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.

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.

Electrospun tube reduces adhesion in rabbit Achilles tendon 12 weeks post-surgery without PAR-2 overexpression.

One great challenge in surgical tendon repair is the minimization of peritendinous adhesions. An electrospun tube can serve as a physical barrier around a conventionally sutured tendon. Six New Zealand White rabbits had one Achilles tendon fully transsected and sutured by a 4-strand suture. Another six rabbits had the same treatment, but with the additional electrospun DegraPol tube set around the sutured tendon. The adhesion formation to the surrounding tissue was investigated 12 weeks post-operation. Moreover, inflammation-related protease-activated receptor-2 (PAR-2) protein expression was assessed. Finally, rabbit Achilles tenocyte cultures were exposed to platelet-derived growth factor-BB (PDGF-BB), which mimicks the tendon healing environment, where PAR-2 gene expression was assessed as well as immunofluorescent staining intensity for F-actin and α-tubulin, respectively. At 12 weeks post-operation, the partially degraded DegraPol tube exhibited significantly lower adhesion formation (- 20%). PAR-2 protein expression was similar for time points 3 and 6 weeks, but increased at 12 weeks post-operation. In vitro cell culture experiments showed a significantly higher PAR-2 gene expression on day 3 after exposure to PDGF-BB, but not on day 7. The cytoskeleton of the tenocytes changed upon PDGF-BB stimulation, with signs of reorganization, and significantly decreased F-actin intensity. An electrospun DegraPol tube significantly reduces adhesion up to twelve weeks post-operation. At this time point, the tube is partially degraded, and a slight PAR-2 increase was detected in the DP treated tendons, which might however arise from particles of degrading DegraPol that were stained dark brown. PAR-2 gene expression in rabbit tenocytes reveals sensitivity at around day 10 after injury.

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

Liver diseases pose a big global health problem and liver failure may result from viral infection, overnutrition or tumors. Studying pathologic liver tissue demands for accurate and specific histological stainings and immunohistochemical labellings, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy liver stainings and labellings are required, to provide a baseline or reference for the pathological situation. Here, we used the liver tissue of a healthy rabbit and compared different histological key steps, such as paraffin embedding after formalin fixation versus cryopreservation; or an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or chromogenic with fluorescent detection system, respectively. Moreover, we provide images of serial sections, where we stained the same morphological structure with different markers, including collagen I, collagen III, fibronectin, α-SMA, elastin, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, ki67 for proliferating cells, and orcein (as negative control for pathological aberrations like Wilson disease). Differences between conditions were quantitatively assessed by measuring the colour intensity. Generally, we observed that cryosections exhibited a stronger signal intensity in immunohistochemically labelled sections than in paraffin sections; however, the strong staining got slurred, which sometimes hampered proper identification of morphological structures at higher magnifications. Moreover, there was a clear increase in signal intensity for paraffin sections when an AR step was performed compared to condition without AR. Results for mouse isotype staining as a negative control clearly supported those findings. Different stainings of the portal triad, the central vein and the bile ducts revealed a clear-cut distribution of extracellular matrix components, with prominent fibronectin and elastin around the lumen of the central vein as well as a patchy PAR-2 expression. As for the bile ducts, complete absence of α-SMA and PAR-2 was found at the margins, however, collagen I expression and elastin were positive and showed a strong signal. Like this, we provide useful and valuable reference images for researchers using the rabbit liver model. It may help to decide which of the immunohistochemical protocols are valuable to reach a certain aim and which protocols lead to the best visualization of the target structure.

Delineation of the healthy rabbit lung by immunohistochemistry - a technical note.

