Daniel Lindhorst

Consequences of seeded cell type on vascularization of tissue engineering constructs in vivo

Implantation of tissue engineering constructs is a promising technique to reconstruct injured tissue. However, after implantation the nutrition of the constructs is predominantly restricted to vascularization. Since cells possess distinct angiogenic potency, we herein assessed whether scaffold vitalization with different cell types improves scaffold vascularization. 32 male balb/c mice received a dorsal skinfold chamber. Angiogenesis, microhemodynamics, leukocyte-endothelial cell interaction and microvascular permeability induced in the host tissue after implantation of either collagen coated poly (L-lactide-co-glycolide) (PLGA) scaffolds (group 4), additionally seeded with osteoblast-like cells (OLCs, group 1), bone marrow mesenchymal stem cells (bmMSCs, group 2) or a combination of OLCs and bmMSCs (group 3) were analyzed repetitively over 14 days using intravital fluorescence microscopy. Apart from a weak inflammatory response in all groups, vascularization was found distinctly accelerated in vitalized scaffolds, indicated by a significantly increased microvascular density (day 6, group 1: 202+/-15 cm/cm(2), group 2: 202+/-12 cm/cm(2), group 3: 194+/-8 cm/cm(2)), when compared with controls (group 4: 72+/-5 cm/cm(2)). This acceleration was independent from the seeded cell type. Immunohistochemistry revealed in vivo VEGF expression in close vicinity to the seeded OLCs and bmMSCs. Therefore, the observed lack of cell type confined differences in the vascularization process suggests that the accelerated vascularization of vitalized scaffolds is VEGF-related rather than dependent on the potential of bmMSCs to differentiate into specific vascular cells.

Effects of VEGF loading on scaffold-confined vascularization

Adequate vascularization of tissue-engineered constructs remains a major challenge in bone grafting. In view of this, we loaded ß-tricalcium-phosphate (ß-TCP) and porous poly(L-lactide-co-glycolide) (PLGA) scaffolds via collagen coating with vascular endothelial growth factor (VEGF) and studied whether the VEGF loading improves scaffold angiogenesis and vascularization. Dorsal skinfold chambers were implanted into 48 balb/c mice, which were assigned to 6 groups (n = 8 each). Uncoated (controls), collagen-coated, and additionally VEGF-loaded PLGA and ß-TCP scaffolds were inserted into the chambers. Angiogenesis, neovascularization, and leukocyte-endothelial cell interaction were analyzed repeatedly during a 14-day observation period using intravital fluorescence microscopy. Furthermore, VEGF release from PLGA und ß-TCP scaffolds was studied by ELISA. Micromorphology was studied from histological specimens. Unloaded ß-TCP scaffolds showed an accelerated and increased angiogenic response when compared with unloaded PLGA scaffolds. In vitro, PLGA released significantly higher amounts of VEGF compared with ß-TCP at the first two days resulting in a rapid drop of the released amount at the following days up to day 7 where the VEGF release was negligible. Nonetheless, in vivo VEGF loading increased neovascularization, especially in ß-TCP scaffolds. This increased vascularization was associated with a temporary leukocytic response with pronounced leukocyte-endothelial cell interaction at days 3 and 6. Histology revealed adequate host tissue response and engraftment of both ß-TCP and PLGA scaffolds. Our study demonstrates that ß-TCP scaffolds offer more suitable conditions for vascularization than PLGA scaffolds, in particular if they are loaded with VEGF.

The Hannover experience: Surgical treatment of tongue cancer - A clinical retrospective evaluation over a 30 years period

ObjectivesIn this retrospective study, we present a clinical review of our experience with tongue cancer in order to obtain valid criteria for therapeutic decision-making.Materials and methodsBetween 1980 and 2009, a total of 341 patients with squamous cell carcinoma of the tongue were treated at our Department. The average follow-up was 5.2 years. 309 patients received surgical treatment, which was combined in nearly 10% with neoadjuvant and in nearly 20% with postoperative radio(chemo)therapy. 32 patients were excluded from surgery and received primary radiation.ResultsLocal and regional failure occurred in 23.9% and 20.4%, leading to a total failure rate of 37.2% after an average duration of 1,6 years. N-Status, extracapsular spread and clear margins were identified as the dominant factors for survival, which was calculated with 54.5% after 5 years.ConclusionsWe recommend categorical bilateral neck dissection in order to reliably remove occult lymph node metastases. Adjuvant treatment modalities should be applied more frequently in controlled clinical trials and should generally be implemented in cases with unclear margins and lymphatic spread.Clinical relevanceThis study provides new treatment strategies for primary tumour disease and for tumour recurrence.

