Adipose-derived stem cells applied in skin diseases, wound healing and skin defects: a review.

Adipose tissue presents a comparably easy source for obtaining stem cells, and more studies are increasingly investigating the therapeutic potential of adipose-derived stem cells. Wound healing, especially in chronic wounds, and treatment of skin diseases are some of the fields investigated. In this narrative review, the authors give an overview of some of the latest studies concerning wound healing as well as treatment of several skin diseases and concentrate on the different forms of application of adipose-derived stem cells.

In Vitro and In Vivo Effects of IGF-1 Delivery Strategies on Tendon Healing: A Review.

Tendon injuries suffer from a slow healing, often ending up in fibrovascular scar formation, leading to inferior mechanical properties and even re-rupture upon resumption of daily work or sports. Strategies including the application of growth factors have been under view for decades. Insulin-like growth factor-1 (IGF-1) is one of the used growth factors and has been applied to tenocyte in vitro cultures as well as in animal preclinical models and to human patients due to its anabolic and matrix stimulating effects. In this narrative review, we cover the current literature on IGF-1, its mechanism of action, in vitro cell cultures (tenocytes and mesenchymal stem cells), as well as in vivo experiments. We conclude from this overview that IGF-1 is a potent stimulus for improving tendon healing due to its inherent support of cell proliferation, DNA and matrix synthesis, particularly collagen I, which is the main component of tendon tissue. Nevertheless, more in vivo studies have to be performed in order to pave the way for an IGF-1 application in orthopedic clinics.

Bioactive and Elastic Emulsion Electrospun DegraPol Tubes Delivering IGF-1 for Tendon Rupture Repair.

Tendon injuries can result in two major drawbacks. Adhesions to the surrounding tissue may limit the range of motion, while fibrovascular scar formation can lead to poor biomechanical outcomes. Prosthetic devices may help to mitigate those problems. Emulsion electrospinning was used to develop a novel three-layer tube based on the polymer DegraPol (DP), with incorporated insulin-like growth factor-1 (IGF-1) in the middle layer. Scanning electron microscopy was utilized to assess the fiber diameter in IGF-1 containing pure DP meshes. Further characterization was performed with Fourier Transformed Infrared Spectroscopy, Differential Scanning Calorimetry, and water contact angle, as well as through the assessment of mechanical properties and release kinetics from ELISA, and the bioactivity of IGF-1 by qPCR of collagen I, ki67, and tenomodulin in rabbit Achilles tenocytes. The IGF-1-containing tubes exhibited a sustained release of the growth factor up to 4 days and showed bioactivity by significantly upregulated ki67 and tenomodulin gene expression. Moreover, they proved to be mechanically superior to pure DP tubes (significantly higher fracture strain, failure stress, and elastic modulus). The novel three-layer tubes intended to be applied over conventionally sutured tendons after a rupture may help accelerate the healing process. The release of IGF-1 stimulates proliferation and matrix synthesis of cells at the repair site. In addition, adhesion formation to surrounding tissue can be reduced due to the physical barrier.

Impact of High-Molecular-Weight Hyaluronic Acid on Gene Expression in Rabbit Achilles Tenocytes In Vitro.

(1) Background: Surgical tendon repair often leads to adhesion formation, leading to joint stiffness and a reduced range of motion. Tubular implants set around sutured tendons might help to reduce peritendinous adhesions. The lubricant hyaluronic acid (HA) is a viable option for optimizing such tubes with the goal of further enhancing the anti-adhesive effect. As the implant degrades over time and diffusion is presumed, the impact of HA on tendon cells is important to know. (2) Methods: A culture medium of rabbit Achilles tenocytes was supplemented with high-molecular-weight (HMW) HA and the growth curves of the cells were assessed. Additionally, after 3, 7 and 14 days, the gene expression of several markers was analyzed for matrix assembly, tendon differentiation, fibrosis, proliferation, matrix remodeling, pro-inflammation and resolution. (3) Results: The addition of HA decreased matrix marker genes, downregulated the fibrosis marker α-SMA for a short time and slightly increased the matrix-remodeling gene MMP-2. Of the pro-inflammatory marker genes, only IL-6 was significantly upregulated. IL-6 has to be kept in check, although IL-6 is also needed for a proper initial inflammation and efficient resolution. (4) Conclusions: The observed effects in vitro support the intended anti-adhesion effect and therefore, the use of HMW HA is promising as a biodegradable implant for tendon repair.