Investigation and studies of pulmonary diseases and injuries require pre-clinical animal models. The rabbit lung model is widely used and allows for a diverse set of readouts. Among them, histology and immunohistochemistry are of invaluable merit because qualitative and quantitative information about tissue morphology and composition can be easily obtained. In this technical note, we performed several histological and immunohistochemical stainings in the rabbit healthy naïve lung tissue. Overnight formalin fixation with subsequent paraffin embedding was compared to cryopreservation with a subsequent 10-minute formalin fixation prior to staining. Antigen retrieval (AR) for paraffin embedded sections proved to enhance the corresponding signals compared to analogous staining without AR. Advantages and disadvantages of chromogenic versus immunofluorescence stainings were discussed. In addition, several morphological structures, such as the intrapulmonary bronchus with its mucosal folds, the pulmonary artery, the alveoli and the lymph nodes, were stained with various stainings at the same site in order to give a comprehensive picture of their composition. Besides Haematoxylin&Eosin and Elastica van Gieson staining, collagen I, collagen III, fibronectin, α-SMA, ki-67 and protease-activated receptor-2 (PAR-2) immunohistochemistry was performed. Collagen I, collagen III and fibronectin expression was positive at the outer rim of the pulmonary arteries, while the inner rim was collagen III positive. Moreover, the fibronectin staining in the intrapulmonary bronchus showed an opposite trend when compared to the collagen III staining. The alveoli exhibited PAR-2 expression, while PAR-2 was not expressed in lymph nodes of the healthy rabbit lung.

Elastic and surgeon friendly electrospun tubes delivering PDGF-BB positively impact tendon rupture healing in a rabbit Achilles tendon model.

A major problem after tendon laceration is the low mechanical strength of the repaired tissue. One viable strategy for improving the functional and biomechanical properties of ruptured and repaired tendons is the delivery of growth factors at the injury site. Here, bioactive and reversibly expandable double-layered emulsion and coaxially electrospun tubes made from biodegradable DegraPol® (DP) (polyester urethane), delivering platelet-derived growth factor BB (PDGF-BB), are explored as implants to improve tendon healing in a rabbit Achilles tendon full laceration model. In vitro studies showed that both emulsion and coaxially electrospun scaffolds allow sustained delivery of bioactive PDGF-BB with similar release kinetics (150-190 pg PDGF-BB/mg of DP scaffold) over a period of 30 days. In vivo assessment after three weeks showed that PDGF-BB delivery through the bioactive DP tubes increased the tensile strength of the treated tendons 2-fold without additional pro-fibrotic effects, i.e., cell hyperproliferation or increase in α-smooth muscle actin expression at the wound site. While no major differences in ECM composition at the wound site were observed for ± PDGF-BB treated samples, collagen I and III were upregulated and fibronectin was downregulated compared to native tendons. In areas away from the wound, increased fibronectin expression was observed qualitatively in regions with lower collagen I and III expression. Both types of bioactive DP tubes provided surgeon-friendly and stable implants to deliver bioactive molecules and positively affected the strength of the repaired tendons after 3 weeks, thus presenting promising bioactive implants for clinical applications in the tendon repair field.

Hybrid nanocomposite as a chest wall graft with improved integration by adipose-derived stem cells.

Surgery of the chest wall is potentially required to cover large defects after  removal of malignant tumours. Usually, inert and non-degradable Gore-Tex serves to replace the missing tissue. However, novel biodegradable materials combined with stem cells are available that stimulate the healing. Based on poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) and pure PLGA, a dual layer biodegradable hybrid nanocomposite was generated. Mouse adipose-derived stem cells were cultered on electrospun disks (ASCs of C57BL/6), and biomechanical tests were performed. The cell-seeded scaffolds were engrafted in C57BL/LY5.1 mice to serve as a chest wall substitute. Cell invasion into the bi-layered material, extent of CD45+ cells, inflammatory response, neo-vascularization and ECM composition were determined at 1 and 2 months post-surgery, respectively. The bi-layered hybrid nanocomposite was stable after a 2-week in vitro culture, in contrast to PLGA/aCaP without a PLGA layer. There was a complete biointegration and good vascularization in vivo. The presence of ASCs attracted more CD45+ cells (hematopoietic origin) compared to cell-free scaffolds. Inflammatory reaction was similar for both groups (±ASCs) at 8 weeks. A bi-layered hybrid nanocomposite fabricated of electrospun PLGA/aCaP and a reinforcing layer of pristine PLGA is an ideal scaffold for chest wall reconstruction. It is stable and allows a proper host tissue integration. If ASCs are seeded, they attract more CD45+ cells, supporting the regeneration process.

Cartilage/bone interface fabricated under perfusion: Spatially organized commitment of adipose-derived stem cells without medium supplementation.

