Raymund E. Horch

Tissue engineering of bone: the reconstructive surgeon's point of view.

Bone defects represent a medical and socioeconomic challenge. Different types of biomaterials are applied for reconstructive indications and receive rising interest. However, autologous bone grafts are still considered as the gold standard for reconstruction of extended bone defects. The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Tissue engineering is, according to its historic definition, an “interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function”. It is based on the understanding of tissue formation and regeneration and aims to rather grow new functional tissues than to build new spare parts. While reconstruction of small to moderate sized bone defects using engineered bone tissues is technically feasible, and some of the currently developed concepts may represent alternatives to autologous bone grafts for certain clinical conditions, the reconstruction of largevolume defects remains challenging. Therefore vascularization concepts gain on interest and the combination of tissue engineering approaches with flap prefabrication techniques may eventually allow application of bone‐tissue substitutes grown in vivo with the advantage of minimal donor site morbidity as compared to conventional vascularized bone grafts. The scope of this review is the introduction of basic principles and different components of engineered bioartificial bone tissues with a strong focus on clinical applications in reconstructive surgery. Concepts for the induction of axial vascularization in engineered bone tissues as well as potential clinical applications are discussed in detail.

Tissue Engineering of Cultured Skin Substitutes

Skin replacement has been a challenging task for surgeons ever since the introduction of skin grafts by Reverdin in 1871. Recently, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue‐engineered living skin replacements. This has been fostered by the dramatically improved survival rates of major burns where the availability of autologous normal skin for grafting has become one of the limiting factors. The ideal properties of a temporary and a permanent skin substitute have been well defined. Tissue‐engineered skin replacements: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts, autologous/allogeneic composites, acellular biological matrices, and cellular matrices including such biological substances as fibrin sealant and various types of collagen, hyaluronic acid etc. have opened new horizons to deal with such massive skin loss. In extensive burns it has been shown that skin substitution with cultured grafts can be a life‐saving measure where few alternatives exist. Future research will aim to create skin substitutes with cultured epidermis that under appropriate circumstances may provide a wound cover that could be just as durable and esthetically acceptable as conventional split‐thickness skin grafts. Genetic manipulation may in addition enhance the performance of such cultured skin substitutes. If cell science, molecular biology, genetic engineering, material science and clinical expertise join their efforts to develop optimized cell culture techniques and synthetic or biological matrices then further technical advances might well lead to the production of almost skin like new tissue‐engineered human skin products resembling natural human skin.

Skeletal muscle tissue engineering

The reconstruction of skeletal muscle tissue either lost by traumatic injury or tumor ablation or functional damage due to myopathies is hampered by the lack of availability of functional substitution of this native tissue. Until now, only few alternatives exist to provide functional restoration of damaged muscle tissues. Loss of muscle mass and their function can surgically managed in part using a variety of muscle transplantation or transposition techniques. These techniques represent a limited degree of success in attempts to restore the normal functioning, however they are not perfect solutions. A new alternative approach to addresssing difficult tissue reconstruction is to engineer new tissues. Although those tissue engineering techniques attempting regeneration of human tissues and organs have recently entered into clinical practice, the engineering of skletal muscle tissue ist still a scientific challenge. This article reviews some of the recent findings resulting from tissue engineering science related to the attempt of creation and regeneration of functional skeletal muscle tissue.

Translating tissue engineering technology platforms into cancer research

•  Introduction •  History of tissue engineering •  Physiological and structural aspects of 2D versus 3D culture in cancer research •  State of the art of 3D culture systems in cancer research •  New tissue engineering‐routed scaffolds for 3D culture •  Endothelial progenitor cells and tumour vasculature •  In vivo models •  Arteriovenous loop isolation chamber for tumour angiogenesis research •  Conclusion

Cultured Human Keratinocytes on Type I Collagen Membranes to Reconstitute the Epidermis

The development of new techniques and modifications to overcome some of the disadvantages in cultured keratinocyte grafting has been motivated by several well-known drawbacks in the use of cultured epithelial autografts such as long culture periods, lack of adherence, difficulty in handling, lack of dermal substrates, and high costs. Two recent insights have influenced further research. On the one hand, it has been shown that the use of undifferentiated proliferative cells in fibrin glue suspensions is effective in epithelial reconstitution. On the other hand, the enzymatic release of cells from the culture surfaces is a critical step leading to at least temporary destruction of anchoring structures of the cultured cells. In this study, we tried to combine these two aspects in an attempt to modify common modalities of keratinocyte transplantation. To avoid dispase dissolving of the cultured cells, keratinocytes were seeded onto bovine collagen type I membranes without feeder layers and under serum-free culture conditions. Subconfluent monolayers of cultured human keratinocytes were transplanted as an upside-down graft on collagen membranes (keratinocyte collagen membrane grafts [KCMG], n = 12) after 3 days of culture or as membrane grafts alone (n = 12) onto standard nude mice full-thickness wounds. Fully differentiated epidermis was found at 21 days after grafting KCMG with persistence of human keratinocytes. This study demonstrates that upside-down grafts of undifferentiated monolayers of keratinocytes on non-cross-linked bovine type I collagen membranes do lead to an early reconstitution of multilayered squamous epithelium with enhanced wound healing compared to the control group. The upside down KCMG grafting technique is able to transfer actively proliferative keratinocytes and simplifies the application compared to conventional epithelial sheet grafting.