Adipose tissue and the vascularization of biomaterials: Stem cells, microvascular fragments and nanofat-a review.

Tissue defects in the human body after trauma and injury require precise reconstruction to regain function. Hence, there is a great demand for clinically translatable approaches with materials that are both biocompatible and biodegradable. They should also be able to adequately integrate within the tissue through sufficient vascularization. Adipose tissue is abundant and easily accessible. It is a valuable tissue source in regenerative medicine and tissue engineering, especially with regard to its angiogenic potential. Derivatives of adipose tissue, such as microfat, nanofat, microvascular fragments, stromal vascular fraction and stem cells, are commonly used in research, but also clinically to enhance the vascularization of implants and grafts at defect sites. In plastic surgery, adipose tissue is harvested via liposuction and can be manipulated in three ways (macro-, micro- and nanofat) in the operating room, depending on its ultimate use. Whereas macro- and microfat are used as a filling material for soft tissue injuries, nanofat is an injectable viscous extract that primarily induces tissue remodeling because it is rich in growth factors and stem cells. In contrast to microfat that adds volume to a defect site, nanofat has the potential to be easily combined with scaffold materials due to its liquid and homogenous consistency and is particularly attractive for blood vessel formation. The same is true for microvascular fragments that are easily isolated from adipose tissue through collagenase digestion. In preclinical animal models, it has been convincingly shown that these vascular fragments inosculate with host vessels and subsequently accelerate scaffold perfusion and host tissue integration. Adipose tissue is also an ideal source of stem cells. It yields larger quantities of cells than any other source and is easier to access for both the patient and doctor compared with other sources such as bone marrow. They are often used for tissue regeneration in combination with biomaterials. Adipose-derived stem cells can be applied unmodified or as single cell suspensions. However, certain pretreatments, such as cultivation under hypoxic conditions or three-dimensional spheroids production, may provide substantial benefit with regard to subsequent vascularization in vivo due to induced growth factor production. In this narrative review, derivatives of adipose tissue and the vascularization of biomaterials are addressed in a comprehensive approach, including several sizes of derivatives, such as whole fat flaps for soft tissue engineering, nanofat or stem cells, their secretome and exosomes. Taken together, it can be concluded that adipose tissue and its fractions down to the molecular level promote, enhance and support vascularization of biomaterials. Therefore, there is a high potential of the individual fat component to be used in regenerative medicine.

Supporting Cell-Based Tendon Therapy: Effect of PDGF-BB and Ascorbic Acid on Rabbit Achilles Tenocytes in Vitro.

Cell-based tendon therapies with tenocytes as a cell source need effective tenocyte in vitro expansion before application for tendinopathies and tendon injuries. Supplementation of tenocyte culture with biomolecules that can boost proliferation and matrix synthesis is one viable option for supporting cell expansion. In this in vitro study, the impacts of ascorbic acid or PDGF-BB supplementation on rabbit Achilles tenocyte culture were studied. Namely, cell proliferation, changes in gene expression of several ECM and tendon markers (collagen I, collagen III, fibronectin, aggrecan, biglycan, decorin, ki67, tenascin-C, tenomodulin, Mohawk, α-SMA, MMP-2, MMP-9, TIMP1, and TIMP2) and ECM deposition (collagen I and fibronectin) were assessed. Ascorbic acid and PDGF-BB enhanced tenocyte proliferation, while ascorbic acid significantly accelerated the deposition of collagen I. Both biomolecules led to different changes in the gene expression profile of the cultured tenocytes, where upregulation of collagen I, Mohawk, decorin, MMP-2, and TIMP-2 was observed with ascorbic acid, while these markers were downregulated by PDGF-BB supplementation. Vice versa, there was an upregulation of fibronectin, biglycan and tenascin-C by PDGF-BB supplementation, while ascorbic acid led to a downregulation of these markers. However, both biomolecules are promising candidates for improving and accelerating the in vitro expansion of tenocytes, which is vital for various tendon tissue engineering approaches or cell-based tendon therapy.

Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration.

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell-material interaction, the material-medium interaction was also important for the stem cell commitment here, affecting the cell-medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge.

Cellular self-assembly into 3D microtissues enhances the angiogenic activity and functional neovascularization capacity of human cardiopoietic stem cells.

While cell therapy has been proposed as next-generation therapy to treat the diseased heart, current strategies display only limited clinical efficacy. Besides the ongoing quest for the ideal cell type, in particular the very low retention rate of single-cell (SC) suspensions after delivery remains a major problem. To improve cellular retention, cellular self-assembly into 3D microtissues (MTs) prior to transplantation has emerged as an encouraging alternative. Importantly, 3D-MTs have also been reported to enhance the angiogenic activity and neovascularization potential of stem cells. Therefore, here using the chorioallantoic membrane (CAM) assay we comprehensively evaluate the impact of cell format (SCs versus 3D-MTs) on the angiogenic potential of human cardiopoietic stem cells, a promising second-generation cell type for cardiac repair. Biodegradable collagen scaffolds were seeded with human cardiopoietic stem cells, either as SCs or as 3D-MTs generated by using a modified hanging drop method. Thereafter, seeded scaffolds were placed on the CAM of living chicken embryos and analyzed for their perfusion capacity in vivo using magnetic resonance imaging assessment which was then linked to a longitudinal histomorphometric ex vivo analysis comprising blood vessel density and characteristics such as shape and size. Cellular self-assembly into 3D-MTs led to a significant increase of vessel density mainly driven by a higher number of neo-capillary formation. In contrast, SC-seeded scaffolds displayed a higher frequency of larger neo-vessels resulting in an overall 1.76-fold higher total vessel area (TVA). Importantly, despite that larger TVA in SC-seeded group, the mean perfusion capacity (MPC) was comparable between groups, therefore suggesting functional superiority together with an enhanced perfusion efficacy of the neo-vessels in 3D-MT-seeded scaffolds. This was further underlined by a 1.64-fold higher perfusion ratio when relating MPC to TVA. Our study shows that cellular self-assembly of human cardiopoietic stem cells into 3D-MTs substantially enhances their overall angiogenic potential and their functional neovascularization capacity. Hence, the concept of 3D-MTs may be considered to increase the therapeutic efficacy of future cell therapy concepts.

Serotonin uptake is required for Rac1 activation in Kras-induced acinar-to-ductal metaplasia in the pancreas.

Pancreatic ductal adenocarcinoma (PDAC), which is the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical for developing alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacological approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin [5-hydroxytryptamine (5-HT)] is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up 5-HT in a transport-mediated manner. 5-HT uptake promoted activation of the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for transdifferentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the 5-HT transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell-autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected the proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of ADM, with the potential to stall the initiation and progression of pancreatic carcinogenesis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Correspondence of high-frequency ultrasound and histomorphometry of healing rabbit Achilles tendon tissue.

OBJECTIVES: Static and dynamic high-frequency ultrasound of healing rabbit Achilles tendons were set in relationship to histomorphometric analyses at three and six weeks post-surgery. MATERIALS AND METHODS: Twelve New Zealand White rabbits received a clean-cut Achilles tendon laceration (the medial and lateral Musculus gastrocnemius) and were repaired with a four-strand Becker suture. Six rabbits got additionally a tight polyester urethane tube at the repair site in order to vary the adhesion extent. Tendons were analysed by static and dynamic ultrasound (control: healthy contralateral legs). The ultrasound outcome was corresponded to the tendon shape, tenocyte and tenoblast density, tenocyte and tenoblast nuclei width, collagen fibre orientation and adhesion extent. RESULTS: The spindle-like morphology of healing tendons (ultrasound) was confirmed by the swollen epitenon (histology). Prediction of adhesion formation by dynamic ultrasound assessment was confirmed by histology (contact region to surrounding tissue). Hyperechogenic areas corresponded to acellular zones with aligned fibres and hypoechogenic zones to not yet oriented fibres and to cell-rich areas. CONCLUSIONS: These findings add new in-depth structural knowledge to the established non-invasive analytical tool, ultrasound.

