Paul Schumann

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.

Sandwich osteotomy for vertical and transversal augmentation of the posterior mandible

The aim of this study is to describe the treatment outcome after alveolar ridge augmentation in the atrophic posterior mandible by segmental sandwich osteotomy combined with an interpositional autograft prior to placement of endosseous implants. Thirteen consecutive patients (five males, mean age 48 years, and eight females, mean age 61 years) were included in this study. The postoperative course was uneventful in six patients. Sensory disturbances in the mental nerve were found in five patients, all of them with hypoaesthesia. None of these patients complained of permanent sensory disturbances. Vertical gain ranged from 2.0 to 7.8mm (mean value 4.61mm). Horizontal gain ranged from 2.0 to 6.3 (mean value 3.42mm). A total of 41 implants were placed in 22 surgical sites, 12 weeks after bone reconstruction. In conclusion, segmental mandibular sandwich osteotomy is recommended to meet the dimensional requirements of preimplant bone augmentation in atrophic posterior mandible.

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.

Forensic imaging of projectiles using cone-beam computed tomography

In patients with gunshot injuries, it is easy to detect a projectile within the body due to the high-density of the object, but artefacts make it difficult to obtain information about the deformation and the exact location of the projectile in surrounding tissues. Cone-beam computed tomography (CBCT) is a new radiological imaging modality that allows radio-opaque objects to be localised and assessed in three dimensions. The full potential of the use of CBCT in forensic medicine has not yet been explored. In this study, three different modern projectiles were fired into the heads of pig cadavers (n=6) under standardised conditions. Tissue destruction and the location of the projectiles were analysed separately using CBCT and multi-slice computed tomography (MDCT). The projectiles had the same kinetic energy but showed considerable differences in deformation behaviour. Within the study groups, tissue destruction was reproducible. CBCT is less severely affected by metallic artefacts than MDCT. Therefore CBCT is superior in visualising bone destruction in the immediate vicinity of the projectile and projectile deformation, whereas MDCT allows soft tissue to be evaluated in more detail. CBCT is an improved diagnostic tool for the evaluation of gunshot injuries. In particular, it is superior to MDCT in detecting structural hard-tissue damage in the immediate vicinity of high-density metal projectiles and in identifying the precise location of a projectile in the body.

En bloc prefabrication of vascularized bioartificial bone grafts in sheep and complete workflow for custom-made transplants

The aim of this pilot study was to determine, in a new experimental model, whether complex bioartificial monoblocs of relevant size and stability can be prefabricated in a defined three-dimensional design, in which the latissimus dorsi muscle serves as a natural bioreactor and the thoracodorsal vessel tree is prepared for axial construct perfusion. Eighteen sheep were included in the study, with six animals in each of three experimental groups. Vitalization of the β-tricalcium phosphate-based constructs was performed by direct application of unmodified osteogenic material from the iliac crest (group A), in vivo application of nucleated cell concentrate (NCC) from bone marrow aspirate (group B), and in vitro cultivation of bone marrow stromal cells (BMSC) in a perfusion bioreactor system (group C). The contours of the constructs were designed digitally and transferred onto the bioartificial bone grafts using a titanium cage, which was bent over a stereolithographic model of the defined subvolume intraoperatively. At the end of the prefabrication process, only the axial vascularized constructs of group A demonstrated vital bone formation with considerable stability. In groups B and C, the applied techniques were not able to induce ectopic bone formation. The presented computer-assisted workflow allows the prefabrication of custom-made bioartificial transplants.

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.

A new model for chronic in vivo analysis of the periosteal microcirculation

OBJECTIVE Microvascular perfusion is indispensable for the growth and remodulation of membrane bone. Trauma, inflammation and surgical interventions may alter periosteal perfusion. However, there is not much known about periosteal perfusion in membrane bones. Therefore, the aim of this study was to establish a new chronic model that permits the repeated in vivo analysis of the microcirculation of periosteum. METHODS A circular skin island with a diameter of 6 mm was excised at the forehead of six Lewis rats. Then a newly developed chamber was implanted, containing a coverglass to protect the periosteum. Intravital microscopy (IVM) enables a quantitative analysis of periosteal microcirculation immediately as well as 3, 5 and 10 days after chamber implantation. At the end of the experiment the calvaria and periosteum were removed for histological examination. Six unoperated Lewis rats served as histological controls. RESULTS The periosteal microcirculation remained stable over 10 days. The implantation of the chamber did not result in any substantial inflammatory response. The functional microvascular density was 131.2+/-19.3 cm/cm(2). The histological examinations revealed a regular anatomical structure of periosteum and bone including an intact interface. CONCLUSION The presented model allows for the first time to conduct a repetitive, quantitative in vivo analysis of the periosteal microcirculation in membrane bone. Future studies may thus evaluate novel strategies to influence the periosteal perfusion.

Osseous alterations at the interface of hydrogel expanders and underlying bone

INTRODUCTION In plastic and reconstructive surgery, self-activating hydrogel expanders are used to augment soft-tissue space. The purpose of this study was to investigate the morphological response of underlying bone to the constant pressure exerted by a hydrogel expander. METHODS Eighteen Lewis rats were randomly divided into three groups. In group 1, a hydrogel expander was placed subperiosteally directly onto the calvaria of the rats. In group 2, the expander and the underlying bone were separated by a polydioxanone (PDS) foil. Group 3 animals served as controls. Before and 14 days after the insertion of the expanders, micro-computed tomography (CT) images were obtained and fused. We analysed hydroxyapatite density beneath and at the periphery of the expander and performed a histomorphometric bone analysis. RESULTS Whereas there were no significant differences (p<0.05) (groups 1 and 2) in bone density at the periphery of the expanders between the study groups, a significant decrease in hydroxyapatite density beneath the expanders was observed in those animals in which the devices were placed directly onto the calvaria (group 1). Whereas bone thickness was unaffected at the periphery of the expanders in all groups, it was significantly decreased beneath the expanders in all implanted animals. A morphological examination revealed resorption lacunae with a diameter of 218.4+/-56 mICROm in those rats in which the expanders had been placed directly onto the calvaria. CONCLUSION This study shows the direct influence of hydrogel expanders on underlying bone. Whereas bone resorption and connective tissue formation also occur underneath hydrogel expanders, these effects can be avoided if the expander and the underlying bone are separated by PDS foil. The key to success is to ensure the appropriate placement of expanders and thus to avoid bone resorption.