Justus P. Beier

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.

Osteochondral tissue engineering: scaffolds, stem cells and applications.

Osteochondral tissue engineering has shown an increasing development to provide suitable strategies for the regeneration of damaged cartilage and underlying subchondral bone tissue. For reasons of the limitation in the capacity of articular cartilage to self‐repair, it is essential to develop approaches based on suitable scaffolds made of appropriate engineered biomaterials. The combination of biodegradable polymers and bioactive ceramics in a variety of composite structures is promising in this area, whereby the fabrication methods, associated cells and signalling factors determine the success of the strategies. The objective of this review is to present and discuss approaches being proposed in osteochondral tissue engineering, which are focused on the application of various materials forming bilayered composite scaffolds, including polymers and ceramics, discussing the variety of scaffold designs and fabrication methods being developed. Additionally, cell sources and biological protein incorporation methods are discussed, addressing their interaction with scaffolds and highlighting the potential for creating a new generation of bilayered composite scaffolds that can mimic the native interfacial tissue properties, and are able to adapt to the biological environment.

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

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.

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.

Collagen matrices from sponge to nano: new perspectives for tissue engineering of skeletal muscle

BackgroundTissue engineering of vascularised skeletal muscle is a promising method for the treatment of soft tissue defects in reconstructive surgery. In this study we explored the characteristics of novel collagen and fibrin matrices for skeletal muscle tissue engineering. We analyzed the characteristics of newly developed hybrid collagen-I-fibrin-gels and collagen nanofibers as well as collagen sponges and OPLA®-scaffolds. Collagen-fibrin gels were also tested with genipin as stabilizing substitute for aprotinin.ResultsWhereas rapid lysis and contraction of pure collagen I- or fibrin-matrices have been great problems in the past, the latter could be overcome by combining both materials. Significant proliferation of cultivated myoblasts was detected in collagen-I-fibrin matrices and collagen nanofibers. Seeding cells on parallel orientated nanofibers resulted in strongly aligned myoblasts. In contrast, common collagen sponges and OPLA®-scaffolds showed less cell proliferation and in collagen sponges an increased apoptosis rate was evident. The application of genipin caused deleterious effects on primary myoblasts.ConclusionCollagen I-fibrin mixtures as well as collagen nanofibers yield good proliferation rates and myogenic differentiation of primary rat myoblasts in vitro In addition, parallel orientated nanofibers enable the generation of aligned cell layers and therefore represent the most promising step towards successful engineering of skeletal muscle tissue.

Successful human long-term application of in situ bone tissue engineering

Tissue Engineering (TE) and Regenerative Medicine (RM) have gained much popularity because of the tremendous prospects for the care of patients with tissue and organ defects. To overcome the common problem of donor‐site morbidity of standard autologous bone grafts, we successfully combined tissue engineering techniques for the first time with the arteriovenous loop model to generate vascularized large bone grafts. We present two cases of large bone defects after debridement of an osteomyelitis. One of the defects was localized in the radius and one in the tibia. For osseus reconstruction, arteriovenous loops were created as vascular axis, which were placed in the bony defects. In case 1, the bone generation was achieved using cancellous bone from the iliac crest and fibrin glue and in case 2 using a clinically approved β‐tricalciumphosphate/hydroxyapatite (HA), fibrin glue and directly auto‐transplanted bone marrow aspirate from the iliac crest. The following post‐operative courses were uneventful. The final examinations took place after 36 and 72 months after the initial operations. Computer tomogrphy (CT), membrane resonance imaging (MRI) and doppler ultrasound revealed patent arterio‐venous (AV) loops in the bone grafts as well as completely healed bone defects. The patients were pain‐free with normal ranges of motion. This is the first study demonstrating successfully axially vascularized in situ tissue engineered bone generation in large bone defects in a clinical scenario using the arteriovenous loop model without creation of a significant donor‐site defect utilizing TE and RM techniques in human patients with long‐term stability.

Engineering skeletal muscle tissue - new perspectives in vitro and in vivo

•  Introduction •  Matrices for skeletal muscle tissue engineering – gaining orientation •  Electrospun nanofibre matrices •  Nanotechnology and smart matrices •  Cell sources for skeletal muscle TE •  Conclusion

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.

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three‐dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long‐term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform‐sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live‐dead assay, and real‐time RT‐PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell‐seeded scaffold product for applications in regenerative medicine.