, 63.8% of male centenarians and Fig. 5, as many as 74.7% of men and 62.5% of 93.2% of female centenarians were never women feel that they are in good health. smokers. However, 30.7% of males and 6% of Those who had a hobby accounted for 46.6% females were past smokers, and 5.6% of males of men and 26.9% of women, which seemed to and 0.8% of females were current smokers.6) be correlated with the degree of independent

342 JMAJ, July 2004—Vol. 47, No. 7 RESEARCH IN CENTENARIANS

(1,341סFemale] (n] (566סMale] (n] (%) (%) 100 80 60 40 20 0 0 20 40 60 80 100 Do you feel that you are in (1,011סgood health? 62.5 (n 74.7 (494סn) (Yes) Do you feel good on (999סmost days? 77.4 (n 83 (489סn) (Yes) Are you often in (1,005סa poor physical condition? 68.5 (n 72.2 (492סn) (No)

Fig. 5 Centenarians self-rated health6)

functioning in life. A total of 43.6% of men and J Gerontol 1979; 34: 94–98. 25.8% of women found their lives worth living, 3) Shinkai, S.: Investigation of health and welfare whereas 28.4% of men and 33.6% of women in centenarians. Report of the Study on Mental did not; 27.9% of men and 40.6% of women and Physical Health Promotion for Elderly People to Live a Free and Independent Life. were unclear. It is difﬁcult to obtain accurate Japan Health Promotion & Fitness Founda- answers from centenarians with intellectual tion, 1999; pp.173–207. (in Japanese) impairment. 4) Shibata, H., Haga, H., Nagai, H. et al.: Nutri- Understandably, few centenarians partici- tion for the Japanese elderly. Nutr Health pate in social activities. Only 20.5% of men and 1992; 8: 165–175. 11.6% of women are involved in activities such 5) Uemura, M., Ogihara, R., Shibata, H. et al.: as work and volunteer service. However, such The effects of experiencing a fall on the activi- active centenarians accounted for a relatively ties of daily living (ADL) in Japanese cente- high proportion among those who are function- narians. Jpn J Prim Care 2002; 25: 8–18. 6) Japan Health Promotion & Fitness Founda- ing independently in life. tion: Lives of Japanese Centenarians; 2001. (in The presence of centenarians contributes to Japanese) society through their embodiment of human 7) Watanabe, S. and Shibata, H.: Centenarians in longevity, their role as living witnesses to his- Japan: Geographic distribution of centenar- tory, and their ability to transmit wisdom to the ians. Geriatr Med 2000; 38: 1269–1276. (in next generation. The 21st century is a time of Japanese) mutual cooperation, in which the contribution 8) Japan Health Promotion & Fitness Foun- of the elderly to society is indispensable, as is dation: 1981 Report of the Health and Nutri- tion Survey on Long-lived People; 1982. (in the support of the younger generation in caring 12) Japanese) for society’s elders. The contribution made by 9) Japan Health Promotion & Fitness Foun- the elderly to society is broad in scope, and the dation: 1993 Report of the Survey on Health way centenarians contribute to society has and Welfare of Long-lived People; 1995. (in great signiﬁcance. Japanese) 10) Tokyo Metropolitan Institute of Gerontology: Report of the Survey on Centenarians; 1975. REFERENCES (in Japanese) 11) Elderly People’s Welfare Development Cen- 1) Shibata, H.: Long Life, Peaceful Death—Live ter: Integrated Study Report of Long-lived long happily and die happily. Hokendojinsha People; 1976. (in Japanese) Co., Ltd., Tokyo, 1994. (in Japanese) 12) Shibata, H.: More than 80% of the Elderly Are 2) Mazess, R.B. and Forman, S.H.: Longevity and Independent! Business-Sha, Tokyo, 2002. (in age exaggeration in Vilcabamba, Ecuador. Japanese)

