Alexander D. Bach

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.

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.

Tissue engineering of injectable muscle : Three-dimensional myoblast-fibrin injection in the syngeneic rat animal model

Background: Surgical treatment of skeletal muscle loss resulting from trauma, tumor ablation, or inborn tissue defects is hampered by the scarcity of functional substitute tissue. By using techniques of tissue engineering, reconstitution of skeletal muscle defects might become a more viable option. However, it is necessary to develop an adequate, practical method for delivering myoblasts within a three-dimensional scaffold in vivo. The aim of this study was to create and evaluate a novel method for the transfer of myoblasts with clinically approved components within a three-dimensional matrix. Methods: The authors injected expanded primary male myoblasts into muscle defects in female syngeneic rats using a two-way syringe (Duploject) within a three-dimensional fibrin matrix. Detection and evaluation were performed using Y chromosome in situ hybridization, antidesmin immunostaining, and hematoxylin and eosin staining. To identify possible differences by means of integration, the injected myoblasts were compared with 7 days of precultivated constructs. Results: Injected myoblasts showed increasing integration into host muscle fibers in a time-dependent manner, exclusively at the injection site. Antidesmin staining revealed a conserved myogenic phenotype of transplanted cells. The fibrin matrix resolved over a period of 12 weeks, with no indication of an inflammatory reaction. No significant difference in the number of detected Y chromosome-positive nuclei was found between the two transplantation groups. Conclusions: The presented technique of myoblast-fibrin injection indicates a clinical potential for reconstruction of skeletal muscle defects in vivo using a ready-to-use device in combination with tissue-engineering methods.

Engineering of muscle tissue

The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in health care. Tissue engineering and regenerative medicine is an emerging interdisciplinary field that applies the principles of biology and engineering to the development of viable substitutes that restore, maintain, or improve the function of human tissues and organs. Tissue engineering science has provided critical new knowledge that will deepen our understanding of the phenotype of an important category of cell types-the muscle cells-and this knowledge may enable meaningful advances in musculoskeletal tissue engineering. There are two principle strategies for the replacement of impaired muscle tissues. One approach uses the application of isolated and differentiated cells (in vivo tissue engineering), using a transport matrix for the cell delivery; the other uses in vitro-designed and pre-fabricated tissue equivalents (in vitro tissue engineering). Future developments and the decision regarding which approach is more promising depend on the elucidation of the relationships among cell growth and differentiation, the three-dimensional environment, the architecture of the cells, and gene expression of the developmental process and the survival of the cells and integration in the host in in vivo experiments. As the techniques of tissue engineering become more sophisticated and as issues such as vascularization and innervation are addressed, the usefulness of these methods for reconstructive surgery may grow significantly.

Impact of electrical stimulation on three‐dimensional myoblast cultures ‐ a real‐time RT‐PCR study

Several focal skeletal muscle diseases, including tumours and trauma lead to a limited loss of functional muscle tissue. There is still no suitable clinical approach for treating such defects. A promising approach could be the tissue engineering of skeletal muscle. However, a clinically reliable differentiation stimulus for three‐dimensional (3‐D) cultures is necessary for this process, and this condition has not yet been established. In order to qunantify and analyze the differentiation potential of electrical cell stimulation, primary myoblasts were stimulated within a 3‐D fibrin‐matrix. Gene expression of MyoD, myogenin and AChR were measured by real‐time RT‐PCR over a time period of eight days, showing immediate down‐regulation of all marker genes. For tissue engineering approaches, cell multiplication is crucial for acquisition of sufficient tissue volumes for reconstruction. Therefore, all experiments were performed with high and low passaged myoblasts, demonstrating higher transcript rates of marker genes in lowpassage cells. Our findings strongly suggest a reconsideration of electrical stimulation in muscle tissue engineering.

The versatility of the distally based peroneus brevis muscle flap in reconstructive surgery of the foot and lower leg

Soft tissue and bone defects of the lower leg, ankle, and heel region often require coverage by local or distant flaps. The authors successfully used the distally based peroneus brevis muscle flap for the treatment of 15 patients with osteomyelitis (n = 5), melanoma (n = 1), Achilles tendon defects (n = 6), posttraumatic bone defects (n = 2), and chronic diabetic heel ulcer (n = 1). The size of the defects ranged from 6 to 60 cm2. All defects were covered successfully without major complications by the muscle flap. The distally based peroneus brevis muscle represents a very reliable flap for coverage of small and moderate defects of the medial and lateral malleolus, the Achilles tendon, and the heel area. This flap offers a convincing alternative for covering defects in the distal leg region and is often preferable to the use of free flaps because the surgery is rapidly performed and does not require microsurgical expertise.

Intrapulmonary and cutaneous siliconomas after silent silicone breast implant failure.

Abstract:  Since the implementation and use of silicone implants in breast surgery the risks are published and discussed. Especially, the incidence of late silicone implant rupture and its potential risk to induce local siliconomas are still under discussion and not sufficiently evaluated. So far literature data offer no information of intrapulmonal or peripheral located cutaneous siliconomas because of systemic migration of silicone after breast augmentation. In light of silicones checkered history, and given the large and growing number of women who choose to undergo breast augmentation surgery each year, the presented clinical findings in our study are likely to be of interest to medical professionals, producers, and consumers alike. We present six female patients with an average age of 55 (±5) years with bilateral rupture of silicone implants after breast augmentation for aesthetic reasons. The average time after operation was 18 (±6) years. In five patients, we identified peripheral located cutaneous siliconomas and one patient suffered from an intrapulmonal siliconoma. The diagnosis of bilateral rupture of the silicone implants was performed preoperatively by MRI‐scans. All five peripheral cutaneous siliconomas and the intrapulmonal siliconoma were validated by histopathologic analysis. Six female patients suffered from bilateral rupture of silicone implants after breast augmentation. In five patients, we identified peripheral located cutaneous siliconomas which were surgically excised. One patient suffered from an intrapulmonal siliconoma. In this unique case a lobectomy with resection of the pulmonal segment 10 had to be performed. Clinical findings of peripheral cutaneous and even intrapulmonary siliconomas after bilateral rupture of silicone breast implants indicate a systemic hematogen or lymphatic pathway of silicone. These findings suggest that it is mandatory to inform the patient about the potential risk of local siliconomas, but also about the potential risk of peripheral cutaneous or even intrapulmonary siliconomas caused by systemic hematogen or lymphatic pathways of silicone after silent implant failure.