Haihua Pan

Yeast cells with an artificial mineral shell: protection and modification of living cells by biomimetic mineralization.

In all living organisms, whether very basic or highly complex, nature provides a multiplicity of materials, architectures, systems and functions. A number of unicellular organisms have an outer-surface proteinaceous membrane as a template for biomineralization. The resultant thin mineral layer is a functional covering. For example, the mineral shell can protect an egg from invasion from the exterior, and the diatom has an ornately patterned silicified shell that evolved as mechanical protection. But most cells in nature cannot make their own hard shells. Here we show a strategy to fashion an artificial shell for the yeast cell so that it has extensive protection. Individual Saccharomyces cerevisiae (S. cerevisiae) cells are coated with a uniform calcium mineral layer by first self-assembly of functional polymers (layer-bylayer technique, LbL) and then in situ mineralization under physiological conditions. The viability of the cells is maintained after the encapsulation. The enclosed cells become inert (stationary phase) and their lifetime can be extended. Furthermore, the mineral shell protects the cell under harsh conditions. The encapsulated S. cerevisiae can even survive the attack of the lytic enzyme zymolyase. The shell can also be used as a scaffold for chemical and biological functionalization. For example, S. cerevisiae becomes magnetic by the incorporation of Fe3O4 nanoparticles in the mineral layer. The present work demonstrates that the artificial shell has a great potential in the storage, protection, delivery, and modification of living cells. Furthermore, insights from systems biology combined with an understanding of the molecular mechanisms of functional shells will facilitate the tailoring of “super cells” through biomimetic mineralization. As S. cerevisiae shares a common life cycle and cell structure with higher eukaryotes, it is a popular and successful model system for understanding eukaryotic biology at the cellular and molecular levels. Like most natural cells, S. cerevisiae cannot induce spontaneous mineralization on its surface. Figure 1a shows a typical scanning electron microscopy (SEM) image of S. cerevisiae. Although the precipitation of calcium phosphate is induced in a supersaturated calcium phosphate solution (concentrations of calcium and phosphate ions are 5.0 mm, ionic strength 0.12m, and pH 6.8), most reactions do not occur on the cell surface. The surface of S. cerevisiae is unchanged during the precipitation and the mineral forms separately (Figure 1b). Although some mineralization can occur on the cell surface, the deposited minerals are not uniform and they cannot form an ideal shell around the cell (Figure 1c). This phenomenon can be explained by the chemical structure of the cell wall of S. cerevisiae, which consists mainly of polysaccharides made up of glucose, mannose, and N-acetylglucosamine. Such a structure hardly induces the templated crystallization of calcium phosphates, which is caused by the relatively low density of electronic charge. Previous studies have already pointed out that the electronic interaction is a key factor in biomineralization. It is generally agreed that proteins that are most active in the mediation of biologically directed mineralization contain regions rich in carboxylates or other charged functional groups. The mineralization skill of living cells can be improved by introducing functional factors onto the cell surface. LbL has been applied as a general approach for the fabrication of multicomponent films on solid supports. We used two polyelectrolytes with opposite charges, poly(diallyldimethylammonium chloride) (PDADMAC) and poly(acrylic sodium) (PAA) are used. PAA has a high density of carboxylate groups, which provide the active nucleation sites for calcium minerals. When the adsorbed PAA molecules are on the outermost layer of the yeast cell, the physicochemical properties of the cells are altered significantly. The carboxylate groups migrate toward the water–polymer interface and bind Ca ions. Upon contact with calcification solution the reorganized surface induces the heterogeneous nucleation of calcium minerals. Besides, it is emphasized the LbL coating does not kill the yeast cells after the immobilization. In situ precipitation of calcium phosphates on the LbLtreated S. cerevisiae cell surface dominates the mineral formation, and the cells can be fully enclosed by the mineral phase (Figure 1d). SEM shows that the surface of S. cerevisiae typically becomes rough and porous and is covered by numerous flakelike nanocrystals. A large-scale SEM view (Figure 1e) shows that practically all of the treated cells have mineral surfaces. The formation of the mineral shell around S. cerevisiae cells is confirmed by transmission electron [*] B. Wang, P. Liu, W. Jiang, Dr. H. Pan, Dr. X. Xu, Prof. R. Tang Center for Biomaterials and Biopathways Department of Chemistry, Zhejiang University Hangzhou, 310027 (China) Fax: (+86)571-8795-3736 E-mail: rtang@zju.edu.cn

