Matthias Mueller

Engineered adipose tissue supplied by functional microvessels.

A volume-persistent culture of adipose tissue under in vivo conditions can be achieved only by early vascularization after cell transplantation. Cotransplantation of autologous preadipocytes with endothelial cells may enable the early formation of a capillary network. Investigations were performed in vivo in a specially adapted chorioallantoic membrane (CAM) model. Fertilized White Leghorn eggs were incubated and opened on day 3 of incubation and human dermal microvascular endothelial cell (HDMVEC) spheroids and preadipocytes were transferred in a fibrin matrix to the CAM. On day 7 after incubation the composites were explanted and immunohistologically investigated. Numerous vessels consisting of HDMVECs could be detected and the lumena of these vessels were perfused by chick erythrocytes. These results show the formation of a capillary network consisting of transplanted HDMVECs. The microcirculation of chick erythrocytes in vessels consisting of human endothelial cells proves the continuity of a newly formed capillary system to the host vessel system. The experiments demonstrate the first patent connection of tissue-engineered microvessels in adipose tissue to a host vessel system without applying exogenous angiogenic growth factors or transient transfection. The cotransplantation of endothelial cell spheroids with angiogenic mesenchymal cells may lead to the engineering of complex three-dimensional implants.

Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model.

Tissue-engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold before in vivo transplantation. After transplantation, vascularization of the scaffold is a principal limiting factor for cell viability for the first 6-8 days posttransplantation. A model for systematic analysis of this process has been developed. Fertilized White Leghorn eggs were incubated (at 37.8 degrees C in 60% relative humidity) and opened on day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening on the surface of the chorioallantoic membrane (CAM) on day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days posttransplantation. The survival rate for embryos receiving transplanted constructs was about 90%. Histology confirmed transplant cell viability at day 4 posttransplantation and vascularization of the constructs by avian endothelial cells began at this time. A new in vivo model to study the effect of angiogenesis in TE constructs, including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties, is presented. Advantages include easy access to the vascular network of the CAM, lack of immunocompetence, low costs, and avoidance of animal experiments.

Adipose precursor cells (preadipocytes) induce formation of new vessels in fibrin glue on the newly developed cylinder chorioallantoic membrane model (CAM)

Successful augmentation of soft tissues by transplantation of preadipocytes within a matrix requires the formation of a new capillary network with connection to the host vessel system. Particularly, cells located centrally within the transplanted cell‐matrix‐construct represent a population with a blood supply questionable for survival. We demonstrated that under in vivo conditions preadipocytes possess the ability to induce and support the vascularization of the implant presumably by expression of several growth factors, such as VEGF (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor). Fertilized White‐Leghorn eggs were incubated under standardized conditions. Opening was performed at day three of incubation and preadipocytes with and without recombinant growth factors were transferred into a fibrin matrix and subsequently placed on the Chorioallantoic Membrane (CAM), respectively. Eight days later, the implanted constructs were explanted, histologically processed and vascularization evaluated by means of a computer‐assisted image analysis program. Matrices containing preadipocytes displayed a significantly higher density of vascularization, whereas in the control group (fibrin without preadipocytes) no vessel ingrowth was observed. Daily application of recombinant growth factors added to the medium did not positively influence vascularization of the implant. Our investigations demonstrate that preadipocytes possess a strong angiogenic potential to induce and support neovascularization of 3D‐fibrin matrices under in vivo conditions. Addition of recombinant growth factors did not result in any stimulatory effect. Neither did the application of fibrin alone demonstrate an angiogenic potential with regard to induction of vascularization.

Implantation of VEGF transfected preadipocytes improves vascularization of fibrin implants on the cylinder chorioallantoic membrane (CAM) model

