Holger Bannasch

Single-Cell Suspensions of Cultured Human Keratinocytes in Fibrin–Glue Reconstitute the Epidermis

To overcome common disadvantages of standard cultured epidermal sheet grafts (CEG) we have developed a new technique of transplanting cultured human keratinocytes suspended as single cells in a fibrin-glue matrix (Keratinocyte-fibrin-glue suspension-KFGS). In an athymic mouse model with reproducible standardized full thickness wounds this new technique was compared directly to CEG. Reepithelialization was similar in both groups, but reconstitution of the dermo-epidermal junction zone, as shown by electron microscopy and immunohistochemistry was significantly enhanced by the fibrin-glue suspension technique. The new KFGS technique is earlier available than sheet grafts, is able to transfer actively proliferative single keratinocytes, and simplifies the application.

Skin tissue engineering

The coverage of extensive wounds with viable autologous keratinocytes remains the only option of treatment if autologous donor skin is not obtainable. There is evidence that proliferating keratinocytes, as suspended cells or as a single layer, are adequate for wound closure. Understanding keratinocyte-matrix interactions not only allows us to influence keratinocyte outgrowth, adhesion, and migration, but may also guide us to modify matrix molecules for enhancing keratinocyte take. Further approaches may include the generation of genetically manipulated keratinocytes, which allow the use of an off-the-shelf epidermal replacement. As surgeons, our goal is to help burn patients with the best quality of skin in the shortest time possible. As tissue engineers, we have not achieved the goal of a universal skin product. By continually reviewing the options and using them, we can at least use the proper material in the adequate situation. Because of the limited resources, the need for comparisons of clinical effectiveness and cost are ever more important. As anatomy and physiology of engineered skin substitutes improve, they will become more similar to native skin autografts. Improvement of skin substitutes will result from inclusion of additional cell types (eg, melanocytes) and from modifications of culture media and scaffolds. Skin-substitute materials may be able to stimulate regeneration rather than repair, and tissue-engineered skin may match the quality of split-skin autografts, our present gold standard.

Complications in abdominoplasty: a risk factor analysis.

INTRODUCTION Abdominoplasty is one of the most popular body-contouring procedures. However, it is associated with a significant number of complications, the most common being seroma and wound-healing problems. In this study, we analysed postoperative complications following abdominoplasty and evaluated the presence of statistically significant correlations between pre-existing risk factors and postoperative complications. METHODS We carried out a retrospective chart review of all patients who underwent abdominoplasty from June 1994 to April 2004 at our institution. Patient demographics, risk factors (smoking, previous abdominal surgery, obesity, and male gender) and complications (minor and major) were noted from the patients medical record. RESULTS In all, 139 patients were included in the study (126 female and 13 male), with a mean age of 42.8 years at the time of surgery. Minor and major complications were seen in 40 (28.8%) and 16 patients (11.5%), respectively. Smoking and previous abdominal surgery were not associated with a significantly increased complication rate. However, patients without previous surgery displayed a significantly increased complication rate (43.2% vs. 22.1% for minor and 25% vs. 5.3% for major complications; p<0.05). Obesity (BMI >30 kg/m(2)) was associated with a significant increase in major complications (20.8% vs. 9.7%; p<0.05). Male gender was predisposed to increased minor and major complications; however, this was not statistically significant. CONCLUSION Despite its popularity, abdominoplasty is still associated with a significant rate of complications. In particular, obese patients and those with no previous history of abdominal surgery seem to be at risk for complications. The role of male gender needs to be evaluated further.

Fibrin Glue as Matrix for Cultured Autologous Urothelial Cells in Urethral Reconstruction

In the present study, we have established a technique to create an artificial urethra in a rat animal model by transplantation of in vitro-expanded urothelial cells onto an in vivo-prefabricated tube formation using tissue engineering methods. Urothelial cells from isogenic rats were harvested for culture. A silicon catheter was used to induce a connective tissue capsule-tube formation underneath the abdominal skin. Two weeks later, the cultivated urothelial cells were seeded onto the lumen of this tube using fibrin glue as delivery matrix. The histomorphological and immunohistochemical studies revealed a viable multilayered urothelium, lining the inner surface of the prior formed connective tissue tube-formation 4 weeks after grafting the cells. We have shown that cultured and in vitro-expanded urothelial cells can be successfully reimplanted onto a prefabricated tube-like structure using fibrin glue as a delivery matrix and native cell expansion vehicle. The results suggest that the creation of an artificial urethra may be achieved in vivo using tissue engineering methods, showing potential for urethral reconstruction and providing autologous urothelium for reconstructive surgery in the genitourinary tract.

