A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues.

Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.

Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.

An in vitro osteosarcoma 3D microtissue model for drug development.

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Therapy today includes surgical removal of the tumour and neoadjuvant and adjuvant chemotherapy. The 5-year survival rates for patients with localised disease are between 50 and 70%, but in patients with metastases the prognosis remains poor (∼ 20%). The aim of this study was the development of a biological relevant OS 3D microtissue model, which is suitable for drug development. Microtissues were formed by the hanging drop method with the established OS cell lines SaOS-2, HOS and MG-63, as well as with cells derived from osteoblastic and chondroblastic OS patient material. Histological characterisation of the microtissues with H/E- and Ki-67-(proliferation), as well as apoptosis staining (TUNEL) revealed the inherent histological heterogeneity of OS. Microtissues from SaOS-2 and HOS cell lines were exposed to doxorubicin, cisplatin, taurolidine, pemetrexed and taxol and the viability was assessed by the CellTiter-GLO(®) Luminescent Cell Viability Assay. The obtained IC50-values for 3D cultures were all higher (1.7 to >16,000-fold) when compared to corresponding cells grown in 2D monolayer culture, except for pemetrexed that was inactive in 2D and 3D cultures. Doxorubicin did not affect the viability of chondroblastic monolayer cultures whereas on 3D microtissues an IC50-value of 2.3 µM was obtained. The 3D microtissues reflect the tissue heterogeneity of OS and are potential suitable tools for drug development towards personalised medicine.

Disease cell models - their use in industry TEDD workshop in Sion on 27 March 2014.

On 27, March 2014, experts met at the first TEDD Workshop 2014, held at the HES-SO Valais/Wallis in Sion, to present innovative cell models for industrial applications. This was the first time that a TEDD event had been organized in French-speaking Switzerland and it offered local network partners an opportunity to showcase their research activities.

The use of skin models in drug development.

Three dimensional (3D) tissue models of the human skin are probably the most developed and understood in vitro engineered constructs. The motivation to accomplish organotypic structures was driven by the clinics to enable transplantation of in vitro grown tissue substitutes and by the cosmetics industry as alternative test substrates in order to replace animal models. Today a huge variety of 3D human skin models exist, covering a multitude of scientific and/or technical demands. This review summarizes and discusses different approaches of skin model development and sets them into the context of drug development. Although human skin models have become indispensable for the cosmetics industry, they have not yet started their triumphal procession in pharmaceutical research and development. For drug development these tissue models may be of particular interest for a) systemically acting drugs applied on the skin, and b) drugs acting at the site of application in the case of skin diseases or disorders. Although quite a broad spectrum of models covering different aspects of the skin as a biologically acting surface exists, these are most often single stand-alone approaches. In order to enable the comprehensive application into drug development processes, the approaches have to be synchronized to allow a cross-over comparison. Besides the development of biological relevant models, other issues are not less important in the context of drug development: standardized production procedures, process automation, establishment of significant analytical methods, and data correlation. For the successful routine use of engineered human skin models in drug development, major requirements were defined. If these requirements can be accomplished in the next few years, human organotypic skin models will become indispensable for drug development, too.

Automation of 3D cell culture using chemically defined hydrogels.

Drug development relies on high-throughput screening involving cell-based assays. Most of the assays are still based on cells grown in monolayer rather than in three-dimensional (3D) formats, although cells behave more in vivo-like in 3D. To exemplify the adoption of 3D techniques in drug development, this project investigated the automation of a hydrogel-based 3D cell culture system using a liquid-handling robot. The hydrogel technology used offers high flexibility of gel design due to a modular composition of a polymer network and bioactive components. The cell inert degradation of the gel at the end of the culture period guaranteed the harmless isolation of live cells for further downstream processing. Human colon carcinoma cells HCT-116 were encapsulated and grown in these dextran-based hydrogels, thereby forming 3D multicellular spheroids. Viability and DNA content of the cells were shown to be similar in automated and manually produced hydrogels. Furthermore, cell treatment with toxic Taxol concentrations (100 nM) had the same effect on HCT-116 cell viability in manually and automated hydrogel preparations. Finally, a fully automated dose-response curve with the reference compound Taxol showed the potential of this hydrogel-based 3D cell culture system in advanced drug development.

