Elias Polykandriotis

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.

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.

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.

Tissue engineering and regenerative medicine -where do we stand?

Introduction Intrinsic and extrinsic vascularization of TE constructs – the AV‐loop model Gene Transfer Techniques Combining mesenchymal stem cells with the AV‐loop model of intrinsic vascularization TE and RM in the context of Cancer Research Newly discovered cells of potential benefit for RM Summary

Axial vascularization of a large volume calcium phosphate ceramic bone substitute in the sheep AV loop model.

Vascularization still remains an obstacle to engineering of bone tissue with clinically relevant dimensions. Our aim was to induce axial vascularization in a large volume of a clinically approved biphasic calcium phosphate ceramic by transferring the arteriovenous (AV) loop approach to a large animal model. HA/β‐TCP granula were mixed with fibrin gel for a total volume of 16 cm3, followed by incorporation into an isolation chamber together with an AV loop. The chambers were implanted into the groins of merino sheep and the development of vascularization was monitored by sequential non‐invasive magnetic resonance imaging (MRI). The chambers were explanted after 6 and 12 weeks, the pedicle was perfused with contrast agent and specimens were subjected to micro‐computed tomography (µ‐CT) scan and histological analysis. Sequential MRI demonstrated a significantly increased perfusion in the HA/β‐TCP matrices over time. Micro‐CT scans and histology confirmed successful axial vascularization of HA/β‐TCP constructs. This study demonstrates, for the first time, successful axial vascularization of a clinically approved bone substitute with a significant volume in a large animal model by means of a microsurgically created AV loop, thus paving the way for the first microsurgical transplantation of a tissue‐engineered, axially vascularized bone with clinically relevant dimensions. Copyright

Automatic quantitative micro-computed tomography evaluation of angiogenesis in an axially vascularized tissue-engineered bone construct.

INTRODUCTION We invented an automatic observer-independent quantitative method to analyze vascularization using micro-computed tomography (CT) along with three-dimensional (3D) reconstruction in a tissue engineering model. MATERIALS AND METHODS An arteriovenous loop was created in the medial thigh of 30 rats and was placed in a particulated porous hydroxyapatite and beta-tricalcium phosphate matrix, filled with fibrin (10 mg/mL fibrinogen and 2 IU/mL thrombin) without (group A) or with (group B) application of fibrin-gel-immobilized angiogenetic growth factors vascular endothelial growth factor (VEGF¹⁶⁵) and basic fibroblast growth factor (bFGF). The explantation intervals were 2, 4, and 8 weeks. Specimens were investigated by means of micro-CT followed by an automatic 3D analysis, which was correlated to histomorphometrical findings. RESULTS In both groups, the arteriovenous loop led to generation of dense vascularized connective tissue with differentiated and functional vessels inside the matrix. Quantitative analysis of vascularization using micro-CT showed to be superior to histological analysis. The micro-CT analysis also allows the assessment of different other, more complex vascularization parameters within 3D constructs, demonstrating an early improvement of vascularization by application of fibrin-gel-immobilized VEGF¹⁶⁵ and bFGF. CONCLUSIONS In this study quantitative analysis of vascularization using micro-CT along with 3D reconstruction and automatic analysis exhibit to be a powerful method superior to histological evaluation of cross sections.

Dose-Finding Study of Fibrin Gel-Immobilized Vascular Endothelial Growth Factor 165 and Basic Fibroblast Growth Factor in the Arteriovenous Loop Rat Model

The angiogenic effects of different concentrations of vascular endothelial growth factor (VEGF) 165 and basic fibroblast growth factor (bFGF) immobilized in a fibrin-based drug-delivery system were quantitatively assessed in the arteriovenous (AV) loop model. An AV loop was created in the medial thigh of 60 rats. The loop was placed in a Teflon isolation chamber and embedded in 500 microL of fibrin gel loaded with VEGF and bFGF in four different concentrations (no growth factor, 100 ng/mL of VEGF, 25 ng/mL of VEGF and bFGF, 100 ng/mL pf VEGF and bFGF). The explantation intervals were 1, 2, and 4 weeks after the initial operation for all groups. Specimens were investigated using (micro-CT) and histological and morphometrical techniques. After 2 weeks, the cross-section area and construct weight were significantly lower with the use of 100 ng/mL of VEGF and bFGF. Micro-CT and histology showed significantly greater vascular density and number of vessels of the constructs at 2 and 4 weeks when 100 ng/mL of VEGF165 and bFGF were applied than in the growth factor-free specimens. The angioinductive effects were dose-dependent, with best results when using 100 ng/mL of VEGF165 and bFGF. The greater tissue formation was accompanied by faster resorption of the fibrin matrix.

De novo generation of axially vascularized tissue in a large animal model

De novo generation of axially vascularized tissue with clinically relevant dimensions in a large animal model and implementation of clinically established imaging modalities for in vivo evaluation of vascularization. To be used for reconstruction of tissue defects, engineered grafts need to be axially vascularized to enable transplantation without graft loss due to hypoxia. Limitations to dimensions in small animal models had not yet been overcome, which is necessary to yield clinical relevance. Anatomical studies of groin and axillary regions in eight merino sheep were followed by microsurgical creation of an arteriovenous loop (AV‐loop), embedded in an isolation chamber filled with fibrin matrix. Constructs were implanted in the groin of six sheep for up to 6 weeks. Course of vascularization in de novo forming tissue was assessed by sequential computed tomography angiography (CTA) and magnetic resonance angiography (MRA) in vivo, as well as by postexplantational micro‐computed tomography and histology. A vascular axis was constantly found epifascially at the medial aspect of all sheeps thighs, which was used for AV‐loop creation. Patency of AV‐loop could be visualized by CTA and MRA scans during 1–6 weeks. Complex 3D‐vessel‐reconstruction revealed increasing axial vascularization of the fibrin matrix and growing connective tissue within the isolation chamber, which was confirmed by micro‐computed tomography and histology postexplantation. De novo formation of axially vascularized tissue was demonstrated for the first time ever in a large animal model, paving the way for the first application of tissue engineering vascularized grafts with clinically relevant dimensions.

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.