BACKGROUND: Peri-implant health depends on the complex interactions between the dental implant, surrounding soft/hard tissues and the oral microbial environment. However, existing 2D and monoculture models fail to replicate this complexity, limiting their clinical relevance. Therefore, this study aimed to develop a clinically relevant 3D in vitro model that integrates oral soft tissue, hard tissue and a titanium implant in a 3D setup to accurately replicate the peri-implant environment. In addition, the model was designed to integrate bacterial biofilms, in order to mimic incipient peri-implant infections. METHODS: As a hard tissue component, osteoblast-covered HA/TCP scaffold structures were developed and merged with peri-implant mucosa, resulting in a 3D in vitro peri-implant bone-mucosa composite model. The composite model was then cultivated for 2, 7 and 14 days. At each time point, histological analysis, live/dead staining and collagen immunofluorescence staining were performed to assess its structural integrity, osteoblast viability and bone ECM characteristics. To demonstrate proof-of-concept for suitability in simulating implant infection, an oral multispecies biofilm was integrated on top of the implant in the peri-implant bone-mucosa model. RESULTS: Cell viability and osteoblastic phenotype were maintained throughout the study period. Microscopic and histological analyses confirmed a homogenous structure, with a stratified epithelium overlying collagen-embedded human gingival fibroblasts closely connected to the underlying scaffold structure interspersed with bone cells. Combined with a living multispecies biofilm, this model represents several essential components observed in peri-implant interaction. CONCLUSIONS: By combining oral soft tissue, hard tissue and a titanium implant in a 3D setup, this model represents the first and most complex model for evaluating innovative implant materials and novel treatment strategies as well as studying the development of peri-implant diseases. Incorporating different biofilms could enhance the model’s clinical relevance, enabling the study of pro-inflammatory responses to bacterial infections in a setting that includes both soft and hard tissue.

IntroductionUnderstanding the biology of implant-associated infections is essential in order to provide adequate detection, prevention and therapeutic strategies. Advanced 3D in vitro models offer valuable insights into the complex interactions between cells and bacteria in the presence of implant materials. This review aims to give a comprehensive overview of current 3D in vitro models that mimic implant-associated infections.MethodsThe structured literature search initially identified 258 publications, seven of which fitted the inclusion criteria.ResultsThe included 3D models were established either to mimic the in vivo situation (organotypic model) or to investigate future implant materials. In three studies, organotypic models for dental implants were created and one study described an organotypic model containing immune cells. In the remaining three studies, biomaterials for constructing future orthopedic implants were developed and tested. All authors included specific cells and bacteria suitable for the respective implants. The dental implant models used fibroblasts and keratinocytes; the orthopedic implant models used stem cells and fibroblast-like cells; the model containing immune cells incorporated co-cultivation of fibroblasts and THP-1 derived macrophages. For bacterial challenge, most authors used Gram positive bacteria, but three studies employed Gram negative bacterial species. A wide variety of analytical methods of different complexity were applied after co-culture of cells and bacteria and between one and five different methods were used.DiscussionAll models could be employed to provide answers to specific scientific questions regarding implant-associated infections. Nonetheless, this review reveals the limitations of current 3D models for the investigation of implant-associated infections and highlights the opportunities for further development in this scientific field.

Animal models are essential for studying tumor pathophysiology; however, most lack the capacity for repeated in vivo observation of tumor growth and vascularization over extended periods. This study aimed to establish a novel in vivo model using the mouse dorsal skinfold chamber. Tumor induction was performed using different membrane types (two polytetrafluoroethylene meshes and a polydioxanone plate), followed by monitoring of tumor vascularization via intravital fluorescence microscopy (IVM). Tumors developed successfully over six weeks, demonstrating sustained vascular supply and enabling, for the first time, the investigation of vascular networks in advanced tumors. Among the membranes tested, the polydioxanone membrane facilitated easier chamber preparation but may negatively affect angiogenesis and promote inflammation. IVM revealed persistent microcirculation in manifested tumors over six consecutive days, allowing detailed assessment of microvascular parameters, leukocyte–endothelial interactions, and functional capillary density. This model enables repetitive, high-resolution visualization of tumor microcirculation dynamics in vivo. In conclusion, this improved mouse dorsal skinfold chamber combined with IVM provides a powerful tool for investigating tumor angiogenesis and evaluating therapeutic interventions in advanced tumors.