JMAJ, July 2004—Vol. 47, No. 7 343 Ⅵ Abdominal Aortic Aneurysm

Forefront of Treatment for Abdominal Aortic Aneurysm

JMAJ 47(7): 344–349, 2004

Hiroshi YASUHARA

Professor, Teikyo University School of Medicine, Ichihara Hospital

Abstract: This paper is a comprehensive review of the latest treatments for abdominal aortic aneurysm, with primary reference to endovascular stent graft placement. While there has already been an established operative procedure for the treatment of abdominal aortic aneurysms, i.e. surgical excision of aortic aneurysm upon laparotomy and vascular replacement with prostheses, further developments are under way toward minimally invasive approaches with emphasis on quality of life of patients. Most noteworthy is the advent of endovascular stent graft placement, which has been introduced into the clinical setting; there are already reports of the short-term results of the procedure. On the other hand, new problems peculiar to endovascular stent graft placement have been emerging, leading to various consequences. The problems may be roughly categorized as: 1) problems related to surgical techniques, 2) problems involving the surgeons conducting the treatment, and 3) medicoeconomical problems. These problems should be resolved by further assessments from the standpoint of patient primacy. Key words: Abdominal aortic aneurysm; Endovascular stent graft placement; Minimally invasive surgery

Introduction the vascular replacement with prostheses currently being undertaken has become estab- The history of surgical operation in the treat- lished as a standard procedure. Materials used ment of abdominal aortic aneurysm began with for vascular prostheses are usually of two types, the attempt made by Charles Dubost et al.1) i.e., the nonwoven fiber ePTFE and the knitted involving vascular allografting upon resection synthetic Dacron. Dacron tubes sealed with of an aortic aneurysm. Through the subsequent such readily decomposed and absorbable sub- improvement of operative techniques and medi- stances as fibrin, albumin, or gelatin in order cal materials over the past five decades or so, to prevent blood leakage have come into fre-

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 128, No. 11, 2002, pages 1651–1655). The Japanese text is a transcript of a lecture originally aired on June 6, 2002, by the Nihon Shortwave Broadcasting Co., Ltd., in its regular program “Special Course in Medicine”.

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quent use recently. Therefore, the need for the conventional process of preclotting has been diminishing. The current surgical procedure for treating abdominal aortic aneurysms may well be said to be almost completed, although the medical materials being used are still being improved little by little. The high degree of perfection of the procedure is reflected in the low postoperative mortality rate; reports documenting postoperative mortality rates of ͨ1% have become standard in recent years, compared to the previous 5 to nearly 10%. Surgery-related Conventional operation Endovascular stent grafting deaths are reportedly nil at some institutions. Fig. 1 Comparison of surgical wounds Nevertheless, this apparently perfected operative procedure has recently been undergoing innovative changes through endovascular stent grafting incorporating endovascular surgical pubes (Fig. 1). techniques. This article will briefly review the A folded prosthetic graft is led, as it stands, endovascular stent grafting process, and con- into the lumen of an abdominal aortic aneu- sider changes currently occurring at the fore- rysm through the exposed femoral artery or front of treatment for abdominal aortic aneu- iliac artery. This article will not pertain to rysms and the problems inherent therein. individual types of stent grafts. The artificial graft is made of thinner knitted Dacron or Stent Graft Endovascular Placement for ePTFE so that it can be folded into a fine Repair of Abdominal Aortic Aneurysm compact form, and is reinforced with a stent to and Its Characteristic Features endow the vessel with strength when unfolded. The graft led into the lumen of an abdominal Endovascular surgery originates from the aortic aneurysm is deployed in an undistended percutaneous transluminal angioplasty (PTA) aortic region proximal to the aneurysm, and is initiated by Dotter and Jedkins2) and is aimed fixed fast with the stents to the sites of origin at treatment of vascular lesions with minimal and terminus of the aortic aneurysm. There- operative invasion, mainly using catheters. The by, the stent graft is implanted inside of the major distinctive feature of stent graft place- aneurysm (Fig. 2). ment with the application of the techniques of Formerly, stent grafts were available only in endovascular surgery, compared to the conven- straight form and, therefore, surgical excision tional surgery for abdominal aortic aneurysm, with grafting was indicated only for repair of consists in its minimal invasiveness, which places aortic aneurysms localized in the abdominal a great deal less pain and stress on the patient. aorta without involving distention of the iliac The surgical wound involved in stent grafting artery. Recently, however, abdominal aortic measures only a few centimeters extending to aneurysms with associated iliac artery disten- expose the unilateral or bilateral femoral artery tion have also become subject to the treatment or iliac artery; whereas in the conventional thanks to methods for combining two stent operative procedure, the wound extends much grafts. A variety of systems of graft anchorage farther, from just below the xiphoid process with stents have been devised, with two differ- down to just above the superior margin of the ent endoluminal fixation systems in current use,

