Alendronate reduces periosteal microperfusion in vivo.

Objectives: Bisphosphonates are known to induce a severe adverse effect known as medication-related osteonecrosis of the jaw (MRONJ). Previous studies have proven the impact of bisphosphonates on microperfusion; therefore, this study aimed to investigate alendronate-induced microcirculatory reactions in the calvarial periosteum of rats. Study design: Bone chambers were implanted into 48 Lewis rats. Microhemodynamics, inflammatory parameters, functional capillary density and defect healing were examined after alendronate treatment for two and six weeks using repetitive intravital fluorescence microscopy for two weeks. Results: Microhemodynamics remained unchanged. In alendronate-treated rats, inflammation was slightly increased, functional capillary density was significantly reduced (day 10: controls 100.45 ± 5.38 cm/cm2, two weeks alendronate treatment 44.77 ± 3.55 cm/cm2, six weeks alendronate treatment 27.54 ± 2.23 cm/cm2) and defect healing was decelerated. The changes in functional capillary density and defect healing were dose-dependent. Conclusion: The bisphosphonate alendronate has a significant negative impact on periosteal microperfusion in vivo. This could be a promising target for the treatment of MRONJ.

Suitability of CD133 as a Marker for Cancer Stem Cells in Melanoma.

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.

Cell seeding accelerates the vascularization of tissue engineering constructs in hypertensive mice.

Rapid blood vessel ingrowth into transplanted constructs represents the key requirement for successful tissue engineering. Seeding three-dimensional scaffolds with suitable cells is an approved technique for this challenge. Since a plethora of patients suffer from widespread diseases that limit the capacity of neoangiogenesis (e.g., hypertension), we investigated the incorporation of cell-seeded poly-L-lactide-co-glycolide scaffolds in hypertensive (BPH/2J, group A) and nonhypertensive (BPN/3J, group B) mice. Collagen-coated scaffolds (A1 and B1) were additionally seeded with osteoblast-like (A2 and B2) and mesenchymal stem cells (A3 and B3). After implantation into dorsal skinfold chambers, inflammation and newly formed microvessels were measured using repetitive intravital fluorescence microscopy for 2 weeks. Apart from a weak inflammatory response in all groups, significantly increased microvascular densities were found in cell-seeded scaffolds (day 14, A2: 192 ± 12 cm/cm2, A3: 194 ± 10 cm/cm2, B2: 249 ± 19 cm/cm2, B3: 264 ± 17 cm/cm2) when compared with controls (A1: 129 ± 10 cm/cm2, B1: 185 ± 8 cm/cm2). In this context, hypertensive mice showed reduced neoangiogenesis in comparison with nonhypertensive animals. Therefore, seeding approved scaffolds with organ-specific or pluripotent cells is a very promising technique for tissue engineering in hypertensive organisms.

TGF-ß3 Loaded Electrospun Polycaprolacton Fibre Scaffolds for Rotator Cuff Tear Repair: An in Vivo Study in Rats.

Biological factors such as TGF-ß3 are possible supporters of the healing process in chronic rotator cuff tears. In the present study, electrospun chitosan coated polycaprolacton (CS-g-PCL) fibre scaffolds were loaded with TGF-ß3 and their effect on tendon healing was compared biomechanically and histologically to unloaded fibre scaffolds in a chronic tendon defect rat model. The biomechanical analysis revealed that tendon-bone constructs with unloaded scaffolds had significantly lower values for maximum force compared to native tendons. Tendon-bone constructs with TGF-ß3-loaded fibre scaffolds showed only slightly lower values. In histological evaluation minor differences could be observed. Both groups showed advanced fibre scaffold degradation driven partly by foreign body giant cell accumulation and high cellular numbers in the reconstructed area. Normal levels of neutrophils indicate that present mast cells mediated rather phagocytosis than inflammation. Fibrosis as sign of foreign body encapsulation and scar formation was only minorly present. In conclusion, TGF-ß3-loading of electrospun PCL fibre scaffolds resulted in more robust constructs without causing significant advantages on a cellular level. A deeper investigation with special focus on macrophages and foreign body giant cells interactions is one of the major foci in further investigations.

Vascularization and biocompatibility of poly(ε-caprolactone) fiber mats for rotator cuff tear repair.

