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Research Group “Applied Molecular Biology”

The aim of the ERC Advanced Investigator Group of Univ.-Prof. Dr. Dr. h. c. Werner E.G. Müller, which includes the groups of Prof. Dr. Xiaohong Wang, Prof. Dr. Dr. Heinz C. Schröder and Dr. Matthias Wiens, is to gain new knowledge in basic research that can be transferred to medical applications.

From biomolecules to biominerals: evolution of medically relevant metabolic pathways

The starting point of our current research is our long-term investigation of the secondary metabolites of sponges, one of the richest sources of new, potentially medically applicable bioactive compounds in the sea. Among others, we succeeded in finding an inhibitor of the Herpes simplex virus DNA polymerase and elucidating its mechanism: araA, a virostatic that is still in clinical use today. The mechanism of action of another nucleoside analogue, araC, which is used in tumor therapy, was also clarified. In the course of this work, we discovered and investigated in sponges metabolic pathways and mechanisms, such as apoptosis, that do not only occur in these animals, which led us also to understand the molecular basis of cell death in human diseases such as Alzheimer's disease, HIV and Creutzfeldt-Jakob disease, as well as the protective effect of certain drugs, such as Memantine and Flupirtine. It became increasingly clear that the basic metabolic processes and stress response mechanisms in sponges are similar to those in humans. This ultimately led to the discovery that all animals are of monophyletic origin. The proof was based on our results of an extensive project for the sequencing of the expressed sponge genome and required the establishment of a previously unavailable method for sponge cell cultivation (“primmorphs”) to examine the metabolic pathways. However, one metabolic pathway remained inexplicable: the formation of the mineral skeleton of the sponges. Surprisingly, a comparison with other organisms such as humans showed that the molecular biochemical processes underlying biomineralization – a basic process in almost all animals – were still largely unknown. This process, which we turned to study in the following, has sparked our research interest until today.

Biomineralization - an enzyme-catalyzed process?

A fundamental question is: How do skeletal organisms overcome the activation energy barriers that also apply to mineralization processes? For example, deep-sea sponges are able to form their silica, i.e. glass skeleton, at temperatures close to freezing point, although technical glass production requires temperatures of over 1000°C. We know from biochemistry that enzymes can reduce the activation energy of chemical reactions. Therefore we asked: Is silica formation also enzyme-catalyzed? In fact, we could demonstrate this. As we have been able to show, the sponge protein silicatein has the ability to synthesize silica (i.e. an inorganic material) from orthosilicate (also a purely inorganic material) via an enzymatic mechanism. Until then, no other enzyme had been able to perform such a reaction – a groundbreaking discovery that was examined in detail in an ERC Advanced Grant project ("BIOSILICA") by the head of the research group, not only for technical (silica is a basic material in nanotechnology), but also of great importance for medical applications.

From Gene to Enzyme to Inorganic Polymer
Genetically controlled formation of the silica skeleton of the siliceous sponges via the expression of silicatein. Top: Mechanism of silicatein catalysis. Bottom left: Controlled formation of a sponge silica spicula (skeletal element) around a central silicatein axial filament. Bottom right: Deep sea sponge.

Next we asked: Is the formation of the calcium carbonate skeleton of the – evolutionarily younger – calcareous sponges enzyme-catalyzed? This could also be affirmed – the enzyme: a carbonic anhydrase. Building on the results obtained with sponges, we now asked the question: is the formation of the human bone mineral, hydroxyapatite (a calcium phosphate mineral), enzyme-catalyzed and, if so, can this knowledge contribute to the understanding of the pathogenesis and possibly for the treatment of human bone diseases? The answer brought a paradigm shift. We found that during bone mineralization, hydroxyapatite is not initially formed, but amorphous calcium carbonate (ACC), synthesized by a carbonic anhydrase. The ACC is then converted to amorphous calcium phosphate (ACP) by carbonate-phosphate exchange, and finally hydroxyapatite. The required phosphate is supplied – again enzymatically – by the hydrolysis of inorganic polyphosphates (polyP), catalyzed by the alkaline phosphatase (ALP), a key enzyme of the osteoblasts.

Steps during human bone formation
Steps during human bone formation.

In addition, we found that only the amorphous biominerals (ACC, ACP, as well as silica and polyP – as nano/microparticles), but not their crystalline forms, are biologically/regeneratively active – a discovery that opened new strategies for tissue engineering and new potential therapeutic targets (ACC, ACP, polyP) for bone diseases.

Tissue regeneration: who supplies the energy extracellularly?