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Table 1 Problems Inherent in Endovascular Stent Grafting

1) Problems related to the operative technique per se 2) Problems related to the doctor conducting the operation 3) Problems related to medicoeconomics

procedures. Reports have documented an earlier start to oral dietary intake after the operation, and earlier leaving of the sickbed and hospital discharge owing to the lower severity of the surgical wound in patients treated with endoluminal stent grafting, as compared with those treated by conventional operative techniques. Consequently, the duration of the patient’s hospital stay is signiﬁcantly reduced.

Fig. 2 Endovascular stent grafting Points at Issue Inherent in Endoluminal Stent Grafting

From what has been described herein, one i.e. the anchoring of the stent onto the arterial might have the impression that endoluminal wall by means of inflating a balloon from inside stent grafting will quite naturally supplant the the stent, and that by the self-expansive force conventional surgery, which is highly invasive. of the stent spring per se. Thus, stent grafting is Unfortunately, however, several problems aimed at treatment of aortic aneurysm by inter- have been pointed out regarding this surgical cepting blood flow in the space between the approach, and may well hinder the spread of external surface of a prosthetic vessel wall and the transluminal procedure. the inner surface of the wall of the abdominal The points at issue in endoluminal stent aortic aneurysm, and by forming thrombosis grafting are summarized in Table 1. They within the blood flow interception space (Fig. 2). include 1) problems related to the operative Although the physical burden upon the patient technique per se, 2) those related to the doctor can be substantially reduced, the degree of conducting the operation, and 3) those related invasiveness into the vasculature itself does not to medicoeconomics. Needless to say, these much differ from that of other procedures, problems are in no way independent, but are since artificial graft and stents are used in the mutually and closely interrelated. However, procedure of endoluminal stent grafting. As summarization in this fashion may facilitate the mentioned above, the operation of endoluminal readers’ understanding of the problems in the stent grafting requires no laparotomy, whilst a practice of endoluminal stent grafting. laparotomy entailing a large surgical wound has to be carried out in usual surgical treatment 1. Problems related to the operative of abdominal aortic aneurysms. The postopera- technique per se tive clinical course differs greatly between the Let me refer first to problems related to the

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Type 2 endoleak due Renal artery is ascribed to collateral blood ﬂow such as that to blood flow from lumbar artery or from the lumbar artery. inferior mesenteric Recently, the ﬁxation system for the proxi- artery mal end of the stent graft has been improved so 0 0 Abdominal aorta Type 1 that the stent may even approach the takeoff of 00 endoleak due 3) Durability of to inadequate the renal arteries, yet the problem of “endo- stent graft graft fixation leak” has not been fully resolved.4) Moreover, it is known that type 1 blood leakage may also