Hepatic tissue engineering: from transplantation to customized cell-based liver directed therapies from the laboratory

•  Introduction •  Development of cell isolation and primary culture for hepatocytes •  Three–dimensional culture using matrices •  Development of bioreactor systems for liver cells •  First clinical application of bioreactors with liver cells •  Development of matrix‐based hepatocyte transplantation •  Outlook: future perspective for the development of successful tissue engineering approaches for transplantation

Gene transfer strategies in tissue engineering

•  Introduction •  Methods of gene delivery ‐  Non‐viral techniques ‐  Naked DNA ‐  Cationic liposomes ‐  Viral techniques ‐  Retrovirus ‐  Adenovirus ‐  Adeno‐associated virus ‐  Herpes simplex virus •  Gene delivery from scaffolds for tissue engineering •  Gene therapy applications in tissue engineering ‐  Skin and wound healing ‐  Cartilage ‐  Bone ‐  Nerve ‐  Liver and endocrine pancreas ‐  Blood vessels •  Outlook

Delayed reverse sural flap for staged reconstruction of the foot and lower leg.

Background: Soft-tissue defects of the foot and lower leg caused by traumatic injury, tumor ablation, or infection associated with osteomyelitis often require coverage by flaps. One excellent option for reconstruction of these defects is the distally based neurofasciocutaneous sural flap. It allows rapid and reliable coverage of defects from the distal third of the lower leg to the forefoot without significant functional donor-site morbidity. However, the maximal size of the flap is limited by the delicate perfusion of the arterial network associated with the superficial sensory nerve. Delay procedures may increase the reliability of large sural flaps. Methods: The authors successfully used delayed sural flaps based on a two-step procedure for the treatment of 11 patients (three women and eight men, age 50.1 ± 20.0 years) with osteomyelitis (n = 3), melanoma (n = 3), sarcoma (n = 1), squamous cell carcinoma (n = 1), posttraumatic defects (n = 2), and recurrent gouty ulcer (n = 1). The delay period ranged from 7 to 15 days (9.7 ± 3.1), the length of the flap was from 9 to 19 (14.8 ± 3.0) cm, and the width of the flap from 7 to 12 (9.2 ± 1.3) cm. Temporary wound coverage was achieved by vacuum-assisted closure during the delay period. Results: All defects were covered successfully without major complications. Conclusions: The delay procedure positively affects the viability of large sural neurofasciocutaneous flaps. The authors recommend this modification for patients with large defects at the distal third of the lower leg or foot, requiring a two-step surgical approach due to the underlying disease.

50 years experience with Dupuytren's contracture in the Erlangen University Hospital – A retrospective analysis of 2919 operated hands from 1956 to 2006

BackgroundDupuytrens disease (DD) is a hand disorder mainly among the northern population. In contrast it is rare in the mediterranean population. Therefore typical habits and dietetic influences have been discussed as well as genetic predisposition. Still, since the first description by Dupuytren in 1834 only little is known about the etiology and pathogenesis of this disease. Some hints were found for a higher prevalence among people with diabetes, alcohol abuse or smoking. Also, intensive manual work or hand injuries have been discussed to have an influence on DD. To our knowledge this is the largest retrospectively evaluated series of symptomatic patients published to date. The study includes patients from the last 50 years. It was performed to show possible correlations between DD and typical risk factors such as diabetes, alcohol consumption, and smoking.MethodsWe retrospectively analysed all patient records with DD documented between 1956 and 2006 in the Surgical University Hospital in Erlangen. Data acquisition was conducted by reviewing the medical records from 1956 to 2006 including data from all patients who were surgically treated because of DD.ResultsWe reviewed 2579 male and 340 female surgically treated patients with DD. More than 80% of the patients were between 40 and 70 years old. In 28.9% only the right hand was effected by DD, in 25.3% only the left hand and in 45.8% both hands. In 10.3% of all Patients suffered from Diabetes mellitus. Statistical analysis revealed no significant correlation between diabetes, alcoholism or smoking on the degree of DD in our patients.ConclusionMost data are consistent with previously published results from smaller, comparable retrospective studies with regard to right- or left handedness. We could not confirm a statistically significant correlation of DD with diabetes mellitus, severe alcohol consumption, heavy smoking or epilepsy and the stage of the disease as described in other studies. However, in the whole cohort of our operated patients during the last 50 years the prevalence of the above mentioned risk factors is slightly higher than in the normal population.

A new approach to tissue engineering of vascularized skeletal muscle.

Tissue Engineering of skeletal muscle tissue still remains a major challenge. Every neo‐tissue construct of clinically relevant dimensions is highly dependent on an intrinsic vascularisation overcoming the limitations of diffusion conditioned survival. Approaches incorporating the arteriovenous‐loop model might bring further advances to the generation of vascularised skeletal muscle tissue. In this study 12 syngeneic rats received transplantation of carboxy‐fluorescine diacetate‐succinimidyl ester (CFDA)‐labelled, expanded primary myoblasts into a previously vascularised fibrin matrix, containing a microsurgically created AV loop. As control cells were injected into fibrin‐matrices without AV‐loops. Intra‐arterial ink injection followed by explantation was performed 2, 4 and 8 weeks after cell implantation. Specimens were evaluated for CFDA, MyoD and DAPI staining, as well as for mRNA expression of muscle specific genes. Results showed enhanced fibrin resorption in dependence of AV loop presence. Transplanted myoblasts could be detected in the AV loop group even after 8 weeks by CFDA‐fluorescence, still showing positive MyoD staining. RT‐PCR revealed gene expression of MEF‐2 and desmin after 4 weeks on the AV loop side, whereas expression analysis of myogenin and MHCembryo was negative. So far myoblast injection in the microsurgical rat AV loop model enhances survival of the cells, keeping their myogenic phenotype, within pre‐vascularised fibrin matrices. Probably due to the lack of potent myogenic stimuli and additionally the rapid resorption of the fibrin matrix, no formation of skeletal muscle‐like tissue could be observed. Thus further studies focussing on long term stability of the matrix and the incorporation of neural stimuli will be necessary for generation of vascularised skeletal muscle tissue.