Arteriogenesis or collateral growth is able to compensate for the stenosis of major arteries. Using differential display RT-PCR on growing and quiescent collateral arteries in a rabbit femoral artery ligation model, we cloned the rabbit full-length cDNA of osteoglycin/mimecan. Osteoglycin was present in the adventitia of collateral arteries as a glycosylated protein without keratan sulfate side chains, mainly produced by smooth muscle cells (SMCs) and perivascular fibroblasts. Northern blot, Western blot, and immunohistochemistry confirmed a collateral artery-specific downregulation of osteoglycin from 6 h to 3 weeks after the onset of arteriogenesis. Treatment of primary SMCs with the arteriogenic protein fibroblast growth factor-2 (FGF-2) resulted in a similar reduction of osteoglycin expression as observed in vivo. Application of the FGF-2 inhibitor polyanethole sulfonic acid (PAS) blocked the downregulation of osteoglycin and interfered with arteriogenesis. From our study we conclude that downregulation of osteoglycin is a fundamental requirement for proper arteriogenesis.
Recent evidence suggests a subset of cells within a tumor with "stem-like" characteristics. These cells are able to transplant tumors in immunodeficient hosts. Distinct from non-malignant stem cells, cancer stem cells (CSC) show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumor cells, and resistance to chemotherapy or radiation. They are often characterized by elevated expression of stem cell surface markers, in particular CD133, and sets of differentially expressed stem cell-associated genes. CSC are usually rare in clinical specimens and hardly amenable to functional studies and gene expression profiling. In this study, a panel of heterogenous melanoma cell lines was screened for typical CSC features.Nine heterogeneous metastatic melanoma cell lines including D10 and WM115 were studied. Cell lines were phenotyped using flow cytometry and clonogenic assays were performed by limiting dilution analysis on magnetically sorted cells. Spheroidal growth was investigated in pretreated flasks. Gene expression profiles were assessed by using real-time rt-PCR and DNA microarrays. Magnetically sorted tumor cells were subcutaneously injected into the flanks of immunodeficient mice. Comparative immunohistochemistry was performed on xenografts and primary human melanoma sections.D10 cells expressed CD133 with a significantly higher clonogenic capacity as compared to CD133- cells. Na8, D10, and HBL cells formed spheroids on poly-HEMA-coated flasks. D10, Me39, RE, and WM115 cells expressed at least 2 of the 3 regulatory core transcription factors SOX2, NANOG, and OCT4 involved in the maintenance of stemness in mesenchymal stem cells. Gene expression profiling on CD133+ and CD133- D10 cells revealed 68 up- and 47 downregulated genes (+/-1.3 fold). Two genes, MGP and PROM1 (CD133), were outstandingly upregulated. CD133+ D10 cells formed tumors in NSG mice contrary to CD133- cells and CD133 expression was detected in xenografts and primary human melanoma sections using immunohistochemistry.Established melanoma cell lines exhibit, to variable extents, the typical features of CSCs. The tumorigenic cell line D10, expressing CD133 and growing in spheroids and might qualify as a potential model of melanoma CSCs.
The Eustachian tube (ET) is a bottleneck when it comes to middle ear (ME) health. If its function is impaired, this can lead to serious consequences for the patient, such as hearing problems or deafness. Therefore, this study investigated a tapered nitinol stent (3-5 mm × 14 mm) for the human ET as a potential new permanent treatment for chronic Eustachian tube dysfunction (ETD) and thus ME ventilation disorders. The self-expanding stent was inserted unilaterally into the ET of 24 sheep with observation periods of 3, 6, and 12 months. Local tissue effects and the safety of the stent insertion were analyzed based on regular endoscopic checks, weekly tympanometry measurements, final imaging, and histological examinations. The animals showed no stent-related health restrictions. However, the individual anatomy and stenting procedure had an influence on the results. The tissue reaction in the endoscopic examinations was mild even though no concomitant antibiotics were administered. After all three monitoring periods, stented ETs had a significantly larger ET lumen than the non-stented contralateral ETs. However, tissue growth was detected in the stent. Overall, the first long-term study on an ET stent showed that the tapered ET stent could be a promising treatment option for ETD.