Tissue engineering of an osteochondral interface demands for a gradual transition of chondrocyte- to osteoblast-prevailing tissue. If stem cells are used as a single cell source, an appropriate cue to trigger the desired differentiation is the use of composite materials with different amounts of calcium phosphate. Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) in weight ratios of 100:0; 90:10, 80:20, and 70:30 were seeded with human adipose-derived stem cells (ASCs) and cultured in DMEM without chemical supplementation. After 2 weeks of static cultivation, they were either further cultivated statically for another 2 weeks (group 1), or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm-2 min-1 (group 2). Markers for stem cell criteria, chondrogenesis, osteogenesis, adipogenesis and angiogenesis were analyzed by quantitative real-time PCR. Cell distribution, Sox9 protein expression and proteoglycans were assessed by histology. In group 2 (perfusion culture), chondrogenic Sox9 was upregulated toward the cartilage-mimicking side compared to pure PLGA. On the bone-mimicking side, Sox9 experienced a downregulation, which was confirmed on the protein level. Vice versa, expression of osteocalcin was upregulated on the bone-mimicking side, while it was unchanged on the cartilage-mimicking side. In group 1 (static culture), CD31 was upregulated in the presence of aCaP compared to pure PLGA, whereas Sox9 and osteocalcin expression were not affected. aCaP nanoparticles incorporated in electrospun PLGA drive the differentiation behavior of human ASCs in a dose-dependent manner. Discrete gradients of aCaP may act as promising osteochondral interfaces. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1833-1843, 2019.

Tissue mechanics of piled critical size biomimetic and biominerizable nanocomposites: Formation of bioreactor-induced stem cell gradients under perfusion and compression.

BACKGROUND: Perfusion bioreactors are used to solve problems in critical size bone tissue engineering. Biominerizable and biocompatible nanocomposites are suitable scaffold materials for this purpose because they offer mineral components in organic carriers. Human adipose derived stem cells (ASCs) can potentially be used to increase bone healing. MATERIALS AND METHODS: Electrospun nanocomposite disks of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/a-CaP) were seeded with ASCs and eight disks were stacked in a bioreactor running with normal culture. Under perfusion and uniaxial cyclic compression, load-displacement curves as a function of time were assessed. Stiffness and energy dissipation were recorded. Moreover, stem cell densities in the layers of the piled scaffold were determined as well as their morphologies and differentiation status. RESULTS: While the stiffness of the cell free constructs increased over time based on the transformation of the a-CaP nanoparticles into flake-like apatite, ASC-seeded constructs showed a constant stiffness. Stem cell density gradients had a linear increase from the bottom to the top of the pile (r(2)>0.95). Stem cells were getting more roundish at higher flow rates. Some osteogenesis was found upon osteopontin immunostaining, while no endothelial cell differentiation and no chondrogenesis was triggered. CONCLUSIONS: The fabrication of a critical size bone graft is presented based on a biominerizable bone-biomimetic nanocomposite with preserved stiffness when seeded with ASCs. The cell densities of ASCs inside the piled construct varied with a linear gradient. Beginning osteogenesis was triggered by the dynamic culture conditions including perfusion and compression.

Synthesis, characterization and histomorphometric analysis of cellular response to a new elastic DegraPol® polymer for rabbit Achilles tendon rupture repair.

Tendon rupture repair is a surgical field where improvements are still required due to problems such as repeat ruptures, adhesion formation and joint stiffness. In the current study, a reversibly expandable and contractible electrospun tube based on a biocompatible and biodegradable polymer was implanted around a transected and conventionally sutured rabbit Achilles tendon. The material used was DegraPol® (DP), a polyester urethane. To make DP softer, more elastic and surgeon-friendly, the synthesis protocol was slightly modified. Material properties of conventional and new DP film electrospun meshes are presented. At 12 weeks post-surgery, tenocyte and tenoblast density, nuclei and width, collagen fibre structure and inflammation levels were analyzed histomorphometrically. Additionally, a comprehensive histological scoring system by Stoll et al. (2011) was used to compare healing outcomes. Results showed that there were no adverse reactions of the tendon tissue following the implant. No differences were found whether the DP tube was applied or not for both traditional and new DP materials. As a result, the new DP material was shown to be an excellent carrier for delivery of growth factors, stem cells and other agents responsible for tendon healing.