Delineation of the healthy rabbit duodenum by immunohistochemistry - A short communication.

The duodenum acts as a vital organ that performs fundamental physiological functions like digestion and nutrient absorption. Situated in the lower abdomen, the duodenum is located between the stomach and the jejunum. Usually, the duodenum is divided into four anatomical portions. We here compare paraffin embedded and cryosections of the healthy rabbit duodenum for research purposes. This analysis evaluates the differential outcomes resulting from the application of these fixation methodologies in conjunction with immunohistochemical assays targeting extracellular matrix markers collagen I, collagen III, fibronectin, α-smooth muscle actin (α-SMA), and proliferation marker ki67 as well as inflammatory marker PAR-2. Subsequent recommendations are provided based on our findings. Furthermore, the advantage of an antigen retrieval step in immunohistochemical labelling of paraffin sections was demonstrated and confirmed with an isotype negative control. Basic classical histological stainings as HE, GT and elastin were also performed. Comparison of different stainings and labellings was performed in serial sections, showing that adjacent to the circular muscle of the duodenum, the connective tissue was composed of collagen I and fibronectin, while the artery and vein walls were predominantly α-SMA positive. Moreover, PAR-2 immunohistochemical staining was performed, where particularly a type of gland adjacent to Brunner's glands showed prominent PAR-2 positive areas, while the Brunner's glands themselves were PAR-2 negative. Proliferating ki67 positive cells facing the lumen were highly abundant in all kinds of glands except for the Brunner's glands. This effort serves to furnish the research community with reference imagery pertinent to scientists opting for the rabbit duodenum model. The diversity of staining techniques employed herein establishes a foundational repository of images, primed for comparative analysis against pathological conditions. Furthermore, these images hold the potential to illustrate inter-species variations. For instance, they can be juxtaposed against murine or rat intestinal tracts, or even offer insights into the human context.

Delineation of the healthy rabbit tongue by immunohistochemistry - A technical note.

In the oral cavity the tongue is an important muscular organ that supports the swallowing of food and liquids. It is responsible for the sense of taste, based on the many different taste buds it contains. Research in the field of tongue diseases demands for suitable preclinical models. The healthy rabbit tongue may therefore serve as baseline and reference for the pathological situation. With this consideration, we covered the fixation and histological stainings as well as the immunohistochemical labelling of the healthy rabbit tongue. In this technical note, initial choice of the fixative is discussed, with a comparison of formalin fixation and subsequent paraffin embedding versus cryopreservation. Moreover, we delineate the effect of an antigen retrieval step for formalin fixation by several examples. Finally, we provide ECM markers collagen I, collagen III, fibronectin, α-SMA and elastin staining as well as ki67 for proliferative status and PAR-2 protein expression as a marker for inflammatory status and nociception in tongue sections, mainly from the tongue body. Technically, we found superiority of paraffin sections for collagen I, collagen III, fibronectin, ki67 and α-SMA labelling, for selected detections systems. As for ECM components, the lamina propria was very rich in collagen and fibronectin, while the muscular body of the tongue showed only collagen and fibronectin positive areas between the muscle fibers. Moreover, α-SMA was clearly expressed in the walls of arteries and veins. The inflammatory marker PAR-2 on the other hand was prominently expressed in the salivary glands and to some extent in the walls of the vessels. Particular PAR-2 expression was found in the excretory ducts of the tongue. This technical note has the aim to provide baseline images that can be used to compare the pathological state of the diseased rabbit tongue as well as for inter-species comparison, such as mouse or rat tongue. Finally, it can be used for the comparison with the human situation.

Ultrasonic Coating of Poly(D,L-lactic acid)/Poly(lactic-co-glycolic acid) Electrospun Fibers with ZnO Nanoparticles to Increase Angiogenesis in the CAM Assay.