The successful substitution or augmentation of soft tissues by implantation of three dimensional cell constructs, consisting of human preadipocytes and fibrin glue as a carrier matrix, requires a rapid and homogeneous vascularization of the whole implant in order to provide a sufficient blood supply of centrally situated cells. Previous investigations have shown that under in vivo conditions primary human preadipocytes induce vascularization of fibrin matrices by secretion of several growth factors, such as VEGF and bFGF. The current study investigates whether vascularization of implants can be improved by transplantation of preadipocytes following transfection with a VEGF‐vector. Transfection was performed by electroporation with an pCMX‐GFP and pCMX‐VEGF165 vector. Transfection efficiency (GFP expression) and VEGF expression were determined in vitro by FACS analysis and VEGF immunoassay, respectively. In vivo investigations to determine the vascularization of the implants were performed on the cylinder chorioallantoic membrane (CAM). Four million VEGF transfected cells were transferred within a fibrin matrix onto the CAM on the 7th day of incubation and after 8 days the vascularization of the implant was histologically examined and evaluated by means of a computer‐assisted image analysis program. Transfection of preadipocytes with the GFP vector by electroporation yielded transfection efficiencies between 12% and 41% of surviving cells. Results of the VEGF immunoassay demonstrated that VEGF expression was significantly higher following transfection. Investigations on the CAM outlined a significantly higher rate of vascularization in the transfected vs. control population. Our investigations demonstrate that primary human preadipocytes can be successfully transfected by electroporation with a VEGF vector. The enhanced VEGF expression on transfected cells results in an increase of vascularization of the cell constructs on the CAM.

Application of Continuous Wound-Infusion Catheters in Lung Transplantation: A Retrospective Data Analysis

Background Lung transplantation is the only treatment option for many patients with end‐stage pulmonary disease. Therefore, postthoracotomy pain therapy is of vital interest. Thoracic epidural analgesia (EPI) is the “gold standard” for postthoracotomy pain, but especially in lung transplantation contraindications, and potential infectious complications limit its advantages. Under these circumstances surgically placed postthoracotomy catheter‐assisted continuous paravertebral intercostal nerve block (PVB) could be of advantage. Methods We performed a retrospective cohort study of patients who underwent lung transplantation between 2005 and 2012. Groups were defined according to the type of postoperative pain therapy: PVB, EPI, and SYS (systemic analgesia). Total 44 patients were eligible. Results Postoperative opioid requirement of the PVB and EPI group was comparable and less than that of the SYS group. Patients of the PVB group were weaned earlier from mechanical ventilation after lung transplantation. Conclusion The potency of postoperative pain therapy of EPI and PVB seemed to be comparable and superior to SYS. Considering the risks and benefits, PVB could be a better choice than EPI for postthoracotomy pain therapy, especially in lung transplantation.

Rapid prototyping of flexible intrafascicular electrode arrays by picosecond laser structuring

OBJECTIVE Interfacing the peripheral nervous system can be performed with a large variety of electrode arrays. However, stimulating and recording a nerve while having a reasonable amount of channels limits the number of available systems. Translational research towards human clinical trial requires device safety and biocompatibility but would benefit from design flexibility in the development process to individualize probes. APPROACH We selected established medical grade implant materials like precious metals and Parylene C to develop a rapid prototyping process for novel intrafascicular electrode arrays using a picosecond laser structuring. A design for a rodent animal model was developed in conjunction with an intrafascicular implantation strategy. Electrode characterization and optimization was performed first in saline solution in vitro before performance and biocompatibility were validated in sciatic nerves of rats in chronic implantation. MAIN RESULTS The novel fabrication process proved to be suitable for prototyping and building intrafascicular electrode arrays. Electrochemical properties of the electrode sites were enhanced and tested for long-term stability. Chronic implantation in the sciatic nerve of rats showed good biocompatibility, selectivity and stable stimulation thresholds. SIGNIFICANCE Established medical grade materials can be used for intrafascicular nerve electrode arrays when laser structuring defines structure size in the micro-scale. Design flexibility reduces re-design cycle time and material certificates are beneficial support for safety studies on the way to clinical trials.

Modified repair of interrupted aortic arch utilizing retroesophageal right subclavian artery based on a neonatal hybrid approach in hypoplastic left heart complex.

OBJECTIVE Interrupted aortic arch (IAA) combined with an aberrant right subclavian artery (ARSA) is frequently associated with a hypoplastic ascending aorta. Neonatal surgical therapy carries a high risk particularly for aortic arch obstructions during the further follow-up. METHODS We performed a modified reconstruction of the aortic arch utilizing the ARSA as a natural substitute in a staged surgical approach. In a novel approach, the distal part of the ARSA is reimplanted into the brachiocephalic trunk. RESULTS In three patients, a novel arch reconstruction was successfully performed during complete biventricular repair. In a follow-up of 60 to 87 months, the reconstructed aortic arch has grown without any signs of obstruction in all three patients. CONCLUSION Utilizing the ARSA for surgical aortic arch repair is a satisfactory solution, when postnatal borderline left heart obstruction associated with IAA and ARSA is postponed by an initial hybrid approach.