Additive effect of mesenchymal stem cells and VEGF to vascularization of PLGA scaffolds

Bone marrow derived mesenchymal stem cells (bmMSCs) are widely used for the generation of tissue engineering constructs, since they can differentiate into different cell types occurring in bone tissues. Until now their use for the generation of tissue engineering constructs is limited. All cells inside a tissue engineering construct die within a short period of time after implantation of the construct because vascularization and establishment of connections to the recipient circulatory system is a time consuming process. We therefore compared the influences of bmMSC, VEGF and a combination of both on the early processes of vascularization, utilizing the mice skinfold chamber model and intravital fluorescence microscopy. Tissue engineering constructs based on collagen coated Poly d,l-lactide-co-glycolide (PLGA) scaffolds, were either functionalized by coating with vascular endothelial growth factor (VEGF) or vitalized with bmMSC. PLGA without cells and growth factor was used as the control group. Functionalized and vitalized tissue engineering constructs showed an accelerated growth of microvessels compared to controls. Only marginal differences in vascular growth were detected between VEGF containing and bmMSC containing constructs. Constructs containing VEGF and bmMSC showed a further enhanced microvascular growth at day 14. We conclude that bmMSCs are well suited for bone tissue engineering applications, since they are a valuable source of angiogenic growth factors and are able to differentiate into the tissue specific cell types of interest. The dynamic process of vascularization triggered by growth factor producing cells can be amplified and stabilized with the addition of accessory growth factors, leading to a persisting angiogenesis, but strategies are needed that enhance the resistance of bmMSC to hypoxia and increase survival of these cells until the tissue engineering construct has build up a functional vascular system.

Accelerating the early angiogenesis of tissue engineering constructs in vivo by the use of stem cells cultured in matrigel

In tissue engineering research, generating constructs with an adequate extent of clinical applications remains a major challenge. In this context, rapid blood vessel ingrowth in the transplanted tissue engineering constructs is the key factor for successful incorporation. To accelerate the microvascular development in engineered tissues, we preincubated osteoblast-like cells as well as mesenchymal stem cells or a combination of both cell types in Matrigel-filled PLGA scaffolds before transplantation into the dorsal skinfold chambers of balb/c mice. By the use of preincubated mesenchymal stem cells, a significantly accelerated angiogenesis was achieved. Compared with previous studies that showed a decisive increase of vascularization on day 6 after the implantation, we were able to halve this period and achieve explicitly denser microvascular networks 3 days after transplantation of the tissue engineering constructs. Thereby, the inflammatory host tissue response was acceptable and low, comparable with former investigations. A co-incubation of osteoblast-like cells and stem cells showed no additive effect on the density of the newly formed microvascular network. Preincubation of mesenchymal stem cells in Matrigel is a promising approach to develop rapid microvascular growth into tissue engineering constructs. After the implantation into the host organism, scaffolds comprising stem cells generate microvascular capillary-like structures exceptionally fast. Thereby, transplanted stem cells likely differentiate into vessel-associated cells. For this reason, preincubation of mesenchymal stem cells in nutrient solutions supporting different steps of angiogenesis provides a technique to promote the routine use of tissue engineering in the clinic.

Virtual 3D tumor marking-exact intraoperative coordinate mapping improve post-operative radiotherapy

The quality of the interdisciplinary interface in oncological treatment between surgery, pathology and radiotherapy is mainly dependent on reliable anatomical three-dimensional (3D) allocation of specimen and their context sensitive interpretation which defines further treatment protocols. Computer-assisted preoperative planning (CAPP) allows for outlining macroscopical tumor size and margins. A new technique facilitates the 3D virtual marking and mapping of frozen sections and resection margins or important surgical intraoperative information. These data could be stored in DICOM format (Digital Imaging and Communication in Medicine) in terms of augmented reality and transferred to communicate patients specific tumor information (invasion to vessels and nerves, non-resectable tumor) to oncologists, radiotherapists and pathologists.