Critical-size bone defects necessitate bone void fillers that should be integrated well and be easily vascularized. One viable option is to use a biocompatible synthetic polymer and sonocoat it with zinc oxide (ZnO) nanoparticles (NPs). However, the ideal NP concentration and size must be assessed because a high dose of ZnO NPs may be toxic. Electrospun PDLLA/PLGA scaffolds were produced with different concentrations (0.5 or 1.0 s of sonocoating) and sizes of ZnO NPs (25 nm and 70 nm). They were characterized by SEM, EDX, ICP-OES, and the water contact angle. Vascularization and integration into the surrounding tissue were assessed with the CAM assay in the living chicken embryo. SEM, EDX, and ICP-OES confirmed the presence of ZnO NPs on polymer fibers. Sonocoated ZnO NPs lowered the WCA compared with the control. Smaller NPs were more pro-angiogenic exhibiting a higher vessel density than the larger NPs. At a lower concentration, less but larger vessels were visible in an environment with a lower cell density. Hence, the favored combination of smaller ZnO NPs at a lower concentration sonocoated on PDLLA/PLGA electrospun meshes leads to an advanced state of tissue integration and vascularization, providing a valuable synthetic bone graft to be used in clinics in the future.

Hyaluronic acid/PEO electrospun tube reduces tendon adhesion to levels comparable to native tendons - An in vitro and in vivo study.

A major problem after tendon injury is adhesion formation to the surrounding tissue leading to a limited range of motion. A viable strategy to reduce adhesion extent is the use of physical barriers that limit the contact between the tendon and the adjacent tissue. The purpose of this study was to fabricate an electrospun bilayered tube of hyaluronic acid/polyethylene oxide (HA/PEO) and biodegradable DegraPol® (DP) to improve the anti-adhesive effect of the implant in a rabbit Achilles tendon full laceration model compared to a pure DP tube. Additionally, the attachment of rabbit tenocytes on pure DP and HA/PEO containing scaffolds was tested and Scanning Electron Microscopy, Fourier-transform Infrared Spectroscopy, Differential Scanning Calorimetry, Water Contact Angle measurements, and testing of mechanical properties were used to characterize the scaffolds. In vivo assessment after three weeks showed that the implant containing a second HA/PEO layer significantly reduced adhesion extent reaching levels comparable to native tendons, compared with a pure DP implant that reduced adhesion formation only by 20 %. Tenocytes were able to attach to and migrate into every scaffold, but cell number was reduced over two weeks. Implants containing HA/PEO showed better mechanical properties than pure DP tubes and with the ability to entirely reduce adhesion extent makes this implant a promising candidate for clinical application in tendon repair.

Pro-angiogenic and antibacterial copper containing nanoparticles in PLGA/amorphous calcium phosphate bone nanocomposites.

Large bone defects after trauma demand for adequate bone substitutes. Bone void fillers should be antibacterial and pro-angiogenic. One viable option is the use of composite materials like the combination of PLGA and amorphous calcium phosphate (aCaP). Copper stimulates angiogenesis and has antibacterial qualities. Either copper oxide (CuO) nanoparticles (NPs) were therefore added to PLGA/aCaP/CuO in different concentrations (1, 5 and 10 w/w %) or copper-doped tricalcium phosphate NPs (TCP with 2% of copper) were electrospun into PLGA/CuTCP nanocomposites. Bi-layered nanocomposites of PLGA/aCaP with different copper NPs (CuO or TCP) and a second layer of pristine PLGA were fabricated. Two clinical bacterial isolates (Staphylococcus aureus and Staphylococcus epidermidis) were used to assess antibacterial properties of the copper-containing materials. For angiogenesis, the chorioallantoic membrane (CAM) assay of the chicken embryo was performed. The higher the CuO content, the higher were the antibacterial properties, with 10 % CuO reducing bacterial adhesion most effectively. Vessel and cell densities were highest in the 5 % CuO containing scaffolds, while tissue integration was more pronounced at lower CuO content. The PLGA/aCaP/CuO (1 % CuO) behaved similar like PLGA/CuTCP in all angiogenic and antibacterial readouts, based on the same copper fraction. We conclude that CuO NPs or CuTCP NPs are useful components to increase angiogenic properties of nanocomposites and at the same time exhibiting antibacterial characteristics.