Synthetic 3D multicellular systems for drug development.

Since the 1970s, the limitations of two dimensional (2D) cell culture and the relevance of appropriate three dimensional (3D) cell systems have become increasingly evident. Extensive effort has thus been made to move cells from a flat world to a 3D environment. While 3D cell culture technologies are meanwhile widely used in academia, 2D culture technologies are still entrenched in the (pharmaceutical) industry for most kind of cell-based efficacy and toxicology tests. However, 3D cell culture technologies will certainly become more applicable if biological relevance, reproducibility and high throughput can be assured at acceptable costs. Most recent innovations and developments clearly indicate that the transition from 2D to 3D cell culture for industrial purposes, for example, drug development is simply a question of time.

Soft tissue volume augmentation by the use of collagen-based matrices in the dog mandible -- a histological analysis.

OBJECTIVES: The aim was to test, whether or not soft tissue volume augmentation with a specifically designed collagen matrix (CM), leads to ridge width gain in chronic ridge defects similar to those obtained by an autogenous subepithelial connective tissue graft (SCTG). MATERIAL AND METHODS: In six dogs, soft tissue volume augmentation was performed by randomly allocating three treatment modalities to chronic ridge defects [CM, SCTG and sham-operated control (Control)]. Dogs were sacrificed at 28 (n = 3) and 84 days (n = 3). Descriptive histology and histomorphometric measurements were performed on non-decalcified sections. RESULTS: SCTG and CM demonstrated favourable tissue integration, and subsequent re-modelling over 84 days. The overall mean amount of newly formed soft tissue (NMT) plus bone (NB) amounted to 3.8 ± 1.2 mm (Control), 6.4 ± 0.9 mm (CM) and 7.2 ± 1.2 mm (SCTG) at 28 days. At 84 days, the mean NMT plus NB reached 2.4 ± 0.9 mm (Control), 5.6 ± 1.5 mm (CM) and 6.0 ± 2.1 mm (SCTG). Statistically significant differences were observed between CM/SCTG and Control at both time-points (p < 0.05). CONCLUSION: Within the limits of this animal model, the CM performed similar to the SCTG, based on histomorphometric outcomes combining NB and NMT.

Development of a novel automated cell isolation, expansion, and characterization platform.

Implementation of regenerative medicine in the clinical setting requires not only biological inventions, but also the development of reproducible and safe method for cell isolation and expansion. As the currently used manual techniques do not fulfill these requirements, there is a clear need to develop an adequate robotic platform for automated, large-scale production of cells or cell-based products. Here, we demonstrate an automated liquid-handling cell-culture platform that can be used to isolate, expand, and characterize human primary cells (e.g., from intervertebral disc tissue) with results that are comparable to the manual procedure. Specifically, no differences could be observed for cell yield, viability, aggregation rate, growth rate, and phenotype. Importantly, all steps-from the enzymatic isolation of cells through the biopsy to the final quality control-can be performed completely by the automated system because of novel tools that were incorporated into the platform. This automated cell-culture platform can therefore replace entirely manual processes in areas that require high throughput while maintaining stability and safety, such as clinical or industrial settings.

A bioreactor test system to mimic the biological and mechanical environment of oral soft tissues and to evaluate substitutes for connective tissue grafts.

Gingival cells of the oral connective tissue are exposed to complex mechanical forces during mastication, speech, tooth movement and orthodontic treatments. Especially during wound healing following surgical procedures, internal and external forces may occur, creating pressure upon the newly formed tissue. This clinical situation has to be considered when developing biomaterials to augment soft tissue in the oral cavity. In order to pre-evaluate a collagen sponge intended to serve as a substitute for autogenous connective tissue grafts (CTGs), a dynamic bioreactor system was developed. Pressure and shear forces can be applied in this bioreactor in addition to a constant medium perfusion to cell-material constructs. Three-dimensional volume changes and stiffness of the matrices were analyzed. In addition, cell responses such as cell vitality and extracellular matrix (ECM) production were investigated. The number of metabolic active cells constantly increased under fully dynamic culture conditions. The sponges remained elastic even after mechanical forces were applied for 14 days. Analysis of collagen type I and fibronectin revealed a statistically significant accumulation of these ECM molecules (P < 0.05-0.001) when compared to static cultures. An increased expression of tenascin-c, indicating tissue remodeling processes, was observed under dynamic conditions only. The results indicate that the tested in vitro cell culture system was able to mimic both the biological and mechanical environments of the clinical situation in a healing wound.