C-reactive protein: How conformational changes influence inflammatory properties

Recent evidence suggests that the prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP belongs to the family of pentraxins and as such consists of five identical non-covalently linked subunits. Contradictory data on the characteristics of CRP as either being pro- or anti-inflammatory may be explained by the existence of two conformations of the protein: the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. In vitro both isoforms exhibit a very distinct inflammatory profile. We recently identified a localized, physiologically relevant pCRP dissociation mechanism by activated platelets and apoptotic cells and showed the deposition of mCRP in inflamed tissue. Here we review the literature on the causal role of p- and mCRP in the light of our findings and critically analyze the current controversies around CRP. The novel understanding of the localized dissociation of circulating pentameric CRP to the distinctively pro-inflammatory monomeric CRP allows for a new view on CRP in inflammatory reactions and further highlights mCRP and the pCRP dissociation process as a potential therapeutic target.

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.

Dissociation of Pentameric to Monomeric C-Reactive Protein Localizes and Aggravates Inflammation In Vivo Proof of a Powerful Proinflammatory Mechanism and a New Anti-Inflammatory Strategy

Background— The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo. Methods and Results— We identified mCRP in inflamed human striated muscle, human atherosclerotic plaque, and infarcted myocardium (rat and human) and its colocalization with inflammatory cells, which suggests a general causal role of mCRP in inflammation. This was confirmed in rat intravital microscopy of lipopolysaccharide-induced cremasteric muscle inflammation. Intravenous pCRP administration significantly enhanced leukocyte rolling, adhesion, and transmigration via localized dissociation to mCRP in inflamed but not noninflamed cremaster muscle. This was confirmed in a rat model of myocardial infarction. Mechanistically, this process was dependent on exposure of lysophosphatidylcholine on activated cell membranes, which is generated after phospholipase A2 activation. These membrane changes could be visualized intravitally on endothelial cells, as could the colocalized mCRP generation. Blocking of phospholipase A2 abrogated C-reactive protein dissociation and thereby blunted the proinflammatory effects of C-reactive protein. Identifying the dissociation process as a therapeutic target, we stabilized pCRP using 1,6-bis(phosphocholine)-hexane, which prevented dissociation in vitro and in vivo and consequently inhibited the generation and proinflammatory activity of mCRP; notably, it also inhibited mCRP deposition and inflammation in rat myocardial infarction. Conclusions— These results provide in vivo evidence for a novel mechanism that localizes and aggravates inflammation via phospholipase A2–dependent dissociation of circulating pCRP to mCRP. mCRP is proposed as a pathogenic factor in atherosclerosis and myocardial infarction. Most importantly, the inhibition of pCRP dissociation represents a promising, novel anti-inflammatory therapeutic strategy.Background— The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo. Methods and Results— We identified mCRP in inflamed human striated muscle, human atherosclerotic plaque, and infarcted myocardium (rat and human) and its colocalization with inflammatory cells, which suggests a general causal role of mCRP in inflammation. This was confirmed in rat intravital microscopy of lipopolysaccharide-induced cremasteric muscle inflammation. Intravenous pCRP administration significantly enhanced leukocyte rolling, adhesion, and transmigration via localized dissociation to mCRP in inflamed but not noninflamed cremaster muscle. This was confirmed in a rat model of myocardial infarction. Mechanistically, this process was dependent on exposure of lysophosphatidylcholine on activated cell membranes, which is generated after phospholipase A2 activation. These membrane changes could be visualized intravitally on endothelial cells, as could the colocalized mCRP generation. Blocking of phospholipase A2 abrogated C-reactive protein dissociation and thereby blunted the proinflammatory effects of C-reactive protein. Identifying the dissociation process as a therapeutic target, we stabilized pCRP using 1,6-bis(phosphocholine)-hexane, which prevented dissociation in vitro and in vivo and consequently inhibited the generation and proinflammatory activity of mCRP; notably, it also inhibited mCRP deposition and inflammation in rat myocardial infarction. Conclusions— These results provide in vivo evidence for a novel mechanism that localizes and aggravates inflammation via phospholipase A2–dependent dissociation of circulating pCRP to mCRP. mCRP is proposed as a pathogenic factor in atherosclerosis and myocardial infarction. Most importantly, the inhibition of pCRP dissociation represents a promising, novel anti-inflammatory therapeutic strategy. # CLINICAL PERSPECTIVE {#article-title-40}