Proliferation of ASC-derived endothelial cells in a 3D electrospun mesh: impact of bone-biomimetic nanocomposite and co-culture with ASC-derived osteoblasts.

BACKGROUND: Fractures with a critical size bone defect are associated with high rates of delayed- and non-union. The treatment of such complications remains a serious issue in orthopaedic surgery. Adipose derived stem cells (ASCs) combined with biomimetic materials can potentially be used to increase fracture healing. Nevertheless, a number of requirements have to be fulfilled; in particular, the insufficient vascularisation of the bone constructs. Here, the objectives were to study the impact of ASC-derived osteoblasts on ASC-derived endothelial cells in a 3D co-culture and the effect of 40wt% of amorphous calcium phosphate nanoparticles on the proliferation and differentiation of ASC-derived endothelial cells when present in PLGA. MATERIALS AND METHODS: Five primary ASC lines were differentiated towards osteoblasts (OBs) and endothelial cells (ECs) and two of them were chosen based on quantitative PCR results. Either a mono-culture of ASC-derived EC or a co-culture of ASC-derived EC with ASC-derived OB (1:1) was seeded on an electrospun nanocomposite of poly-(lactic-co-glycolic acid) and amorphous calcium phosphate nanoparticles (PLGA/a-CaP; reference: PLGA). The proliferation behaviour was determined histomorphometrically in different zones and the expression of von Willebrand Factor (vWF) was quantified. RESULTS: Independently of the fat source (biologic variability), ASC-derived osteoblasts decelerated the proliferation behaviour of ASC-derived endothelial cells in the co-culture compared to the mono-culture. However, expression of vWF was clearly stronger in the co-culture, indicating further differentiation of the ASC-derived EC into the EC lineage. Moreover, the presence of a-CaP nanoparticles in the scaffold slowed the proliferation behaviour of the co-culture cells, too, going along with a further differentiation of the ASC-derived OB, when compared to pure PLGA scaffolds. CONCLUSIONS: This study revealed significant findings for bone tissue-engineering. Co-cultures of ASC-derived EC and ASC-derived OB stimulate each other's further differentiation. A nanocomposite with a-CaP nanoparticles offers higher mechanical stability, bioactivity and osteoconductivity compared to mere PLGA and can easily be seeded with pre-differentiated EC and OB.

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.

2D motion analysis of rabbits after Achilles tendon rupture repair and histological analysis of extracted tendons: can the number of animals be reduced by operating both hind legs simultaneously?

BACKGROUND: Considering the 3Rs principle in animal experiments, there is a demand to perform research experiments with the fewest number of animals possible while warranting the welfare of the animals. Orthopaedic experimental studies involving operations on the hind legs of rabbits are either performed on one hind leg with the second hind leg serving as control or on both hind legs simultaneously (control: rabbits with no operations at all). METHODS: The Achilles tendon of rabbits was transected and sutured, and the two-dimensional motion pattern of animals having only one leg operated was compared to rabbits having both hind legs operated (control: non-treated animals). Step length, maximum ankle angle, minimum ankle angle and the resulting range of motion of both hind legs were determined weekly over a time span from 3 weeks to 12 weeks post-operation. The results were fitted by a linear mixed effects model including time dependency. Moreover, all tendon specimen were analysed histologically. Tenocyte and tenoblast density, tenocyte and tenoblast nuclei width, inflammation level and collagen fibre alignment were determined. RESULTS: Statistically significant differences in the motion pattern were found when one-leg treated and two-leg treated animals were compared. However, the absolute differences were on average less than 20%. Histologically, 1-leg treated animals had tendon tissue with higher cell density, but lower inflammation and less ondulated collagen fibres compared to 2-leg treated animals; the nuclei width was the same for both groups. With regard to welfare, all animals were fine during the experiments. CONCLUSIONS: While comparative studies should be performed with one-leg treated animals due to interaction effects, for proof-of-principle studies, operating two legs per animal may be justified as the welfare of the animals is warranted. This is a great benefit in the sense of the 3Rs because up to 50% of animals can be spared.