Gabriella Meier Bürgisser

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

BACKGROUNDnFractures 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.nnnMATERIALS AND METHODSnFive 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.nnnRESULTSnIndependently 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.nnnCONCLUSIONSnThis study revealed significant findings for bone tissue-engineering. Co-cultures of ASC-derived EC and ASC-derived OB stimulate each others 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.

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 12u2009weeks 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. Copyright

Prevention of peritendinous adhesions using an electrospun DegraPol polymer tube: a histological, ultrasonographic, and biomechanical study in rabbits

Purpose. One of the great challenges in surgical tendon rupture repair is to minimize peritendinous adhesions. In order to reduce adhesion formation, a physical barrier was applied to a sutured rabbit Achilles tendon, with two different immobilization protocols used postoperatively. Methods. Thirty New Zealand white rabbits received a laceration on the Achilles tendon, sutured with a 4-strand Becker suture, and half of the rabbits got a DegraPol tube at the repair site. While fifteen rabbits had their treated hind leg in a 180° stretched position during 6 weeks (adhesion provoking immobilization), the other fifteen rabbits were recasted with a 150° position after 3 weeks (adhesion inhibiting immobilization). Adhesion extent was analysed macroscopically, via ultrasound and histology. Inflammation was determined histologically. Biomechanical properties were analysed. Results. Application of a DegraPol tube reduced adhesion formation by approximately 20%—independently of the immobilization protocol. Biomechanical properties of extracted specimen were not affected by the tube application. There was no serious inflammatory reaction towards the implant material. Conclusions. Implantation of a DegraPol tube tightly set around a sutured tendon acts as a beneficial physical barrier and prevents adhesion formation significantly—without affecting the tendon healing process.

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

Abstract 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.

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

BACKGROUNDnPerfusion 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.nnnMATERIALS AND METHODSnElectrospun 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.nnnRESULTSnWhile 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.nnnCONCLUSIONSnThe 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.

History and performance of implant materials applied as peritendinous antiadhesives.

Peritendinous fibrotic adhesions after tendon surgery are still a problem up-to-date. Approaches to overcome or at least minimize adhesion formation include implantation of barrier materials, application of lubricants or combinations of materials and functionalized drugs that are controllably released and support the healing tendon to glide and achieve the full range of motion after regeneration. Although a huge amount of different materials have been experimentally tested, the optimal strategy with respect to material and method has not yet been determined. In this review, we present a historical overview of physical barriers as well as liquid agents that have been used in order to prevent peritendinous adhesion formation. The materials are divided according to their first publication into two time frames; before and after 1980. There is no claim to include all materials tested neither will the best material be chosen; however, we present several materials that were experimentally tested in different animal trials as well as in clinical trials in contrast to other materials that were only tested once and disappeared from the assortment of anti-adhesives; which as such is a valuable information about its applicability for this purpose.

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?

BACKGROUNDnConsidering 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).nnnMETHODSnThe 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.nnnRESULTSnStatistically 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.nnnCONCLUSIONSnWhile 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.

Structure and function of tendon and ligament tissues

Tendons and ligaments are connective tissues that serve as the force transmitting entities and enable musculoskeletal motion. Typical features of normal tendon tissue are parallel-aligned collagen I fibers and tenocytes. Moreover, the extracellular matrix (ECM) is composed of proteoglycans, glycoproteins, and elastin. The tissue has almost no vessels and the nutrition as well as oxygen are supplied at the vascularized myotendinous and osteotendinous junctions. Growth factors such as transforming growth factor beta are important for tendon development, homeostasis, and regeneration. Structural changes upon aging and tendinopathy include the extent of vascularization (aging causes less tendinopathy and more vascularization), the ECM (age-related lower collagen content and in tendinopathy collagen disorganization), and the proteoglycan content (older tendons having less, tendinopathic tendons more proteoglycans), which will be addressed in detail in this chapter.

Modification of silicone elastomers with Bioglass 45S5® increases in ovo tissue biointegration: MODIFICATION OF SILICONE ELASTOMERS WITH BIOGLASS 45S5®

Silicone is an important material family used for various medical implants. It is biocompatible, but its bioinertness prevents cell attachment, and thus tissue biointegration of silicone implants. This often results in constrictive fibrosis and implant failure. Bioglass 45S5® (BG) could be a suitable material to alter the properties of silicone, render it bioactive and improve tissue integration. Therefore, BG micro- or nanoparticles were blended into medical-grade silicone and 2D as well as 3D structures of the resulting composites were analyzed in ovo by a chick chorioallantoic membrane (CAM) assay. The biomechanical properties of the composites were measured and the bioactivity of the composites was verified in simulated body fluid. The bioactivity of BG-containing composites was confirmed visually by the formation of hydroxyapatite through scanning electron microscopy as well as by infrared spectroscopy. BG stiffens as prepared non-porous composites by 13% and 36% for micro- and nanocomposites respectively. In particular, after implantation for 7 days, the Youngs modulus had increased significantly from 1.20 ± 0.01 to 1.57 ± 0.03 MPa for microcomposites and 1.44 ± 0.03 to 1.69 ± 0.29 MPa to for nanocpmosites. Still, the materials remain highly elastic and are comparably soft. The incorporation of BG into silicone overcame the bioinertness of the pure polymer. Although the overall tissue integration was weak, it was significantly improved for BG-containing porous silicones (+72% for microcomposites) and even further enhanced for composites containing nanoparticles (+94%). These findings make BG a suitable material to improve silicone implant properties.

Autograft, allograft, and xenograft scaffolds for tendon and ligament repair

In order to regenerate lacerated tendons and ligaments, one viable option is to transplant autologous tendon or ligament tissue (autograft). The problem going along with this approach is primarily limited availability. Therefore, also allogeneic tendon and ligament (T&L) tissue is in use, however, the tissue has to be decellularized before transplanting it to the defect site in order to avoid tissue rejection. Such decellularized T&L grafts can also be reseeded by autologous cells in order to facilitate later biointegration. These two processing steps, however, prolong the time window to have a graft ready. Finally, also xenografts are in use, again decellularized to avoid rejection by the host. In the following chapter, auto-, allo-, and xenografts are discussed and possible processing options such as in vitro preseeding, static versus dynamic cultivation, and incorporation of growth factors such as basal fibroblast growth factor, platelet-derived growth factor, or epidermal growth factor among others are presented.