Rotator cuff tear is the most frequent tendon injury in the adult population. Despite current improvements in surgical techniques and the development of grafts, failure rates following tendon reconstruction remain high. New therapies, which aim to restore the topology and functionality of the interface between muscle, tendon and bone, are essentially required. One of the key factors for a successful incorporation of tissue engineered constructs is a rapid ingrowth of cells and tissues, which is dependent on a fast vascularization. The dorsal skinfold chamber model in female BALB/cJZtm mice allows the observation of microhemodynamic parameters in repeated measurements in vivo and therefore the description of the vascularization of different implant materials. In order to promote vascularization of implant material, we compared a porous polymer patch (a commercially available porous polyurethane based scaffold from Biomerix™) with electrospun polycaprolactone (PCL) fiber mats and chitosan-graft-PCL coated electrospun PCL (CS-g-PCL) fiber mats in vivo. Using intravital fluorescence microscopy microcirculatory parameters were analyzed repetitively over 14 days. Vascularization was significantly increased in CS-g-PCL fiber mats at day 14 compared to the porous polymer patch and uncoated PCL fiber mats. Furthermore CS-g-PCL fiber mats showed also a reduced activation of immune cells. Clinically, these are important findings as they indicate that the CS-g-PCL improves the formation of vascularized tissue and the ingrowth of cells into electrospun PCL scaffolds. Especially the combination of enhanced vascularization and the reduction in immune cell activation at the later time points of our study points to an improved clinical outcome after rotator cuff tear repair.

Angiogenic effects of mesenchymal stem cells in combination with different scaffold materials.

Tissue survival in regenerative tissue engineering requires rapid vascularization, which is influenced by scaffold material and seeded cell selection. Poly-l-lactide-co-glycolide (PLGA) and beta-tricalcium phosphate (ß-TCP) are well-established biomaterials with angiogenic effects because of their material properties. Given the importance of the seeded cell type as a co-factor for vascularization, mesenchymal stem cells (MSCs) are known to have high angiogenic potential. We hypothesized that PLGA and ß-TCP scaffolds seeded with MSCs would effectively induce a potent angiogenic response. Therefore, we studied the angiogenic effects after implanting PLGA and ß-TCP scaffolds seeded with isogeneic MSCs in vivo. Fifty-six BALB/c mice were equally divided into seven groups and underwent implantation of the dorsal skinfold chambers. Two MSC groups were seeded on collagen-coated PLGA or ß-TCP scaffolds, whereas groups 3-6 received collagen-coated or uncoated scaffolds without MSCs. No scaffold implantation was performed for group 7, which served as the control. Angiogenesis was assessed in vivo via intravital fluorescence microscopy. Angiogenic responses were noted on all scaffolds, whereupon MSC angiogenic response was significantly enhanced on days 6 and 10. Additionally, a comparison of biomaterials indicated increased angiogenic activity for ß-TCP scaffolds compared with PLGA scaffolds. In conclusion, seeding ß-TCP scaffolds with MSCs can accelerate vitalization and a combination of both significantly improves angiogenesis.

Possibilities and limitations of electrospun chitosan-coated polycaprolactone grafts for rotator cuff tear repair.

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.

CD44(+) tumor cells promote early angiogenesis in head and neck squamous cell carcinoma.

The role of CD44 in progression of head and neck squamous cell carcinoma (HNSCC) has been controversial. The goal of this study was to study the effects of CD44(+) tumor cells on the initial stages of tumor angiogenesis and to evaluate CD44 as a potential marker of tumor angiogenesis. The CD44 gene expression was studied using the Cancer Genome Atlas (TCGA) Head and Neck Cancer data base. Expression levels of CD44 and of microvascular density (MVD) markers were assessed by immunohistochemistry performed with tissue microarrays in a cohort of 49 HNSCC patients, 11 patients with dysplasia and 12 control oral mucosa tissues. The 4-nitroquinoline-1-oxide oral carcinogenesis mouse model was used to study CD44 expression during carcinogenesis. Gelatin sponges seeded with CD44(+), CD44(-) and unsorted cancer cells suspended in Matrigel were implanted in NOD/SCID mice into a dorsal skinfold chamber and compared to non-seeded sponges as controls. Angiogenic response was assessed by intravital microscopy. In the TCGA analysis, CD44 gene expression correlated with various pro-angiogenic genes. In human HNSCC tissues, CD44 expression was upregulated and was associated with blood vessels, although no correlation between MVD and CD44 expression was found. During oral carcinogenesis CD44 expression was upregulated. In dorsal skinfold chambers, CD44(+) cells showed a significantly higher MVD than CD44(-) or unsorted cells (p < 0.001). The results indicate that CD44(+) cells contain pro-angiogenic factors and stimulate tumor angiogenesis in HNSCC. Thus, CD44 might emerge as a potential angiogenic biomarker and a therapeutic target for anti-angiogenic therapies.

The Impact of Different Augmentative Methods on the Expression of Inflammatory Factors.

