Andreas Kampmann

Poly-ε-caprolactone Coated and Functionalized Porous Titanium and Magnesium Implants for Enhancing Angiogenesis in Critically Sized Bone Defects.

For healing of critically sized bone defects, biocompatible and angiogenesis supporting implants are favorable. Murine osteoblasts showed equal proliferation behavior on the polymers poly-ε-caprolactone (PCL) and poly-(3-hydroxybutyrate)/poly-(4-hydroxybutyrate) (P(3HB)/P(4HB)). As vitality was significantly better for PCL, it was chosen as a suitable coating material for further experiments. Titanium implants with 600 µm pore size were evaluated and found to be a good implant material for bone, as primary osteoblasts showed a vitality and proliferation onto the implants comparable to well bottom (WB). Pure porous titanium implants and PCL coated porous titanium implants were compared using Live Cell Imaging (LCI) with Green fluorescent protein (GFP)-osteoblasts. Cell count and cell covered area did not differ between the implants after seven days. To improve ingrowth of blood vessels into porous implants, proangiogenic factors like Vascular Endothelial Growth Factor (VEGF) and High Mobility Group Box 1 (HMGB1) were incorporated into PCL coated, porous titanium and magnesium implants. An angiogenesis assay was performed to establish an in vitro method for evaluating the impact of metallic implants on angiogenesis to reduce and refine animal experiments in future. Incorporated concentrations of proangiogenic factors were probably too low, as they did not lead to any effect. Magnesium implants did not yield evaluable results, as they led to pH increase and subsequent cell death.

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.

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.

HMGA1 and HMGA2 expression and comparative analyses of HMGA2, Lin28 and let-7 miRNAs in oral squamous cell carcinoma

BackgroundHumans and dogs are affected by squamous cell carcinomas of the oral cavity (OSCC) in a considerably high frequency. The high mobility group A2 (HMGA2) protein was found to be highly expressed in human OSCC and its expression was suggested to act as a useful predictive and prognostic tool in clinical management of oral carcinomas. Herein the expression of HMGA2 and its sister gene HMGA1 were analysed within human and canine OSCC samples. Additionally, the HMGA negatively regulating miRNAs of the let-7 family as well as the let-7 regulating gene Lin28 were also comparatively analysed. Deregulations of either one of these members could affect the progression of human and canine OSCC.MethodsExpression levels of HMGA1, HMGA2, Lin28, let-7a and mir-98 were analysed via relative qPCR in primary human and canine OSCC, thereof derived cell lines and non-neoplastic samples. Additionally, comparative HMGA2 protein expression was analysed by immunohistochemistry.ResultsIn both species, a significant up-regulation of the HMGA2 gene was found within the neoplastic samples while HMGA1 expression did not show significant deregulations. Comparative analyses showed down-regulation of mir-98 in human samples and up-regulation of let-7a and mir-98 in canine neoplastic samples. HMGA2 immunostainings showed higher intensities within the invasive front of the tumours than in the centre of the tumour in both species.ConclusionsHMGA2 could potentially serve as tumour marker in both species while HMGA1 might play a minor role in OSCC progression. Comparative studies indicate an inverse correlation of HMGA2 and mir-98 expression in human samples whereas in dogs no such characteristic could be found.

Putative CD133 + melanoma cancer stem cells induce initial angiogenesis in vivo

Tumor angiogenesis is essential for tumor growth and metastasis, and is regulated by a complex network of various types of cells, chemokines, and stimulating factors. In contrast to sprouting angiogenesis, tumor angiogenesis is also influenced by hypoxia, inflammation, and the attraction of bone-marrow-derived cells. Recently, cancer stem cells have been reported to mimic vascularization by differentiating into endothelial cells and inducing vessel formation. In this study, the influence of cancer stem cells on initial angiogenesis was evaluated for the metastatic melanoma cell line D10. Following flow cytometry, CD133+ and CD133- cells were isolated using magnetic cell separation and different cell fractions were transferred to porcine gelatin sponges, which were implanted into the dorsal skinfold chamber of immunocompromised mice. Angiogenesis was analyzed based on microvessel density over a 10-day period using in vivo fluorescence microscopy, and the results were verified using immunohistology. CD133+ D10 cells showed a significant induction of early angiogenesis in vivo, contrary to CD133- D10 cells, unsorted D10 cells, and negative control. Neovascularization was confirmed by visualizing endothelial cells by immunohistology using an anti-CD31 antibody. Because CD133+ cells are rare in clinical specimens and hardly amenable to functional assays, the D10 cell line provides a suitable model to study the angiogenic potential of putative cancer stem cells and the leukocyte-endothelial cell interaction in the dorsal skinfold chamber in vivo. This cancer stem cell model might be useful in the development and evaluation of therapeutic agents targeting tumors.

