Ulrich Kneser

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.

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

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

Long-term differentiated function of heterotopically transplanted hepatocytes on three-dimensional polymer matrices.

Hepatocyte transplantation using porous matrices is under investigation as an alternative therapy for certain liver diseases. For this purpose, long-term function of transplanted hepatocytes is mandatory. This problem has not been sufficiently investigated yet. In this study Lewis rats were used as donors and recipients. Stimulated (group A, portocaval shunt) or unstimulated (group B) hepatocytes were transplanted into prevascularized polyvinyl-alcohol matrices. Cell-free matrices served as controls (group C). Matrices were harvested between 1 h and 1 year after implantation and analyzed by morphometry; albumin RNA in situ hybridization; and cytokeratin-, actin-, desmin-, and macrophage-specific antigen immunohistology. The hepatocyte number significantly decreased within the first week following implantation. Between 1 month and 1 year after transplantation a significant increase in hepatocyte number was noted in groups A and B. Albumin transcripts of transplanted hepatocytes were at normal levels at all times except for group B after 1 year. The immunohistology suggested engraftment of nonparenchymal liver cells. We conclude that 3-dimensional matrices provide a sufficient environment for long-term engraftment of transplanted liver cells. The hepatocytes are able, despite suboptimal initial engraftment, to repopulate the scaffold for at least half of the recipients life span and maintain cell-specific function after sufficient stimulation.

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.

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.

Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered “sheet grafts” has been shown to vary extremely. Adherence of transplanted cells to the wound bed — especially in the presence of potential wound contamination — is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal—epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.

Evaluation of processed bovine cancellous bone matrix seeded with syngenic osteoblasts in a critical size calvarial defect rat model.

Introduction: Biologic bone substitutes may offer alternatives to bone grafting procedures. The aim of this study was to evaluate a preformed bone substitute based on processed bovine cancellous bone (PBCB) with or without osteogenic cells in a critical size calvarial defect rat model. Methods: Discs of PBCB (Tutobone®) were seeded with second passage fibrin gel‐immobilized syngenic osteoblasts (group A, n = 40). Cell‐free matrices (group B, n = 28) and untreated defects (group C; n=28) served as controls. Specimens were explanted between day 0 and 4 months after implantation and were subjected to histological and morphometric evaluation. Results: At 1 month, bone formation was limited to small peripheral areas. At 2 and 4 months, significant bone formation, matrix resorption as well as integration of the implants was evident in groups A and B. In group C no significant regeneration of the defects was observed. Morphometric analysis did not disclose differences in bone formation in matrices from groups A and B. Carboxyfluorescine‐Diacetate‐Succinimidylester (CFDA) labeling demonstrated low survival rates of transplanted cells. Discussion: Osteoblasts seeded into PBCB matrix display a differentiated phenotype following a 14 days cell culture period. Lack of initial vascularization may explain the absence of added osteogenicity in constructs from group A in comparison to group B. PBCB is well integrated and represents even without osteogenic cells a promising biomaterial for reconstruction of critical size calvarial bone defects.