Many animal studies show that an intact periosteum plays an important role in osseous regeneration. The potential effect of an in vivo periosteal barrier membrane on the expression of specific proteins has not been examined sufficiently. The aim of the present study is to investigate the influence of the flap preparation method and collagen membrane on the emission of inflammatory factors. This study examines 20 patients with dental implants who had previously undergone an augmentation. A soft tissue sample was taken during augmentation and 3 months later from the same location. Samples were always taken from the margins of a previously prepared mucoperiosteal flap. The flap was raised with a conventional periosteal elevator in the control group and with a piezoelectric device in the test group. In both groups, we covered half of the augmented bone with a native collagen membrane (NCM; Geistlich Bio-Gide). This allowed us to examine the same incision area with and without a membrane. An immunohistochemical analysis was performed for collagen IV, fibronectin, and inflammatory factors such as cluster of differentiation 31 (CD31), cyclooxygenase-2 (COX-2), and interleukin 6 (IL-6). There was a clear difference in the expression of specific proteins after the piezoelectric device and the periosteal elevator were used. The expression of fibronectin, IL-6, and COX-2 was higher after preparation with the periosteal elevator than after piezoelectric periosteum dissection. The expression of collagen IV was higher after the piezoelectric procedure. No difference was observed for CD31. The membrane had no effect on the expression of collagen IV, fibronectin, IL-6, and COX-2. The type of periosteal preparation influences the expression of specific proteins. With regard to the factors examined here, NCM did not appear to influence the wound healing cascade.

In vivo analysis of vascularization and biocompatibility of electrospun polycaprolactone fibre mats in the rat femur chamber.

In orthopaedic medicine, connective tissues are often affected by traumatic or degenerative injuries, and surgical intervention is required. Rotator cuff tears are a common cause of shoulder pain and disability among adults. The development of graft materials for bridging the gap between tendon and bone after chronic rotator cuff tears is essentially required. The limiting factor for the clinical success of a tissue engineering construct is a fast and complete vascularization of the construct. Otherwise, immigrating cells are not able to survive for a longer period of time, resulting in the failure of the graft material. The femur chamber allows the observation of microhaemodynamic parameters inside implants located in close vicinity to the femur in repeated measurements in vivo. We compared a porous polymer patch (a commercially available porous polyurethane-based scaffold from Biomerix™) with electrospun polycaprolactone (PCL) fibre mats and chitosan (CS)-graft-PCL modified electrospun PCL (CS-g-PCL) fibre mats in vivo. By means of intravital fluorescence microscopy, microhaemodynamic parameters were analysed repetitively over 20 days at intervals of 3 to 4 days. CS-g-PCL modified fibre mats showed a significantly increased vascularization at Day 10 compared with Day 6 and at Day 14 compared with the porous polymer patch and the unmodified PCL fibre mats at the same day. These results could be verified by histology. In conclusion, a clear improvement in terms of vascularization and biocompatibility is achieved by graft-copolymer modification compared with the unmodified material.

Femtosecond laser-based nanosurgery reveals the endogenous regeneration of single Z-discs including physiological consequences for cardiomyocytes.

A highly organized cytoskeleton architecture is the basis for continuous and controlled contraction in cardiomyocytes (CMs). Abnormalities in cytoskeletal elements, like the Z-disc, are linked to several diseases. It is challenging to reveal the mechanisms of CM failure, endogenous repair, or mechanical homeostasis on the scale of single cytoskeletal elements. Here, we used a femtosecond (fs) laser to ablate single Z-discs in human pluripotent stem cells (hPSC) -derived CMs (hPSC-CM) and neonatal rat CMs. We show, that CM viability was unaffected by the loss of a single Z-disc. Furthermore, more than 40% of neonatal rat and 68% of hPSC-CMs recovered the Z-disc loss within 24 h. Significant differences to control cells, after the Z-disc loss, in terms of cell perimeter, x- and y-expansion and calcium homeostasis were not found. Only 14 days in vitro old hPSC-CMs reacted with a significant decrease in cell area, x- and y-expansion 24 h past nanosurgery. This demonstrates that CMs can compensate the loss of a single Z-disc and recover a regular sarcomeric pattern during spontaneous contraction. It also highlights the significant potential of fs laser-based nanosurgery to physically micro manipulate CMs to investigate cytoskeletal functions and organization of single elements.

Stenting the Eustachian tube to treat chronic otitis media - a feasibility study in sheep.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Examination of Local Periosteal Microcirculation After Application of Absorbable and Nonabsorbable Membranes.