Subperiosteal preparation using a periosteal elevator leads to disturbances of local immunohistochemistry and periosteal histology due to a microtrauma. Usually soft-tissue damage can be considerably reduced by using piezoelectric technology. For this reason, the effects of a novel piezoelectric device on immunohistochemistry and periosteal histology were examined and compared to conventional preparation of the periosteum using a periosteal elevator.Lewis rats were randomly assigned to one of five groups (n=50). Subperiosteal preparation was performed using either a piezoelectric device or a periosteal elevator. Immunohistochemical and histological analyses were performed immediately after preparation as well as three and eight days postoperatively. A statistical analysis of the histological colouring was performed offline using analysis of variance (ANOVA) on ranks (p<0.05).At all times, immunohistochemical and histological analysis demonstrated a significantly more homogenous tissue structure in the group of rats that underwent piezosurgery than in the group of rats that underwent treatment with a periosteal elevator.The use of a piezoelectric device for subperiosteal preparation is associated with more harmonious immunohistochemical and histological results for the periosteum than the use of a conventional periosteal elevator. As a result, piezoelectric devices can be expected to have a positive effect primarily on soft tissue, in particular of the periosteal as well as on surrounding tissues.Einleitung: Subperiostale Präparation mit einem konventionellen Raspatorium führt zu Störungen der lokalen Mikrozirkulation und Immunhistochemie aufgrund von Mikrothromben im Gefäßssystem. Normalerweise kann die Beschädigung von Weichgewebe durch die Anwendung der Piezochirurgie vermeiden werden. Aus diesem Grund wurde der Effekt eines neuen piezoelektrischen Ansatzes auf die Immunhistochemie und die Histologie des Periostes untersucht und mit der konventionellen Präparation mit dem Raspatorium verglichen. Material und Methode: Lewis-Ratten (n=50) wurden randomisiert auf eine von fünf Gruppen aufgeteilt. Die subperiostale Präparation wurde entweder mit dem neuen piezoelektrischen Ansatz oder mit einem konventionellen Raspatorium durchgeführt. Immunhistochemische und histologische Analysen wurden direct, sowie am Tag drei und acht durchgeführt. Eine statistische Auswertung der angefärbten histologischen Schnitte erfolgte offline unter Nutzung einer Varianzanalyse ANOVA (p>0.05).Ergebnisse: Zu allen Zeiten zeigte sich in der immunhistochemischen Betrachtung sowie in der histologischen Betrachtung eine homogenere Struktur bei den Ratten, welche mit dem neuen piezolektrischen Ansatz operiert wurden, im Vergleich zu den Tieren, welche mit dem Raspatorium operiert wurden. Diskussion: Die Anwendung des neuen piezolekzrischen Ansatzes zur subperiostalen Präparation geht einher mit einem harmonischen Bild in den immunhistochemischen und histologischen Schnitten, im Vergleich zur subperiostalen Präparation mit dem Raspatorium. Infolgedessen, wie man erwarten kann, hat der neue piezoelektrische Ansatz eine positive Wirkung in erster Linie auf das Weichgewebe, das Periost sowie auf das Umgebungsgewebe.
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.