Scaffolds/matrices that are suitable for tissue regeneration/repair must offer the invading stem cells a suitable niche that enables them to proliferate and differentiate into functionally active tissues/cells. In the best case, materials that are used for their fabrication are themselves regenerative, without the need to add cytokines/growth factors – this is exactly what our amorphous biomineral-based materials fulfill. There is, however, a second requirement that has so far been neglected and unsolved: the need for metabolic energy. This applies in particular to poorly perfused, bradytrophic tissues such as cartilage with only a few cells and an extensive extracellular matrix. An extracellular energy source was unknown – ATP, the universal energy carrier, is only released from cells in minimal amounts. We therefore asked: Where does this energy come from, who supplies it? The answer is: polyP. These polymers, which consist of numerous phosphate residues linked together via high-energy phosphoanhydride bonds, are not only a phosphate source for bone mineralization, but, as we found, generally serve as an extracellular storage and donor of metabolic energy. During their hydrolysis, a multiple of the free energy of the ATP hydrolysis is released. We were able to show that the energy stored in polyP can be converted extracellularly into biochemically usable energy, in the form of ATP, through the combined action of two enzymes, the cell membrane-bound ALP and the adenylate kinase (ADK).

PolyP: Energy storage and Energy release
PolyP as an energy donor in the extracellular space. Left: The step-wise degradation of polyP catalyzed by the enzyme alkaline phosphatase (ALP) is highly exergonic. The released energy of the phosphoanhydride linkage can either be dissipated in the form of heat or used for the synthesis of ATP, which is then available for energy-consuming processes (example: cartilage formation and cell migration/microvascularization). Right: During the complete hydrolysis of polyP (example: polyP with a chain length of 40), a multiple of the free energy of the hydrolysis of ADP or ATP is released.
Cleavage of the energy-rich phosphoanhydride bonds of polyP
Cleavage of the energy-rich phosphoanhydride bonds of polyP (complexed with Mg ions) and phosphotransfer to AMP catalyzed by the combined action of cell membrane-bound alkaline phosphatase (green) and adenylate kinase (blue), resulting in the formation of ATP in the extracellular space.

Development of medical applications

The amorphous inorganic biomaterials developed by us offer optimal conditions for use in regenerative medicine. Not only are they regeneratively active and biodegradable, they also serve, in the case of polyP, as a source of metabolic energy – an extraordinary combination of properties that has not previously been shown by any other tissue engineering/repair material. Applying a bio-inspired process and different divalent metal ions (counterions) we succeeded in fabricating amorphous (non-crystalline, i.e. biologically active) nano- and microparticles of polyP with different biological properties. These nano/microparticles are stable and become biologically active after conversion into a coacervate in the presence of protein-containing body fluids (e.g. wound secretions): for example, Ca-polyP nano/microparticles, after coacervate conversion, show a strong stimulating effect on bone mineralization.

PolyP-based nano/micro biomaterials
PolyP-based nano/micro biomaterials. Left: Production of amorphous nano/microparticles of polyP and different cations for wound healing and cartilage regeneration (Mg-polyP) as well as bone regeneration (Ca-polyP) and mineralization (Sr-polyP). PolyP can also serve as a "cage" for encapsulating drugs, e.g. for the treatment of bone tumors and metastases. Right: Use of various polyP-based materials in tissue engineering.

By combining polyP or polyP nano/microparticles with hydrogel-forming polymers such as alginate, hyaluronic acid, chitosan derivatives or chondroitin sulfate, we were able to develop hybrid materials (even 3D-printable) for a number of medical applications, from tooth sealing to wound healing materials, to stimulate microvascularization and bone and cartilage regeneration, as well as to manufacturing of artificial blood vessels and corneas. With suitable hydrogels, it was also possible to develop a polyP-based bio-ink for 3D cell printing (3D bioplotting) – the embedded cells remained proliferatively active. The first regeneratively active implant materials have already been tested in animal experiments and are to be brought into clinical use.

3D printed polycaprolactone (PCL) scaffold
3D printed polycaprolactone (PCL) scaffold (A and B) and scaffold made with PCL/Ca-polyP microparticles (B and D). Adhesion and growth of SaOS-2 cells to the PCL scaffold (E and F) and the PCL/Ca-polyP-MP scaffold are shown (G and H). Staining with calcein for living cells (E and G) and with DRAQ5 for the total number of cells (F and H).

In addition to this work funded by three ERC proof-of-concept grants ("Si-Bone-PoC", "MorphoVES-PoC" and "ArthroDUR") and several other EU projects coordinated by us, we are currently developing, in a German-Chinese project, novel wound healing materials, especially for chronic, difficult to heal wounds. Like other regenerative processes, wound healing requires a lot of energy – a need that is met by the polyP component of these materials. In addition, polyP induces microvascularization. Further research in an ongoing EU Horizon 2020 project ("InnovaConcrete") is using the stabilizing properties of polyP on ACC to give cements (both technical and medically applicable) self-healing properties. Recently, we were also able to show that in model systems polyP has a potentially protective effect against the SARS CoV-2 coronavirus.

Artificial blood vessels
Artificial blood vessels. (A) Biomimetic fabrication procedure. The hydrogel is pressed through an extruder into a Ca-containing hardening solution. (B to D) Human umbilical vein endothelial cells (HUVEC) grown on the scaffolds. Staining with DRAQ5 (blue fluorescence) and actin antibodies (red).
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