The use of different membranes is common in dentoalveolar surgery. Absorbable and nonabsorbable membranes are used, often beneath the periosteum, to fulfil different functions (as barriers, patches, or spacers). It is still unclear to what extent such membranes affect the biology of the periosteum and what role is played by piezoelectric devices during preparation of the periosteum. We placed two different membranes (absorbable and nonabsorbable) underneath the periosteum of rat calvaria. We prepared the periosteum using different methods (piezoelectric device vs mechanical device). We then examined and analyzed periosteal microcirculation over a period of 28 days. A clear difference was observed between the two methods when used with absorbable membranes: The piezoelectric device offered advantages. Absorbable membranes maintain considerably more local periosteal microcirculation and should be given preference. In addition, we observed an advantage to using a piezoelectric device for periosteal dissection. Therefore, this method should also be used more widely.

CD24+ tumor-initiating cells from oral squamous cell carcinoma induce initial angiogenesis in vivo.

BACKGROUND: In oral squamous cell carcinoma (OSCC), a minor subset of cancer stem cells has been identified using the surface marker CD24. The CD24+ cell population is involved in initiating, maintaining, and expanding tumor growth, but has not been reported to be involved in angiogenesis to date. METHODS: NOD/SCID mice were equipped with dorsal skinfold chambers and gelatin sponges seeded with CD24+, CD24-, and unsorted cancer cells suspended in Matrigel® were implanted. Following intravital fluorescence microscopy, specimens were examined by immunohistology. RESULTS: Sponges seeded with CD24+ cells showed a significantly higher functional capillary density than those seeded with CD24- cells. The presence of endothelial cells was confirmed by immunohistochemistry for CD31. CONCLUSION: For the first time, CD24+ tumorigenic cells with angiogenic potential, which were isolated from OSCC, were characterized. Our findings provide a promising in vivo model to facilitate the development of therapeutic agents against cancer stem cells and their angiogenic pathways.

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.

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.

Decelerated vascularization in tissue-engineered constructs in association with diabetes mellitus in vivo.

AIMS: Rapid blood vessel ingrowth in transplanted tissue engineering constructs is the key factor for successful incorporation, but many potential patients who may use engineered tissues suffer from widespread diseases that limit the capacity of neovascularization (e.g. diabetes). Thus, in vivo vascularization analyses of tissue-engineered constructs in angiogenically affected organisms are required. METHODS: We therefore investigated the in vivo incorporation of collagen-coated and cell-seeded poly-L-lactide-co-glycolide scaffolds in diabetic B6.BKS(D)-Lepr(db)/J mice using repetitive intravital fluorescence microscopy over a time period of two weeks. For this purpose, scaffolds were seeded with osteoblast-like or bone marrow mesenchymal stem cells and implanted into the dorsal skinfold chambers of diabetic and non-diabetic (C57BL/6) mice. RESULTS: Apart from slightly increased inflammatory parameters, diabetic mice showed significantly reduced capillary densities compared with non-diabetic animals from day 6 onward. In line with previous studies, more densely meshed microvascular networks were demonstrated in cell-seeded than in collagen-coated scaffolds from day 6 onward within the single groups (diabetic and control). CONCLUSIONS: A large number of patients who suffer from systemic diseases that affect angiogenesis would profit from tissue engineering. Therefore, the challenge for the clinical introduction of tissue-engineered constructs will be to overcome the decreased angiogenesis in diabetic organisms.

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.

SLM produced porous titanium implant improvements for enhanced vascularization and osteoblast seeding.

To improve well-known titanium implants, pores can be used for increasing bone formation and close bone-implant interface. Selective Laser Melting (SLM) enables the production of any geometry and was used for implant production with 250-µm pore size. The used pore size supports vessel ingrowth, as bone formation is strongly dependent on fast vascularization. Additionally, proangiogenic factors promote implant vascularization. To functionalize the titanium with proangiogenic factors, polycaprolactone (PCL) coating can be used. The following proangiogenic factors were examined: vascular endothelial growth factor (VEGF), high mobility group box 1 (HMGB1) and chemokine (C-X-C motif) ligand 12 (CXCL12). As different surfaces lead to different cell reactions, titanium and PCL coating were compared. The growing into the porous titanium structure of primary osteoblasts was examined by cross sections. Primary osteoblasts seeded on the different surfaces were compared using Live Cell Imaging (LCI). Cross sections showed cells had proliferated, but not migrated after seven days. Although the cell count was lower on titanium PCL implants in LCI, the cell count and cell spreading area development showed promising results for titanium PCL implants. HMGB1 showed the highest migration capacity for stimulating the endothelial cell line. Future perspective would be the incorporation of HMGB1 into PCL polymer for the realization of a slow factor release.

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

BACKGROUND: Humans 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. METHODS: Expression 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. RESULTS: In 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. CONCLUSIONS: HMGA2 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.