Cartilage Repair . Bone Repair . Stem Cells . Bioreactors . 3D Culture Models Engineered Stromal Tissues

Tissue Engineering

From 3D culture models to regenerative surgery

The common denominator of the research projects in the group is related to the establishment of 3D cell culture systems, combining interdisciplinary efforts in cell biology, engineering technologies, and materials science. These systems are used as models to investigate fundamental aspects of tissue development and as grafts to induce tissue regeneration. Several collaborations have been established within the DBM, with the Institute of Pathology, and with the D-BSSE, ETH Zürich to employ the developed tools for 3D culture of tumor cells (Prof. M. Bentires-Aji, Prof. G. Hutter, Prof. Y. Benenson, PD Dr. med. Berger, Dr. Le Magnen), endothelial cells (Prof. A. Banfi), thymic epithelial cells (Prof. G. Holländer), glial cells (Prof. R. Guzman), omental-derived cells (Prof. V. Heinzelmann), pancreatic cells (Prof. M. Fusseneger), and cardiac cells (PD Dr. A. Marsano). However, the main focus has been maintained around the development of cartilage and bone/bone marrow tissues. Following is a short summary of recent achievements in these research areas.

Nasal chondrocytes and environmental plasticity (PD Dr. A. Barbero)
We found that chondrocytes from the nasal septum, as compared to articular chondrocytes typically used for cell-based cartilage repair, have superior and more reproducible chondrogenic capacity. This led to the first clinical trial demonstrating the suitability and safety of engineered nasal cartilage grafts for reconstructive purposes in the nose. We also found that nasal chondrocytes–which are of neural crest origin–display a profile of HOX gene expression distinct from articular chondrocytes–which are of mesoderm origin. However, upon implantation in a joint, they can be reprogrammed by the recipient site and acquire the HOX “signature” typical of articular chondrocytes. These results have led to the treatment of 17 patients with traumatic cartilage injuries in the knee (Fig. 1). Positive clinical findings have been instrumental to receive EU funding for a multicenter phase II study.

Bone, Bone marrow and Vascularized bone (PD Dr. A. Scherberich)
The use of mesenchymal stromal/stem cells (MSC) from bone marrow has been proposed to generate osteogenic grafts, but with limited efficiency and reproducibility. We hypothesized that the robustness of the process could be increased by mimicking events of bone embryonic development. Intermediate templates of hypertrophic cartilage generated by MSCs could robustly and autonomously remodel into bone tissue. A similar outcome could also be achieved with engineered and decellularized tissues, where the cocktail of factors necessary to initiate the process is embedded in the deposited extracellular matrix. The bone organ developed by this approach is capable of hosting fully functional hematopoietic stem cells and is thus being investigated in collaboration with Prof. R. Skoda and Prof. C. Lengerke as a humanized environment to study interaction of leukemic cells with a 3D stromal niche. MSCs are also found within the stromal vascular fraction (SVF) of adipose tissue, mixed with endothelial lineage cells. After demonstrating that SVF cells can self-assemble into osteogenic and vasculogenic structures, we used them in an intraoperative clinical setting to enhance humerus fracture healing in elderly patients (Fig. 2). Studies are ongoing to combine the strategy of engineered and decellularized matrices (see above) with intraoperative “re-activation” by SVF cells to enhance bone and vascular repair.

Engineering platforms
One main challenge in engineering 3D culture models or tissue grafts is to ensure efficient nutrition and oxygen supply through thick constructs. This has been addressed by developing bioreactor systems to perfuse cell suspensions/culture medium directly through the developing structures. The devices are used in a variety of settings for lab discovery and are being further developed for the streamlined GMP manufacturing of cellular grafts for clinical use. More recently, we have also validated a microfluidic system allowing the formation of 3D cellular structures and their exposure to different combinations and concentrations of regulatory molecules. The system is in use to investigate in a relatively higher throughput the signals supporting the recapitulation of developmental programs by mesenchymal progenitors (Fig. 3).

Fig. 1: Engineering of autologous nasal cartilage grafts. (A) Collection of a nasal cartilage biopsy from a patient, this procedure is performed under local anaesthesia and results in minimal donor site morbidity. (B) Biopsy of nasal cartilage septum. (C-D) Tissue engineered cartilage graft: macroscopic (C) and histological appearance (Safranin-O staining specific for sulfated glycosaminoglycans (D).

Fig. 2: Intraoperative engineering of autologous Stromal Vascular Fraction-based grafts for the clinical treatment of humerus fractures in osteoporotic individuals.

Fig. 3: Upper panel: Direct perfusion system to support efficient cell seeding into 3D porous scaffolds and uniform in vitro tissue development. Lower panel: Microfluidic-based system to allow high-throughput test of compounds within culture chambers, where cells can organize into microtissues and monitored by fluorescence microscopy online.