Patient-specific biodegradable implant in pediatric craniofacial surgery

Surgical correction of premature fusion of calvarial sutures involving the fronto-orbital region can be challenging due to the demanding three-dimensional (3D) anatomy. If fronto-orbital advancement (FOA) is necessary, surgery is typically performed using resorbable plates and screws that are bent manually intraoperatively. A new approach using individually manufactured resorbable implants (KLS Martin Group, Tuttlingen, Germany) is presented in the current paper. Preoperative CT scan data were processed in iPlan (ver. 3.0.5; Brainlab, Feldkirchen, Germany) to generate a 3D reconstruction. Virtual osteotomies and simulation of the ideal outer contour with reassembled bony segments were performed. Digital planning was transferred with a cutting guide, and an individually manufactured resorbable implant was used for rigid fixation. A resorbable patient-specific implant (Resorb X-PSI) allows precise surgery for FOA in craniosynostosis using a complete digital workflow and should be considered superior to manually bent resorbable plates.

Comparably accelerated vascularization by preincorporation of aortic fragments and mesenchymal stem cells in implanted tissue engineering constructs

The demanding need for tissue replacement resulted in manifold approaches for the construction of different tissues. One common problem which hampers the clinical usage of tissue engineering constructs is a limited vascularization. In an attempt to accelerate the vascularization of tissue engineering constructs we compared the usage of bone marrow mesenchymal stem cells (bmMSCs) and fragments derived from the aorta in vivo. Tissue engineering constructs composed of PLGA scaffolds containing Matrigel (n = 8), aortic fragments embedded in Matrigel (n = 8), bmMSCs embedded in Matrigel (n = 8), and aortic fragments embedded in Matrigel combined with bmMSCs (n = 8) were implanted into dorsal skinfold chambers of balb/c mice and analyzed repetitively over 14 days. In all groups a weak inflammatory response was transiently apparent. Vascularization was significantly (p = 0.05) accelerated in bmMSC and aortic fragments containing constructs compared with Matrigel alone, demonstrated by a distinctly increased microvascular density throughout the whole experiment. The combination of bmMSCs and aortic fragments showed no additional effect compared with bmMSCs and aortic fragments alone. The accelerated vascularization and microvascular density of tissue engineering constructs triggered by bmMSCs and aortic fragments is comparable. Thus aortic fragments provide a new promising source for clinical relevant tissue engineering constructs.

Optic nerve monitoring.

Orbital and anterior skull base surgery is generally performed close to the prechiasmatic visual pathway, and clear strategies for detecting and handling visual pathway damage are essential. To overcome the common problem of a missed clinical examination because of an uncooperative or unresponsive patient, flash visual evoked potentials and electroretinograms should be used. These electrophysiologic examination techniques can provide evidence of intact, pathologic, or absent conductivity of the visual pathway when clinical assessment is not feasible. Visual evoked potentials and electroretinograms are thus essential diagnostic procedures not only for primary diagnosis but also for intraoperative evaluation. A decision for or against treatment of a visual pathway injury has to be made as fast as possible due to the enormous importance of the time elapsed with such injuries; this can be achieved additionally using multislice spiral computed tomography. The first-line conservative treatment of choice for such injuries is megadose methylprednisolone therapy. Surgery is used to decompress the orbital compartment by exposure of the intracanalicular part of the optic nerve in the case of optic canal compression. Modern craniomaxillofacial surgery requires detailed consideration of the diagnosis and treatment of traumatic visual pathway damage with the ultimate goal of preserving visual acuity.

Nonradiological Method for 3-dimensional Implant Position Assessment Using an Intraoral Scan: New Method for Postoperative Implant Control

Introduction: This study presents a method to verify the position of dental implants after insertion without the repeated x-ray exposure. Material and Methods: An implant was inserted into the natural gap between the canines and premolars of 8 domestic pigs and 1 human patient. A scanbody was then connected to the implants and a digital intraoral impression of the jaw segment was acquired using a handheld scanner. In addition, the implant position was radiologically detected by cone beam computed tomography. The position of the implant based on both techniques was compared by digital matching. Results: The position of the dental implants determined by the scanner accurately represents the position in the radiograph in the pigs and also in the human patient. Conclusion: Evaluating the position of implants using intraoral scans is a straightforward, accurate, and radiation-free method of 3-dimensional implant position determination.