Injury to artery Iliac artery occur from the peripheral fixation site or from on graft the graft junction site of a combined Y-shaped insertion stent graft, besides blood flow persistence from Fig. 3 Problems in endovascular stent grafting for the proximal end of the stent graft (Fig.3). abdominal aortic aneurysm The various problems described here pertain particularly to operative techniques and early postoperative complications. Due to the relatively short history of endoluminal stent graft- operative technique per se. In the conventional ing (which covers about a decade), long-term standard procedure of replacement of an aortic results are yet to be scrutinized. In fact, some aneurysm with a synthetic graft, as mentioned cases have been reported in which a stent graft above, the aortic blood flow is intercepted and placed in a markedly tortuous abdominal aortic then the aortic aneurysmal wall is incised, fol- aneurysm caused a gradual further advance in lowed by placing sutures on the lumbar artery tortuosity, eventuating in aortic perforation by for hemostasis. No such hemostasis by suturing the stent; and other cases have been reported is required in the endoluminal stent grafting; where an aneurysm continued to expand due to hence, in some cases, blood flow from the lum- “endoleak” postoperatively, resulting in rup- bar artery remains in the space surrounded by ture of the aneurysm.5,6) It is considered that the the stent graft and the wall of the abdominal durability of the synthetic vascular graft must aortic aneurysm where the blood flow should be fully assessed in the future, because a thin be intercepted as intended. graft material is utilized so as to facilitate inser- Such blood flow persistence may occur due tion of a stent graft through small arteries.6) to other causes as well. Aneurysms of the abdominal aorta arise in most cases in the seg- 2. Problems related to the doctor conducting ment inferior to the takeoff of the renal arteries the treatment and, as the site of aneurysmal origin may be Now I would like to discuss the problems distal to that of the takeoff, the proximal end of related to the doctors’ specialty conducting endo- the inserted graft is fixed with the stent onto vascular stent grafting. The status quo is that the undistended site of the aorta. In a case the treatment is undertaken by cardiovascular where the distance is too short, however, the surgeons or medical radiologists with their own graft may cause occlusion of the renal artery, respective procedures, without active mutual or fixation of the stent may become unstable, intercommunication. Endovascular stent graft- resulting in persistent blood flow into the ing for treatment of abdominal aortic aneu- aneurysm by the side of the stent. Such blood rysm is likewise performed by vascular sur- flow persistence within the aneurysm is termed geons as well as by medical radiologists in the “endoleak.” It is classified into two types: United States, where there is an advanced medi- “type 1 endoleak”, which is attributable to the cal specialty board system. Even in the United stent graft itself, and “type 2 endoleak”, which States, however, surgeons formerly regarded it

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as heretical that this treatment was not per- itself directly indicate a low degree of perfec- formed by one of themselves. Among radiolo- tion of the treatment, but what we ought to gists, on the other hand, this treatment was take note of here is that there are also other generally recognized as being performed by factors contributory to the use of various medi- radiologists, since they had gained substantial cal materials in this treatment. In other words, results with treatments involving catheteriza- the stent graft to be used in the present endo- tion as an interventional radiological approach. vascular stent grafting must be individualized/ With the improvement in therapeutic outcomes tailored for a given patient, so that the charac- with endovascular stent grafting achieved dur- teristics and disease state of the patient, such ing the last several years, however, the recog- as the build, size and tortuosity of the aorta, nition that this treatment should be carried patency of the lumbar artery, and so forth out by vascular surgeons is increasingly being largely influence the therapeutic outcome. Endo- accepted among vascular surgeons, as they have vascular stent grafting is basically a tailor-made long-standing experience in operations on therapy, and consequently the medical materi- abdominal aortic aneurysms. Recently, cardio- als used and the medical fees involved are vascular medical interventionalists experienced highly expensive. in PTCA and stenting of the coronary artery With endovascular stent grafting, a signifi- have also been participating in this realm. The cant reduction in the duration of the hospital situation as to what type of professional train- stay can be anticipated as compared to lapa- ing is required for a physician to conduct this rotomic corrective procedures, and this may treatment is now chaotic. help to reduce the treatment fee. It is difficult, Such steering of patients from the viewpoint however, to estimate the medico-economical of the physician, however, is not by any means implications of endovascular stent grafting, favorable to the patient. Many surgeons feel because the procedure involves the use of that the procedure should be performed by expensive, cumbersome medical materials. physicians adequately trained in surgery, just in Although it is unquestioned that the cost of the case of laparoscopic cholecytectomy, which medical materials will gradually decrease with came to be performed by surgeons versed in the future spread of this treatment method, it the knowledge and techniques of laparotomic remains to be clarified to what extent the cost cholecystectomy. From an entirely different will be reduced. viewpoint, however, there are surgeons who point out the possible advent of endovascular Other New Treatments for Abdominal surgeons distinct from the conventional con- Aortic Aneurysm cept of the cardiovascular surgeon. Solution of this problem regarding the training required I have reviewed endovascular stent graft- for this new treatment modality should thus be ing as a therapy for abdominal aortic aneu- sought urgently after candid discussions. rysm, and considered the problems inherent in the therapy. Now, from a different angle, 3. Problems related to medicoeconomics we would like to briefly review other attempts Finally, I would like to discuss the problems towards surgical treatment for abdominal related to medicoeconomics as a subject im- aortic aneurysms. plicit in endovascular stent grafting. In the field of surgery, attempts at minimally In this therapeutic approach, medical mate- invasive surgical interventions utilizing laparo- rials that have not yet been standardized are scopy or robots have been actively undertaken used as mentioned above. The fact that a wide in recent years. These attempts are designed variety of medical materials are used does in to perform operative manipulations beyond