Acute and chronic rotator cuff tears remain challenging for therapy. A wide range of therapeutic approaches were developed but re-tears and postoperative complications occur regularly. Especially in elderly people, the natural regeneration processes are decelerated, and graft materials are often necessary to stabilize the tendon-to-bone attachment and to improve the healing process. We here investigated in a small animal model a newly developed electrospun polycaprolactone fiber implant coated with a chitosan-polycaprolactone graft copolymer and compared these implants biomechanically and histologically with either a commercially available porous polyurethane implant (Biomerix 3D Scaffold) or suture-fixed tendons. Fifty-one rats were divided into three groups of 17 animals each. In the first surgery, the left infraspinatus tendons of all rats were detached, and the animals recovered for 4 weeks. In the second surgery, the tendons were fixed with suture material only (suture-fixed group; n = 17), whereas in the two experimental groups, the tendons were fixed with suture material and the polyurethane implant (Biomerix scaffold group; n = 17) or the modified electrospun polycaprolactone fiber implant (CS-g-PCL scaffold group; n=17), respectively. The unaffected right infraspinatus tendons were used as native controls. After a recovery of 8 weeks, all animals were clinically inconspicuous. In 12 animals of each group, repaired entheses were biomechanically tested for force at failure, stiffness, and modulus of elasticity, and in five animals, repaired entheses were analyzed histologically. Biomechanically, all parameters did not differ statistically significant between both implant groups, and the entheses failed typically at the surgical site. However, with respect to the force at failure, the median values of the two implant groups were smaller than the median value of the suture-fixed group. Histologically, the modified polycaprolactone fiber implant showed no acute inflammation processes, a good infiltration with cells, ingrowth of blood vessels and tendinous tissue, and a normal fibrous ensheathment. Further improvement of the implant material could be achieved by additional implementation of drug delivery systems. Therewith, the used CS-g-PCL fiber mat is a promising basic material to reach the goal of a clinically usable graft for rotator cuff tear repair.
Untreated chronic otitis media severely impairs quality of life in affected individuals. Local destruction of the middle ear and subsequent loss of hearing are common sequelae, and currently available treatments provide limited relief. Therefore, the objectives of this study were to evaluate the feasibility of the insertion of a coronary stent from the nasopharynx into the Eustachian tube in-vivo in sheep and to make an initial assessment of its positional stability, tolerance by the animal, and possible tissue reactions. Bilateral implantation of bare metal cobalt-chrome coronary stents of two sizes was performed endoscopically in three healthy blackface sheep using a nasopharyngeal approach. The postoperative observation period was three months. Stent implantation into the Eustachian tube was feasible with no intra- or post-operative complications. Health status of the sheep was unaffected. All stents preserved their cylindrical shape. All shorter stents remained in position and ventilated the middle ear even when partially filled with secretion or tissue. One of the long stents became dislocated toward the nasopharynx. Both of the others remained fixed at the isthmus but appeared to be blocked by tissue or secretion. Tissue overgrowth on top of the struts of all stents resulted in closure of the tissue-lumen interface. Stenting of the Eustachian tube was successfully transferred from cadaver studies to an in-vivo application without complications. The stent was well tolerated, the middle ears were ventilated, and clearance of the auditory tube appeared possible. For fixation, it seems to be sufficient to place it only in the cartilaginous part of the Eustachian tube.
Objectives: CD133 is considered a cancer stem cell (CSC) marker in various malignancies; however, its role as a biomarker of malignant melanoma remains controversial. The present study was conducted to evaluate the suitability of CD133 surface antigen as a CSC marker in melanoma. Methods: Human melanoma cells were fractionally separated by magnetic cell separation depending on the CD133 phenotype and transplanted into immunodeficient mice to evaluate their tumorigenic capacity. Furthermore, the time until the development of a palpable tumor and the growth rate were measured, and the final tumor volume was assessed after 8 weeks. The immunohistochemical expression of CD133 in the induced neoplasia was then compared using histomorphometry. Results: Notably, neoplasms were induced in all the groups (n = 48), including in the CD133-negative group. Tumors induced by unsorted cells had the largest volume (p = 0.014) but were detected significantly later in this group (p ≤ 0.001). Interestingly, all explanted tumors expressed CD133, with no significant differences among groups. Conclusions: In contrast to the results obtained in prior studies, the suitability of CD133 as a CSC marker could not be demonstrated. The current encouraging progress in targeted therapy for malignant melanoma highlights the need to identify more effective targets.
