BMP-technology and Cell Based Implants: A Dream Team for Bone Regeneration.

Bone fracture healing is a complex, orchestrated, regenerative process that involves a critical number of progenitor cells required to form the new bone tissue. The concerted action of these cells are strictly regulated by a crucial interplay of biochemical, physical and mechanical factors, the former known to largely recapitulate phenomenological events of embryonic skeletal development. In the majority of the fractures, a fully regenerated organ will be restored with the original shape, size and strength of the bone. However, large and in particular long bone defects with a severely damaged surrounding environment result in 10 % of the cases in a delayed or non-union. Since no reliable treatment approaches are available for these today, the necessity for extended and painful treatment by osteodistraction or ultimately limb amputation may be the final outcome. Subsequently, there is a strong medical need for alternative strategies to treat these large complex bone defects. In order to reach success, a biomimetic, developmentally inspired approach has been suggested as essential. Gradually, these biomimetic alternatives are emerging, but the need for an effective and reliable solution, based on a fundamental understanding of the underlying process, is still unmet.

According to the developmental engineering approach, the research presented in this dissertation used healthy fracture healing as a blueprint to guide the engineering of a cell-based construct for the healing of a critically sized long bone defect. Since the cells provide the driving force for tissue formation, human periosteum derived cells (hPDCs), the main contributing cells during long bone fracture repair, were used as the basis of the construct. Furthermore, by combining an experimental, computational and reverse engineering approach, it was hypothesized that an effective and reliable treatment-alternative could be developed.

In order to define optimal conditions for osteochondrogenic differentiation of hPDCs, the early molecular events preceding hPDC-mediated ectopic bone formation were studied. A model based of bone formation using ceramic carriers provided insights into the cellular and molecular cascade of events. It was shown, through a computational approach, that sufficient Ca2+-release from the biomaterials led to a robust activation of the Bone morphogenetic protein (BMP)-, Wingless-related integration site (WNT)- and Protein Kinase C (PKC)-signalling pathways. On the other hand, excessive Ca2+-release led to imbalanced BMP-signalling and PKC-mediated inhibition of the WNT-signalling pathway.

Since the bone forming capacity was slow and limited in the aforementioned model of CaP-induced hPDC-mediated bone formation, it was hypothesized that a more clinically relevant outcome could be achieved by a reverse engineering approach. Robustly activated BMP-signalling displayed crucial for the bone forming capacity with hPDCs, therefore, BMP ligands involved in fracture repair were investigated for their ability to induce in vitro differentiation of hPDCs. The resulting most potent ligands were then coated onto CaP-scaffolds prior to cell seeding. Depending on the particular BMP-ligand as well as the biomaterial characteristics, specific in vitro activation of intracellular signalling were observed. In vivo, these resulted in construct-specific skeletal tissue formation where the synergistic stimulatory effect led to successful bone ossicle formation.

These findings confirmed BMP-ligands as promising stimulatory factors in hPDC-based constructs developed for the healing of critical long bone defects. However, these constructs involve the active delivery of recombinant growth factors, an additional challenge in terms of safety issues and regulatory requirements. In order to overcome this hurdle, it was hypothesised that proper in vitro priming of the cells may eleminate the need for growth factor-coating of the biomaterials. However, the presence of cytokines, proteins and inhibitors in serum, standardly used in cell culture medium, may interfere with stimulatory factors such as BMPs. Consequently, a serum free, chemically defined medium (CDM) was defined, capable of maintaining cell viability without inducing proliferation.

In vitro stimulation of monolayer cultures of hPDCs in BMP-2-supplemented CDM led to an enhanced osteo-chondrogenic response. When seeded onto a CaP-scaffold, elevated bone forming capacity was seen in vivo. Since standard in vitro culture of hPDCs occurs in the presence serum, it was hypothesised that the improved capacity could be further enhanced by a serum free culture period prior to BMP-stimulation. Upon pre-conditioning in CDM, hPDCs displayed an adapted phenotype including elevated expression of CD34 and BMP type 1 and type 2 receptors. Following in vitro BMP-stimulation, these cells displayed a more potent osteochondrogenic differentiation profile in vitro together with an elevated production of cartilaginous matrix in vivo.

Finally, the CDM pre-conditioned cells were assembled as aggregates resembling the developmental cellular condensations preceding cell differentiation. This led to enhanced chondrocyte specification, which, when followed by BMP-stimulation and subsequent ectopic in vivo implantation, resulted in ectopic tissue development resembling early endochondral bone formation. Assessment of the construct orthotopically in a critical sized long bone fracture displayed complete bridging of the defect within four weeks post transplantation.

In conclusion, the results presented within this dissertation displays the development of a manufacturing process for a biomimetic cell-based construct via a biomimetic reverse engineering approach. Importantly, presented findings demonstrate the modularity of the process with a specific serum free growth factor enriched regimen for the generation of a cell based implant with a predictable in vivo outcome.