In Vitro Assessment of Bacterial Adhesion and Biofilm Formation on Novel Bioactive, Biodegradable Electrospun Fiber Meshes Intended to Support Tendon Rupture Repair.

The surgical repair of a ruptured tendon faces two major problems: specifically increased fibrous adhesion to the surrounding tissue and inferior mechanical properties of the scar tissue compared to the native tissue. Bacterial attachment to implant materials is an additional problem as it might lead to severe infections and impaired recovery. To counteract adhesion formation, two novel implant materials were fabricated by electrospinning, namely, a random fiber mesh containing hyaluronic acid (HA) and poly(ethylene oxide) (PEO) in a ratio of 1:1 (HA/PEO 1:1) and 1:4 (HA/PEO 1:4), respectively. Electrospun DegraPol (DP) treated with silver nanoparticles (DP-Ag) was developed to counteract the bacterial attachment. The three novel materials were compared to the previously described DP and DP with incorporated insulin-like growth factor-1 (DP-IGF-1), two implant materials that were also designed to improve tendon repair. To test whether the materials are prone to bacterial adhesion and biofilm formation, we assessed 10 strains of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Enterococcus faecalis, known for causing nosocomial infections. Fiber diameter, pore size, and water contact angle, reflecting different degrees of hydrophobicity, were used to characterize all materials. Generally, we observed higher biofilm formation on the more hydrophobic DP as compared to the more hydrophilic DP-IGF-1 and a trend toward reduced biofilm formation for DP treated with silver nanoparticles. For the two HA/PEO implants, a similar biofilm formation was observed. All tested materials were highly prone to bacterial adherence and biofilm formation, pointing toward the need of further material development, including the optimized incorporation of antibacterial agents such as silver nanoparticles or antibiotics.

Yield and proliferation rate of adipose-derived stromal cells as a function of age, body mass index and harvest site-increasing the yield by use of adherent and supernatant fractions?

BACKGROUND AIMS: Adipose-derived stem cells are easily accessed and have a relatively high density compared with other mesenchymal stromal cells. Isolation protocols of adipose-derived stem cells (ASC) rely on the cell's ability to adhere to tissue culture plastic overnight. It was evaluated whether the floating ASC fractions are also of interest for cell-based therapies. In addition, the impact of age, body mass index (BMI) and harvest site was assessed. METHODS: The surface protein profile with the use of flow cytometry, the cell yield and the doubling time of passages 4, 5 and 6 of ASC from 30 donors were determined. Adherent and supernatant fractions were compared. The impact of age, BMI and harvest site on cell yield and doubling times was determined. RESULTS: Both adherent and supernatant fractions showed high mean fluorescence intensities for CD13, CD29, CD44, CD73, CD90 and CD105 and comparatively low mean fluorescence intensities for CD11b, CD62L, intracellular adhesion molecule-1 and CD34. Doubling times of adherent and supernatant fractions did not differ significantly. Whereas the old age group had a significantly lower cell yield compared with the middle aged group, BMI and harvest site had no impact on cell yield. Finally, doubling times for passages 4, 5 and 6 were not influenced by the age and BMI of the donors, nor the tissue-harvesting site. CONCLUSIONS: The floating ASC fraction is an equivalent second cell source just like the adherent ASC fraction. Donor age, BMI and harvest site do not influence cell yield and proliferation rate.

Transition metals in angiogenesis - A narrative review.

The aim of this paper is to offer a narrative review of the literature regarding the influence of transition metals on angiogenesis, excluding lanthanides and actinides. To our knowledge there are not any reviews up to date offering such a summary, which inclined us to write this paper. Angiogenesis describes the process of blood vessel formation, which is an essential requirement for human growth and development. When the complex interplay between pro- and antiangiogenic mediators falls out of balance, angiogenesis can quickly become harmful. As it is so fundamental, both its inhibition and enhancement take part in various diseases, making it a target for therapeutic treatments. Current methods come with limitations, therefore, novel agents are constantly being researched, with metal agents offering promising results. Various transition metals have already been investigated in-depth, with studies indicating both pro- and antiangiogenic properties, respectively. The transition metals are being applied in various formulations, such as nanoparticles, complexes, or scaffold materials. Albeit the increasing attention this field is receiving, there remain many unanswered questions, mostly regarding the molecular mechanisms behind the observed effects. Notably, approximately half of all the transition metals have not yet been investigated regarding potential angiogenic effects. Considering the promising results which have already been established, it should be of great interest to begin investigating the remaining elements whilst also further analyzing the established effects.