Development of a single-sided Parylene C based intrafascicular multichannel electrode for peripheral nerves

As cleanroom fabricated polyimide based electrodes are commonly used in clinical trials due to their small dimensions and high flexibility we want to translate these advantages to a maskless manufacturing technology and another substrate material with FDA approval: Parlyene C. Using a picosecond laser (355 nm Nd:YVO4) an established laser fabrication process (1064 nm Nd:YAG nansecond laser) for silicone rubber electrodes was modified to allow the fabrication of thin parylene C electrodes for intrafascicular application. The process utilizes a 25 μm thick platinum iridium foil that is placed between two 10 μm thick parylene C substrate layers. Using the laser for thinning down the metal, increasing the active surface and cutting the complete electrode array a new fabrication process is developed. Adhesion parameters of the involved materials are measured and tailored to fit each other. The single electrode arrays feature 4 intrafascicular contacts as well as a ground electrode and fixation openings outside the nerve. Functionality of the electrode array was measured and a first assessment of its usability has been performed. The mechanical and electrochemical parameters are promising for intrafascicular implantation, successful stimulation and recording application in a peripheral nerve.

Fabrication of flat electrodes utilizing picosecond laser manufacturing technology: Preliminary study for fabrication of a novel transverse intrafascicular multichannel electrode

Over the last decade we developed methods for the fabrication of laser-structured electrode arrays for neural engineering. For these electrode arrays a metal foil was structured with a 1064 nm Nd:YAG laser in the nanosecond pulse regime and placed within a silicone rubber substrate. Due to process restrictions the individual electrode sites are not in plane with the upper layer of the silicone rubber. Here, a new laser in the picosecond regime (355 nm Nd:YVO4) was used for laser-structuring. This allowed the fabrication of a novel electrode array out of a 25 μm thick sheet of MP35N metal with thinned-down and buried tracks as well as fixations for the electrode sites and contact pads. For the opening of the single electrodes two different processes, hatching and cutting, have been tested. Due to the interaction of the laser with the metal hatched electrodes had an increased surface area which was investigated with electrochemical measurements. The individual thicknesses of the layers were measured with a novel way of directly laser cutting the electrodes and measuring under a light microscope.

The COSTA Study: Sternal Closure in High-Risk Patients - A Prospective Randomized Multicenter Trial

Background Median sternotomy in patients with risk factors for wound healing is associated with high rates of postoperative wound infections and sternum instability. Methods A total of 338 patients with elective first median sternotomy and at least four predefined risk factors were randomized between Sternal Talon (Gebrüder Martin GmbH & Co. KG—KLS Martin Group, Tuttlingen, Germany) and wire cerclage. The primary end point was mediastinitis and/or sternal instability within 30 ± 5 days, and the secondary end points were mediastinitis and/or sternal instability within 60 ± 5 days; incidence of pneumonia during hospitalization within the first 30 (±5) days and chest pain intensity. Results The primary end point was reached in 10 Sternal Talon and 7 wire cerclage patients (6.2 vs. 4.7%, odds ratio [OR]: 1.3, 95% confidence interval [CI]: 0.5‐3.6, p = 0.57) from 338 randomized patients. Sternal Talon group, n = 170 patients versus wire cerclage group, n = 168 patients. The differences between treatment groups with regard to the incidence of mediastinitis/sternum instability within the first 60 (±5) days after the primary sternum closure and the incidence of pneumonia during the hospitalization within the first 30 (±5) days were not statistically significant, either. We observed comparable rates of superficial surgical site infection (SSI) in Sternal Talon and wire cerclage patients (16.1 vs. 12.1%, OR: 1.4, 95% CI: 0.7‐2.7, p = 0.31). Conclusion According to these data, there is no statistically significant difference between Sternal Talon closure and wire cerclage in reducing the incidence of mediastinitis and superficial SSI after primary closure of median sternotomy in high‐risk patients.