Repair of enamel by using hydroxyapatite nanoparticles as the building blocks

The application of calcium phosphates and their nanoparticles have been received great attention. However, hydroxyapatite (HAP) is not suggested in dental therapy to repair the damaged enamel directly although this compound has a similar chemical composition to enamel. We note that the size-effects of HAP are not taken into account in the previous studies as these artificial particles frequently have sizes of hundreds of nanometres. It has recently been revealed that the basic building blocks of enamel are 20–40 nm HAP nanoparticles. We suggest that the repair effect of HAP can be greatly improved if its dimensions can be reduced to the scale of the natural building blocks. Compared with conventional HAP and nano amorphous calcium phosphate (ACP), our in vitro experimental results demonstrate the advantages of 20 nm HAP in enamel repairs. The results of scanning electron microscopy, confocal laser scanning microscopy, quantitative measurement of the adsorption, dissolution kinetics, and nanoindentation, show the strong affinity, excellent biocompatibility, mechanical improvement, and the enhancement of erosion-free by using 20 nm particles as the repairing agent. However, these excellent in vitro repair effects cannot be observed when conventional HAP and ACP are applied. Clearly, nano HAP with a size of 20 nm shares similar characteristics to the natural building blocks of enamel so that it may be used as an effective repair material and anticaries agent. Our current study highlights the analogues of nano building blocks of biominerals during biomedical applications, which provide a novel pathway for biomimetic repair.

Effect of crystallinity of calcium phosphate nanoparticles on adhesion, proliferation, and differentiation of bone marrow mesenchymal stem cells

The biomedical materials research community frequently accepts that amorphous calcium phosphate (ACP) can be adsorbed and assimilated more readily by living organisms to produce new bone tissue than crystallized calcium phosphates such as hydroxyapatite (HAP). Previous studies also confirm that ACP has improved bioactivity compared to HAP since more adhesion and proliferation of osteogenic cells are observed on the ACP substrates. However, we note that the different size -effects of calcium phosphates are not taken into account in these studies and the used ACP are always smaller than the HAP. Our recent study reveals that the dimensions of nanoparticles are directly related to the bioactivities of calcium phosphates, e.g. the smaller nanocrystallites have a greater promotion effect on the proliferation of bone marrow mesenchymal stem cells (BMSCs). In order to understand the influence of crystallinity of calcium phosphate on the osteogenic cells correctly, it is critical to use ACP and HAP nanoparticles which have the same size distribution in such comparisons. In the present work, ∼20 nm ACP and HAP particles are synthesized and the effects of crystallinity of calcium phosphates are studied. The adhesion, proliferation, and differentiation of BMSCs are measured on ACP and HAP films, which are compared at the same size scale. It is surprising that more cells adsorb and proliferate on the film of well crystallized HAP than those on the ACP film. Alkaline phosphatase (ALP) activity assay and reverse transcription-polymerase chain reaction (RT-PCR) assay are also used to evaluate the differentiation of BMSCs. The results show that the differentiation of BMSCs to osteoblasts is promoted significantly by NanoHAP. The current experimental phenomena clearly demonstrate that the crystallized phase of calcium phosphate, HAP, provides a better substrate for BMSCs than the amorphous one, ACP, when the factor of size effect is removed. A new view on the relationship between the crystallinity of calcium phosphate and the responses of BMSCs indicates the importance of size and phase controls in the application of biomedical materials.

Bio‐Inspired Enamel Repair via Glu‐Directed Assembly of Apatite Nanoparticles: an Approach to Biomaterials with Optimal Characteristics

As the outermost layer, enamel is susceptible to caries due to the products of bacteria metabolism or acidic foods. [ 4 ] It is critically serious that the enamel damage is permanent. [ 4 ] Different form the synthesized apatite phase, enamel is uniquely composed of highly elongated and organized apatite rods and bundles. During enamel generation, the apatite crystals are well assembled into parallel arrays under strict biological controls. It is believed that ameloblast cells secrete the enamelins such as amelogenins and amelobastins to mediate the construction. [ 5 ]

Atomic force microscopy reveals hydroxyapatite-citrate interfacial structure at the atomic level.

An approach to organic-inorganic interfacial structure at the atomic level is a great challenge in the studies of biomineralization. We demonstrate that atomic force microscopy (AFM) is powerful tool to discover the biomineral interface in detail. By using a model system of (100) hydroxyapatite (HAP) face and citrate, it reveals experimentally that only a side carboxylate and a surface calcium ion are involved in the binding effect during the citrate adsorption, which is against the previous understandings by using Langmuir adsorption and computer simulation. Furthermore, the adsorbed citrate molecules can use their free carboxylate and hydroxyl groups to be self-assembled on the HAP surface. AFM examination also finds that the presence of citrate molecules on the HAP crystal faces can enhance the adhesion force of the HAP surface. We suggest that the established AFM method can be used for a precise and direct understanding of biointerfaces at the atomic level.