Delineation of the healthy rabbit tonsil by immunohistochemistry - A short communication.

Situated in the oral cavity, the rabbit palatine tonsils are part of the mucosal immune system and help to defend the body against foreign pathogens. Expressed as two oval protrusions in the wall of the oropharynx, the rabbit palatine tonsils are characterized by excretory ducts and trabeculae. We here compare paraffin embedded and cryosections of the healthy rabbit tonsils. This analysis centers on evaluating the differential outcomes resulting from the application of these fixation methodologies in conjunction with immunohistochemical assays targeting collagen I, collagen III, fibronectin, α-smooth muscle actin (α-SMA), and ki67. Subsequent recommendations are provided based on our findings. Furthermore, we demonstrate the advantage of an antigen retrieval step in immunohistochemical labeling of paraffin sections. Basic classical histological stainings as HE, GT and elastin were also performed. Comparison of different stainings and labelings was furthermore performed in serial sections, showing that adjacent to the excretory ducts, the tonsillar tissue was particularly composed of collagen I and fibronectin, while the vessel walls were predominantly α-SMA positive. Moreover, PAR-2 immunohistochemical staining was performed, where a small fraction of the cells found in the tonsillar connective tissue were PAR-2 positive (probably a subpopulation of mast cells), as well as the lumen of some excretory ducts and trabeculae. Collagen III on the other hand was only weakly expressed in the tonsils. Proliferating ki67 positive cells were rare. This endeavor serves to furnish the scientific community with reference imagery pertinent to researchers opting for the rabbit palatine tonsil model. The diversity of staining techniques employed herein establishes a foundational repository of images, primed for comparative analysis against pathological conditions. Furthermore, these images hold the potential to illustrate inter-species variations. For instance, they can be juxtaposed against murine or rodent tonsils, or even offer insights into the human context.

Delineation of the healthy rabbit heart by immunohistochemistry - A technical note.

Heart failure poses a big health problem and may result from obesity, smoking, alcohol and/or growing age. Studying pathological heart tissue demands accurate histological and immunohistochemical stainings in animal models, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy heart stainings and labeling are required, in order to provide a reference for the pathological situation. Heart and brain tissue of a healthy rabbit were collected, and different histological key steps were compared, such as paraffin embedding after formalin fixation versus cryopreservation; an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or a chromogenic with a fluorescent detection system, respectively. Using serial sections, we stained the same morphological structure with classic approaches (HE, Masson Goldner Trichrome (GT) and Elastica van Gieson (EL)) and with different markers, including collagen I, collagen III, fibronectin, α-SMA, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, and ki67 for proliferating cells. Differences between conditions were quantitatively assessed by measuring the color intensity. Generally, cryosections exhibited a more prominent signal intensity in immunohistochemically labeled sections than in paraffin sections, but the strong staining was slurry, which sometimes impeded proper identification of morphological structures, particularly at higher magnifications. In addition, the advantage of an AR step was observed when compared to the condition without AR, where signal intensities were significantly lower. Different stainings of the heart arteries and the myocardium revealed a clear distribution of extracellular matrix components, with prominent collagen III in the artery wall, but an absence of collagen III in the myocardium. Moreover, paraffin-embedded sections provided more distinct structures compared to cryosections after collagen III, ki67, fibronectin, and α-SMA labeling. As for the Purkinje cells that were depicted in the heart and the cerebellum (Purkinje neurons), we found GT staining most suitable to depict them in the heart, while HE as well as EL staining was ideal to depict Purkinje neurons in the cerebellum. In sum, we provide useful reference images with different stainings for researchers using the rabbit heart or brain model. Such images can help to decide which of the immunohistochemical protocols are valuable to reach a specific aim. Recommendations are given for the best visualization of the target structures and specific (immunohistochemical) staining.