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human capability, with the aid of surgical in- altering the conventional surgical treatment, struments to minimize surgical invasiveness. and the treatment for abdominal aortic aneu- Laparoscopes and robots are capable of enter- rysms is considered to be continuing to prog- ing an incised wound which is so fine and small ress. It is also anticipated that, in the future, that human fingers cannot pass through it, and abdominal aortic aneurysms be prevented by enable operations within the body. Operations medicinal and dietary therapy upon elucida- such as endoscopic gastric mucosectomy are tion of the pathogenetic mechanisms of athero- considered an ultimate target, and can be con- sclerosis based on analysis of genetic infor- ducted without leaving any wound on the body mation, rather than by placing emphasis on surface. treatments involving surgical intervention. In the surgery for abdominal aortic aneurysms, as well, attempts at minimal incision REFERENCES surgery to carry out conventional vascular replacement with a synthetic prosthesis through a 1) Dubost, C., Allary, M. and Oeconomos, N.: smaller surgical wound are being made with Resection of an aneurysm of the abdominal aorta. Arch Surg 1952; 64: 405–408. the improvement of the retractor or with the 2) Dotter, C.T. and Jedkins, M.P.: Transluminal application of laparoscopic surgical instru- treatment of atherosclerotic obstruction: De- ments. Although intra-abdominal operations scription of a new technique and preliminary remain the same as with conventional proce- report of this application. Circulation 1969; 30: dures and may therefore differ little from the 645–670. latter in surgical invasiveness in the strict sense 3) Bove, P.G., Long, G.W., Zelenock, G.B. et al.: of the term, it seems highly likely that the Transrenal fixation of aortic stent-grafts for physical burden on the patient may be sub- the treatment of infrarenal aortic aneurysmal stantially reduced by minimizing laparotomic disease. J Vasc Surg 2000; 32(4): 697–703. 4) Wisselink, W., Cuesta, M.A. and Berends, F.J. manipulations, as in the case of laparoscopic et al.: Retroperitoneal endoscopic ligation of cholecystectomy. For those surgeons skilled in lumbar and inferior mesenteric arteries as a conventional operative procedures, further- treatment of persistent endoleak after endo- more, the treatment is considered to have the luminal aortic aneurysm repair. J Vasc Surg advantage of being more acceptable. 2000; 31(6): 1240–1244. 5) Guidoin, R., Marois, Y., Douville, Y. et al.: First-generation aortic endografts: analysis of Conclusion explanted Stentor devices from the EURO- STAR Registry. J Endovasc Ther 2000; 7(2): I have reviewed surgical treatments such as 105–122. endovascular surgery with stent grafting in 6) Beebe, H.G., Kritpracha, B., Serres, S. et al.: their roles at the forefront of therapy for ab- Endograft planning without preoperative dominal aortic aneurysms. Endovascular sur- arteriography: a clinical feasibility study. J gery with stent grafting has been dramatically Endovasc Ther 2000; 7(1): 8–15.

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