Soft tissue volume augmentation by the use of collagen-based matrices: a volumetric analysis.

OBJECTIVES: The aim was to test whether or not soft tissue augmentation with a newly developed collagen matrix (CM) leads to volume gain in chronic ridge defects similar to those obtained by an autogenous subepithelial connective tissue graft (SCTG). MATERIAL AND METHODS: In six dogs, soft tissue volume augmentation was performed by randomly allocating three treatment modalities to chronic ridge defects (CM, SCTG, sham-operated control). Impressions were taken before augmentation (baseline), at 28, and 84 days. The obtained casts were optically scanned and the images were digitally analysed. A defined region of interest was measured in all sites and the volume differences between the time points were calculated. RESULTS: The mean volume differences per area between baseline and 28 days amounted to a gain of 1.6 mm (CM; SD+/-0.9), 1.5 mm (SCTG; +/-0.1), and a loss of 0.003 mm (control; +/-0.3). At 84 days, the mean volume differences per area to baseline measured a gain of 1.4 mm (CM; +/-1.1), 1.4 mm (SCTG; +/-0.4), and a loss of 0.3 mm (control; +/-0.3). The differences between CM and SCTG were statistically significant compared with control at 28 and 84 days (p<0.001). CONCLUSION: Within the limits of this animal study, the CM may serve as a replacement for autogenous connective tissue.

Tissue engineering--the gateway to regenerative medicine.

Tissue engineering as an emerging biotechnology sector aims at the in vitro regeneration of diseased tissues and promises to profoundly change medical practice, offering the possibility of regenerating tissues and organs instead of just repairing them (regenerative medicine). Improved healing processes and a higher quality of life are the expected results. This article gives an overview of different technologies for regenerative medicine and presents results of our own current applied research and development. A recent project was successfully closed with the development of a natural biomaterial for soft tissue oral defects. The establishment of an in vitro bioreactor system enabled us to simulate the mechanical and biological environment in a healing wound and to investigate the suitability of different implant materials for the oral tissue regeneration. Moreover, focusing the attention on an alternative method for the intervertebral disc (IVD) regeneration, we established a new tissue engineered approach, based on the three-dimensional (3D) culture of autologous human IVD cells into a polyurethane (PU)-fibrin composite. IVD cells were able to proliferate and, thanks to the 3D conditions, to differentiate expressing the typical native tissue markers. The development of an automated platform was the goal of an additional project, to standardize the cell culture technology, increase the bio-safety and reduce the production costs, moving tissue engineering nearer to clinical application.

Clinical applications of glass-ceramics in dentistry.

Glass-ceramics featuring special properties can be used as a basis to develop biomaterials. It is generally differentiated between highly durable biomaterials for restorative dental applications and bioactive glass-ceramics for medical use, for example, bone replacements. In detail, this paper presents one biomaterial from each of these two groups of materials. In respect to the restorative dental biomaterials, the authors give an overview of the most important glass-ceramics for clinical applications. Leucite, leucite-apatite, lithium disilicate and apatite containing glass-ceramics represent biomaterials for these applications. In detail, the authors report on nucleation and crystallization mechanisms and properties of leucite-apatite glass-ceramics. The mechanism of apatite nucleation is characterized by a heterogeneous process. Primary crystal phases of alpha - and beta -NaCaPO4 were determined. Rhenanite glass-ceramics represent biomaterials with high surface reactivity in simulated body fluid, SBF, and exhibit reactive behaviour in tests with bone cells. Cell adhesion phenomena and cell growth were observed. Suitable colonization and proliferation and differentiation of cells as a preliminary stage in the development of a material for bone regeneration applications was established. The authors conclude that the processes of heterogeneous nucleation and crystallization are important for controlling the required reactions in both biomaterial groups.