Direct observation of mineral–organic composite formation reveals occlusion mechanism

Manipulation of inorganic materials with organic macromolecules enables organisms to create biominerals such as bones and seashells, where occlusion of biomacromolecules within individual crystals generates superior mechanical properties. Current understanding of this process largely comes from studying the entrapment of micron-size particles in cooling melts. Here, by investigating micelle incorporation in calcite with atomic force microscopy and micromechanical simulations, we show that different mechanisms govern nanoscale occlusion. By simultaneously visualizing the micelles and propagating step edges, we demonstrate that the micelles experience significant compression during occlusion, which is accompanied by cavity formation. This generates local lattice strain, leading to enhanced mechanical properties. These results give new insight into the formation of occlusions in natural and synthetic crystals, and will facilitate the synthesis of multifunctional nanocomposite crystals.

Biomimetically Triggered Inorganic Crystal Transformation by Biomolecules: A New Understanding of Biomineralization

Phase transformation is an important strategy in biomineralization. However, the role of biomolecules in the mineral transition is poorly understood despite the fact that the biomineralization society greatly highlights the organic controls in the formation of the inorganic phase. Here, we report an induced biomimetic phase transformation from brushite (a widely used calcium phosphate precursor in biological cement) to hydroxyapatite (main inorganic composition of skeletal mineral) by citrate (a rich organic component in bone tissue). The transformation in the absence of the organic additive cannot be spontaneously initiated in an aqueous solution with a pH of 8.45 (no phase transition is detected in 4 days), which is explained by a high interfacial energy barrier between brushite-solution and hydroxyapatite-solution interfaces. Citrate can oppositely regulate these two interfaces, which decreases and increases the stabilities of brushite and hydroxyapatite surfaces in the solution, respectively. Thus, the interfacial energy barrier can be greatly reduced in the presence of citrate and the reaction is triggered; e.g., at 1 mM citrate, the total transformation from brushite to hydroxyapatite can be completed within 3 days. The relationship between the transition kinetics and citrate concentration is also studied. The work reveals how the organic components direct solid-solid phase transformation, which can be understood by an energetic control of the interfacial barrier. It is emphasized that the terms of interfacial energy must be taken into account in the studies of phase transformation. We suggest that this biomimetic approach may provide an in-depth understanding of biomineralization.

Unique roles of acidic amino acids in phase transformation of calcium phosphates.

Although phase transformation is suggested as a key step in biomineralization, the chemical scenario about how organic molecules mediate inorganic phase transformations is still unclear. The inhibitory effect of amino acids on hydroxyapatite (HAP, the main inorganic component of biological hard tissues such as bone and enamel) formation was concluded by the previous biomimetic modeling based upon direct solution crystallization. Here we demonstrate that acidic amino acids, Asp and Glu, could promote HAP crystallization from its precursor crystal, brushite (DCPD). However, such a promotion effect could not be observed when the nonacidic amino acids were applied in the transformation-based HAP formation. We found that the specific modification of acidic amino acid on crystal-solution interfaces played a key role in the phase transition. The distinct properties between DCPD and HAP in the solution resulted in an interfacial energy barrier to suppress the spontaneous formation of HAP phase on DCPD phase. Different from the other amino acids, the carboxylate-rich amino acids, Asp and Glu, could modify the interfacial characteristics of these two calcium phosphate crystals to make them similar to each other. The experiments confirmed that the involvement of Asp or Glu reduced the interfacial energy barrier between DCPD and HAP, leading to a trigger effect on the phase transformation. An in-depth understanding about the unique roles of acidic amino acids may contribute to understanding phase transformation controls druing biomineralization.

Stabilizing amorphous calcium phosphate phase by citrate adsorption

The regulation of citrate on amorphous calcium phosphate (ACP)-mediated crystallization of hydroxyapatite (HAP) is revealed in this work. The surface associated citrate on ACP plays the key role in controlling the nucleation of HAP by inhibiting the reaction of surface nucleation, and the effect of embedded citrate inside ACP or citrate in solution is weak.

Guarding Embryo Development of Zebrafish by Shell Engineering: A Strategy to Shield Life from Ozone Depletion

Background The reduced concentration of stratospheric ozone results in an increased flux of biologically damaging mid-ultraviolet radiation (UVB, 280 to 320 nm) reaching earth surfaces. Environmentally relevant levels of UVB negatively impact various natural populations of marine organisms, which is ascribed to suppressed embryonic development by increased radiation. Methodology/Principal Findings Inspired by strategies in the living systems generated by evolution, we induce an extra UVB-adsorbed coat on the chorion (eggshell surrounding embryo) of zebrafish, during the blastula period. Short and long UV exposure experiments show that the artificial mineral-shell reduces the UV radiation effectively and the enclosed embryos become more robust. In contrast, the uncoated embryos cannot survive under the enhanced UVB condition. Conclusions We suggest that an engineered shell of functional materials onto biological units can be developed as a strategy to shield lives to counteract negative changes of global environment, or to provide extra protection for the living units in biological research.