Visual Universitätsmedizin Mainz

Research Group “Applied Molecular Biology”

The focus of the ERC Advanced Investigator Grant research group of 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, concerns the following Topics:

Molecular Biomineralization

From the evolutionary oldest animals up to humans, we are studying the molecular mechanisms underlying biomineralization. We succeeded to show that the formation of all principle biominerals (silica, calcium carbonate and calcium phosphate), including bone hydroxyapatite (HA), proceeds via enzymatic processes.

Figure 1
Morphogenetically active inorganic biomaterials in human bone formation.

These discoveries allowed the development of novel strategies in tissue engineering and repair of bone and cartilage. We could show that the mineralisation of human bone is initiated by a carbonic anhydrase (CA IX) mediated calcium carbonate “bioseed” formation. The initially formed amorphous calcium carbonate (ACC) deposits are then converted into amorphous calcium phosphate (ACP, HA precursor) by carbonate-phosphate exchange. Thereby the phosphate is provided, again enzymatically, by hydrolysis of inorganic polyphosphate (polyP) via the alkaline phosphatase (ALP), a key enzyme of osteoblasts. ACC also stimulates bone formation, but only as long as it is present in the amorphous form. The amorphous ACC rapidly transforms into the more stable crystalline polymorphs vaterite, aragonite, and calcite. We could show that ACC can be stabilized by polyP. The resulting material strongly stimulates bone formation both in vitro and in vivo.

Inorganic Polyphosphate, an Energy-rich Biopolymer

Figure 2
Basic discoveries and applications of polyP, especially in human therapy. The development of morphogenically active amorphous polyP nano/microparticles and the introduction of 3D printing technologies resulted in a number of innovative applications ranging from dental sealants to wound healing materials, stimulation of angiogenesis and a new generation of bone and cartilage implants.

Polyphosphates (polyP) consist of numerous, up to 100 or more phosphate residues which are linked together by high-energy phosphoanhydride bonds. Originally found only in bacteria and yeasts, we later identified polyP also in animals, including humans. We found that comparably high amounts of polyP are present in human osteoblasts and platelets, but also extracellularly in the blood plasma. In particular, we discovered that polyP serves as an extracellular energy storage/signaling molecule. We demonstrated that extracellular ATP can be generated from polyP via the combined action of the cell membrane bound ALP and the adenylate kinase (AK) which catalyzes the reaction 2 ADP ↔ AMP + ATP. This makes polyP interesting especially when used for energy-dependent regeneration of bradytrophic tissues like cartilage.

Figure 3
The inorganic biopolymer polyP as an energy-delivering molecule in the extracellular space. The polyP-driven phosphorylation of AMP to ATP via ADP is shown. (A) In the presence of the AK inhibitor A(5?)P5(5?)A polyP undergoes extracellular hydrolysis via the ALP under simultaneous increase of the ADP pool. (B) In the absence of A(5?)P5(5?)A the metabolic energy polymer released during degradation of polyP by ALP is stored in ADP which is then converted to ATP and AMP.

Using a newly developed procedure, we were able to produce amorphous nano- and microparticles from the calcium salt of polyP. These amorphous Ca-polyP nano/microparticles are stable and become morphogenetically active and biodegradable after transformation into a coacervate in the presence of protein. They show a strong stimulating effect on bone HA synthesis. By combining polyP with other hydrogel-forming polymers, it was possible to develop 3D printable hybrid materials curable by the formation of Ca2+ bridges. These scaffolds are morphogenetically active both in vitro and in vivo.

Figure 4
Amorphous Ca-polyP nanoparticles. Left: Morphology and electric potential of the nanoparticles. Right: Transition of polyP nano/microparticles into a coacervate in the presence of protein. During this process the zeta-potential decreases, allowing the formation of coacervate aggregates that increase the growth/differentiation and mineralization potency of mesenchymal stem cells and bone-forming SaOS-2 cells.

Bio-inspired Materials for Regenerative Medicine

Building upon our results in basic research, we are developing novel materials for medical applications (bone and cartilage implants, artificial blood vessels and corneas), in particular for 3D-printing/bioplotting. The main focus is on morphogenetically active inorganic polymers, especially energy-rich and smart amorphous polyP nano/microparticles with various metal ions, as well as their combination with other, bioinert materials. This research is funded, among others, by three ERC Proof-of-Concept grants (“Si-Bone-PoC”, “MorphoVES-PoC” and “ArthroDUR”; coordinator: W.E.G. Müller). First regeneratively active implant materials have already been tested in animal experiments and shall be introduced into clinical application. These biocompatible and biodegradable materials allow a fast and complete replacement by the body’s own tissue, without the need of (stem) cells or cytokine/growth factor supplementation.

Figure 5
PolyP as a smart nano/micro-biomaterial.
Left: Our technology enables the fabrication of amorphous nano/microparticles of polyP with different cations that accelerate wound healing and cartilage formation (Mg-polyP), induce bone formation (Ca-polyP) or ? stimulate mineralization (Sr-polyP). Furthermore, polyP can act as a “cage” for encapsulation of drugs, e.g. for treatment of bone tumors/metastases.
Right: Application of various polyP-based materials in tissue engineering/repair.

For example, a morphogenetically active bio-ink with amorphous Ca-polyP microparticles and poly-ε-caprolactone (PCL) has been developed for the potential repair of larger bone defects, as well as Mg-polyP microparticles with viscoelastic properties similar to cartilage for potential treatment of osteoarthritis/arthrosis. Even more effective than Ca-polyP in bone healing are the microparticles obtained with the strontium salt of polyP that not only increased the expression of ALP and BMP-2 in osteoblasts and MSC, but also inhibited the expression of sclerostin, a negative regulator of Wnt signaling and inhibitor of bone cell differentiation and mineralization.

Figure 6
3D printed PCL scaffold (A and B) and scaffold fabricated with PCL/Ca-polyP-MP (B and D). The attachment and growth of SaOS-2 cells onto the PCL scaffold (E and F) and onto the PCL/Ca-polyP-MP scaffold are shown (G and H). Staining with calcein for living cells (E and G) and with DRAQ5 for total cell number (F and H).

Polyphosphate in Wound Healing and Microvascularisation

Moreover, with Ca-polyP, we are developing novel strategies for stimulating microvascularization/angiogenesis and for wound treatment. For example, topical application of morphogenetically active polyP microparticles in animal experiments was found to improve wound healing in normal and diabetic mice showing delayed wound healing.

Figure 7
Effect of polyP on cell migration during the initial phase of microvascularization.
(A) Scheme: In the extracellular space the polyP nanoparticles are processed via the ALP and AK to ADP/ATP that serve as a metabolic fuel and modulate the P2Y13 or the P2Y1 receptor signaling pathway. In concert with VEGF the cells upregulate the glucose import into cells via GLUT1. The ATP produced by glycolysis builds up an intracellular ATP gradient. The cell migration occurs along the direction of the intra/extracellular ATP gradient.
(B-D) Stimulation of the initial phase of microvascularization by Ca-polyP-MP in the “tube formation assay”. The cells (HUVEC) are stained with calcein and visualized by fluorescence microscopy. (B and C) Controls without Ca-polyP-MP and (D and E) Ca-polyP-MP supplemented assays after an incubation period for 4 h (B, D) or 8 h (F, E).
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