Dentin as a suitable bone substitute comparable to ß-TCP—an experimental study in mice

Microvascular supply is of fundamental importance to the survival and integration of grafting. Since the autogenous bone is still the gold standard for osseous augmentation, the aim of this study was to analyze the initial osseous, angiogenic and inflammatory response and subsequent osseointegration after implantation of dentin and beta-tricalcium phosphate (ß-TCP) scaffolds into the calvaria chamber of balb/c mice comparing with bone. The vascularisation of perforated implants of dentin (n=8), ß-TCP (n=8) and isogenic calvarial bone (n=8) displaying pores similar in size and structure was analyzed in vivo using intravital fluorescence microscopy. In additional animals (n=24) the osseointegration of dentin, ß-TCP and bone implants was assessed by fluorochrome sequential labelling of growing bone for up to 12 weeks. Animals without implants served as controls. Intravital fluorescence microscopy revealed that implantation of bone substitutes caused an only mild inflammatory response. Comparable to isogenic bone both dentin and ß-TCP scaffolds were found nearly completely vascularized by day 22 and osseointegrated within 12 weeks. In conclusion, dentin and ß-TCP scaffolds are similar to isogenic bone in terms of inflammatory and neovascularization response, highlighting their potential utility in regeneration of bone defects.

Comparison of different harvesting methods from the flat and long bones of rats

Different harvesting methods have been developed for bony augmentation before implantation. The aim of the present study was to assess the viability of endochondral (femoral) and membranous (mandibular) bone cells harvested by different methods under standard conditions in an animal model, and to investigate the surface of the bone in the harvested area. Samples of mandibular and femoral bone were harvested using a drilling burr, a piezoelectrical device, or a Safescraper(®). Blocks of bone that had been harvested with cutting forceps were used as controls. The size of the samples was measured and they were examined by conventional microscopy and immunohistochemical analysis; osteoblast-like cells were also cultured. The surface of the harvested area was analysed with scanning and conventional microscopy. There was no significant difference between mandibular and femoral bone in the size of particles harvested, but bone chips were significantly smaller when a drilling device had been used in both harvesting areas. Viability of cells in these smaller particles was significantly less than among cells harvested with a piezoelectrical device or Safescraper(®). Scanning microscopy showed a smooth bony surface where a drilling burr or piezoelectrical device had been used, whereas small disruptions were observed after the Safescraper(®) had been used. Harvesting of particulate bone is feasible using a drilling burr, piezoelectrical device, or Safescraper(®) from mandibular and femoral bone. The piezoelectrical device and the Safescraper(®) gave comparable results concerning the viability of osteoblast-like cells, and so are preferred to a drilling burr.

Comparison of Selective Laser Melted Titanium and Magnesium Implants Coated with PCL

Degradable implant material for bone remodeling that corresponds to the physiological stability of bone has still not been developed. Promising degradable materials with good mechanical properties are magnesium and magnesium alloys. However, excessive gas production due to corrosion can lower the biocompatibility. In the present study we used the polymer coating polycaprolactone (PCL), intended to lower the corrosion rate of magnesium. Additionally, improvement of implant geometry can increase bone remodeling. Porous structures are known to support vessel ingrowth and thus increase osseointegration. With the selective laser melting (SLM) process, defined open porous structures can be created. Recently, highly reactive magnesium has also been processed by SLM. We performed studies with a flat magnesium layer and with porous magnesium implants coated with polymers. The SLM produced magnesium was compared with the titanium alloy TiAl6V4, as titanium is already established for the SLM-process. For testing the biocompatibility, we used primary murine osteoblasts. Results showed a reduced corrosion rate and good biocompatibility of the SLM produced magnesium with PCL coating.