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.

IntroductionEustachian tube dysfunction can cause inadequate ventilation of the middle ear and is therefore one of the main reasons for the development of chronic middle ear infections. This can significantly impact the quality of life. There is a variety of treatment options, but in most cases long-term success is limited. For this reason, stenting the Eustachian tube (ET) may be a safe and effective alternative. Biodegradable stents have been developed that support the impaired ET function until natural ventilation of the middle ear via the ET is restored, and dissolve after their function has been fulfilled.ObjectiveThe purpose of the current study was to investigate feasibility of implantation and degradation kinetics of biodegradable polymer stents in the ET.Study designThe study was set up as an orientation study.MethodsBiodegradable stents (diameter: 3.5 mm; length: 15 mm) were made from Poly-L-lactic acid (PLLA) in a dip-coating process and subsequent laser cutting and were crimped on a balloon catheter. Stent implantation into the ET was performed unilaterally in nine healthy female blackface sheep under endoscopic control with a dedicated insertion tool and an inflation pressure of 10 bar for 2 min. Ventilation of the middle ear was controlled by tympanometry weekly throughout the observation period of 3, 6, or 12 months (three animals each). After the final examination, cone beam computed tomography scans and histological examinations of the ETs were performed.ResultsAll stents could be inserted as planned. Pharyngeal orifices of all sheep were closed at half-time and final follow-up inspections. After 3 months, all stents were functional, but already showed first fragment formation. Six months after insertion, the stents had largely collapsed but still held open a residual lumen and after 12 months, the ETs were completely collapsed close to their natural shape. Fragments of various sizes were found distributed in the tissue beneath an epithelialized ET.ConclusionThe polymeric ET stent can be reliably positioned without generating a patulous ET. It seems to support ET function for about 3 months before functionality is lost.

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 alendronateinduced 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.

Abstract Implant-associated infections are still a major issue in implant surgery. Biofilms tend to form on implant surfaces, like titanium, and can hardly be reached by the immune system. Additionally, systemic treatments with antibiotics often fail and cause severe side effects for the patient. Direct delivery of the antibiotic from the implant surface to the surrounding tissue is one approach to solve this problem. To realize this, the application of pH-sensitive coatings on implant surfaces, which release their cargo only if an infection arises, is a promising option. This can be triggered by the decrease of the pH value occurring in infected tissue. For such pH-sensitive systems with integrated drug loading capacity layer-by-layer coatings with weak polyelectrolytes can be used. Here, we present a coating applied on titanium substrates by dip-coating. As negatively charged polyelectrolyte polyacrylic acid, as positively charged poly(allylamine hydrochloride) is used. As an effective drug, the antibiotic ciprofloxacin is incorporated into the coating and the release profiles are recorded.

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.

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.

Biological factors such as TGF–β3 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–β3 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–β3-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–β3-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.

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/cm 2 , A3: 194 ± 10 cm/cm 2 , B2: 249 ± 19 cm/cm 2 , B3: 264 ± 17 cm/cm 2 ) when compared with controls (A1: 129 ± 10 cm/cm 2 , B1: 185 ± 8 cm/cm 2 ). 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.

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.

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.

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 (β-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 β-TCP scaffolds seeded with MSCs would effectively induce a potent angiogenic response. Therefore, we studied the angiogenic effects after implanting PLGA and β-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 β-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 β-TCP scaffolds compared with PLGA scaffolds. In conclusion, seeding β-TCP scaffolds with MSCs can accelerate vitalization and a combination of both significantly improves angiogenesis.

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.

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.

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.

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.

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.

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 (R) 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. (C) 2017 Elsevier Inc. All rights reserved.