A proteomic analysis of C-reactive protein stimulated THP-1 monocytes

BackgroundC-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP.MethodsProtein cell lysates from control and mCRP, pCRP or LPS-treated THP-1 cells were displayed using 2-dimensional SDS PAGE and compared. Differentially expressed proteins were identified by MALDI-TOF MS and confirmed by Western blotting.ResultsmCRP significantly up-regulates ubiquitin-activating enzyme E1, a member of the ubiquitin-proteasome system in THP-1 monocytes. Furthermore, HSP 70, alpha-actinin-4 (ACTN4) and alpha-enolase/enolase 1 were upregulated. The proteomic profile of LPS and pCRP treated monocytes differ significantly from that of mCRP.ConclusionThe data obtained in this study support the hypothesis that isoform-specific effects of CRP may differentially regulate the phenotype of monocytes.

Cultured keratinocytes in fibrin with decellularised dermis close porcine full-thickness wounds in a single step

The purpose of this study was to assess the feasibility of combined keratinocyte and dermal scaffold transplantation performed in a single step for treatment of full-thickness wounds. Cultured autologous keratinocytes were suspended in fibrin and grafted together with decellularised human dermis (Alloderm) in a porcine animal model, involving 10 animals over 4 weeks. Wound healing was evaluated by planimetry. Histology included morphological analysis as well as immunohistochemistry at regular intervals (1, 2 and 4 weeks). The results showed both successful histo-integration of the in vivo composite grafts and reduced wound contraction, compared with the control group (plain epithelial grafts). Histologically a neo-epithelium originated from the grafted cells on top of the decellularised dermis, as well as a reconstituted basement membrane. After 4 weeks cellular ingrowth into the dermal matrix could be observed. The successful combination of a keratinocyte-fibrin suspension and acellular dermis applied in a single step onto full-thickness wounds resulted in closure.

Negative pressure wound therapy reduces the ischaemia/reperfusion-associated inflammatory response in free muscle flaps☆

BACKGROUND We recently established negative pressure wound therapy (NPWT) as a safe postoperative care concept for free muscle flaps; however, the molecular effects of NPWT on free muscle flaps remain elusive. Here we investigated the effects of NPWT on pathological changes associated with ischaemia/reperfusion injury in free flap tissue. METHODS From July 2008 to September 2010, 30 patients receiving skin-grafted free muscle transfer for defect coverage were randomly assigned to two treatment groups: In one group the skin-grafted free flap was covered by a vacuum dressing (NPWT); in the second group, flaps were covered by conventional petroleum gauze dressings (conv). Biopsies were taken intra-operatively prior to clipping of the pedicle and on postoperative day 5. Samples were analysed by immunohistochemistry for infiltration of inflammatory cells, real-time polymerase chain reaction (RT-PCR) for the analysis of expression levels of interleukin-1β (IL-1β) and tumour necrosis factor (TNF)-alpha as markers of inflammation. Histological samples were also examined for interstitial oedema formation, and apoptosis was detected by a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. RESULTS NPWT leads to a significantly reduced tissue infiltration of CD68 + macrophages and reduced expression of the inflammatory cytokines IL-1β and TNFα. None of these parameters was significantly elevated in the pre-ischaemic biopsies. Furthermore, NPWT reduced the interstitial oedema formation and the number of apoptotic cells in free flap tissue. CONCLUSION NPWT of skin-grafted free muscle flaps leads to a reduced inflammatory response following ischaemia/reperfusion, resulting in reduced oedema formation improving the microcirculation and ultimately reduced tissue damage. We thereby deliver new insight into the effects of NPWT.