Altered tooth morphogenesis after silencing the planar cell polarity core component, Vangl2.

Vangl2, one of the core components of the planar cell polarity (PCP) pathway, has an important role in the regulation of morphogenesis in several tissues. Although the expression of Vangl2 has been detected in the developing tooth, its role in tooth morphogenesis is not known. In this study, we show that Vangl2 is expressed in the inner dental epithelium (IDE) and in the secondary enamel knots (SEKs) of bell stage tooth germs. Inhibition of Vangl2 expression by siRNA treatment in in vitro-cultured tooth germs resulted in retarded tooth germ growth with deregulated cell proliferation and apoptosis. After kidney transplantation of Vangl2 siRNA-treated tooth germs, teeth were observed to be small and malformed. We also show that Vangl2 is required to maintain the proper pattern of cell alignment in SEKs, which maybe important for the function of SEKs as signaling centers. These results suggest that Vangl2 plays an important role in the morphogenesis of teeth.

Evolving marine biomimetics for regenerative dentistry.

New products that help make human tissue and organ regeneration more effective are in high demand and include materials, structures and substrates that drive cell-to-tissue transformations, orchestrate anatomical assembly and tissue integration with biology. Marine organisms are exemplary bioresources that have extensive possibilities in supporting and facilitating development of human tissue substitutes. Such organisms represent a deep and diverse reserve of materials, substrates and structures that can facilitate tissue reconstruction within lab-based cultures. The reason is that they possess sophisticated structures, architectures and biomaterial designs that are still difficult to replicate using synthetic processes, so far. These products offer tantalizing pre-made options that are versatile, adaptable and have many functions for current tissue engineers seeking fresh solutions to the deficiencies in existing dental biomaterials, which lack the intrinsic elements of biofunctioning, structural and mechanical design to regenerate anatomically correct dental tissues both in the culture dish and in vivo.

A nanostructured anti-biofilm surface widens the efficacy against spindle-shaped and chain-forming rod-like bacteria.

Current control of pathogenic bacteria at all biomaterial interfaces is poorly attuned to a broad range of disease-causing pathogens. Leading antimicrobial surface functionalization strategies with antimicrobial peptides (AMPs), defensins, have not shown their promised efficacy. One of the main problems is the lack of stability and swift clearance from the surface. Surface nanotopography bearing sharp protrusions is a non-chemical solution that is intrinsically stable and long-lasting. Previously, the geometrically ordered arrays of nanotipped spines repelled or rapidly ruptured bacteria that come into contact. The killing properties so far work on cocci and rod-like bacteria, but there is no validation of the efficacy of protrusional surfaces on pathogenic bacteria with different sizes and morphologies, thus broadening the utility of such surfaces to cover increasingly more disease entities. Here, we report a synthetic analogue of nanotipped spines with a pyramidal shape that show great effectiveness on species of bacteria with strongly contrasting shapes and sizes. To highlight this phenomenon in the field of dental applications where selective bacterial control is vital to the clinical success of biomaterial functions, we modified the poly(methyl)-methacrylate (PMMA) texture and tested it against Streptococcus mutans, Enterococcus faecalis, Porphyromonas gingivalis, and Fusobacterium nucleatum. These nanopyramids performed effectively at levels well above those of normal and roughened PMMA biomaterials for dentistry and a model material for general use in medicine and disease transmission in hospital environments.

Differential Mechanism of Periodontitis Progression in Postmenopause.

Over the past four decades, it has become accepted that periodontal disease is caused by specific bacterial infections and that individuals are uniformly susceptible neither to these infections nor to the damage caused by them. The specific bacterial infections and the composition of the environment in which these bacteria easily settle cause an immune response. The immune cells involved in pathogenesis of periodontitis migrate into the periodontitis lesion and advance the disease. The purpose of the present study is to investigate the correlation between immune cell migration and progression of periodontal disease by inducing estrogen deficiency through ovariectomy (OVX) to mimic postmenopausal women and treatment with lipopolysaccharide (LPS). The LPS derived from Porphyromonas gingivalis induced periodontitis and absorption of the alveolar bone dose-dependently. However, the alveolar crest level reduction after LPS injection between OVX and Sham operated mice did not show a significant difference. Matrix metallopeptidase-9 (MMP-9), which is known to be able to detect the progression of periodontitis in general, was not significantly different between OVX and Sham groups. However, immune cells such as T-lymphocytes and neutrophils migrated less overall in OVX groups than Sham operated groups. These findings can be a topic of debate on the old controversy regarding the relationship between periodontal disease and hormonal change. Currently, in clinical practice, menopause is not a major consideration in the treatment of periodontal disease. This study suggests that treatment methods and medication should be considered in the treatment of infectious periodontal disease in postmenopausal women.

An ex vivo model for chondrogenesis and osteogenesis.

Loss of bone and cartilage are major healthcare issues. At present, there is a paucity of therapies for effectively repairing these tissues sustainably in the long term. A tissue engineering approach using advanced functional scaffolds may provide a clinically acceptable alternative. In this study, an innovative mineralized alginate/chitosan scaffold was used to provide tailored microenvironments for driving chondrogenesis and osteogenesis from single and mixed populations of human articular chondrocytes and human bone marrow stromal cells. Polysaccharide capsules were prepared with combinations of these cell types with the addition of type I or type II collagen to augment cell-matrix interactions and promote the formation of phenotypically distinct tissues and placed in a rotating (Synthecon) bioreactor or held in static 2D culture conditions for up to 28 days. Significant cell-generated matrix synthesis was observed in human bone marrow bioreactor samples containing type I collagen after 21-28 days, with increased cell proliferation, cell activity and osteocalcin synthesis. The cell-generated matrix was immuno-positive for types I and II collagen, bone sialoprotein and type X collagen, a marker of chondrogenic hypertrophy, demonstrating the formation of a mature chondrogenic phenotype with areas of new osteoid tissue formation. We present a unique approach using alginate/collagen capsules encapsulated in chitosan to promote chondrogenic and osteogenic differentiation and extracellular matrix formation and the potential for tissue-specific differentiation. This has significant implications for skeletal regeneration and application.

Natural and Synthetic Coral Biomineralization for Human Bone Revitalization.

Coral skeletons can regenerate replacement human bone in nonload-bearing excavated skeletal locations. A combination of multiscale, interconnected pores and channels and highly bioactive surface chemistry has established corals as an important alternative to using healthy host bone replacements. Here, we highlight how coral skeletal systems are being remolded into new calcified structures or synthetic corals by biomimetic processes, as places for the organized permeation of bone tissue cells and blood vessels. Progressive technologies in coral aquaculture and self-organization inorganic chemistry are helping to modify natural corals and create synthetic coral architectures able to accelerate bone regeneration with proper host integration at more skeletal locations, adapted to recent surgical techniques and used to treat intrinsic skeletal deformities and metabolic conditions.

Bio-implant as a novel restoration for tooth loss.

A dental implant is used to replace a missing tooth. Fixing the implant in its natural position requires the engineering of a substantial amount of conformal bone growth inside the implant socket, osseointegration. However, this conventional implant attachment does not include the periodontal ligament (PDL), which has a fundamental role in cushioning high mechanical loads. As a result, tooth implants have a shorter lifetime than the natural tooth and have a high chance of infections. We have engineered a "bio-implant" that provides a living PDL connection for titanium implants. The bio-implant consists of a hydroxyapatite coated titanium screw, ensheathed in cell sheets made from immortalized human periodontal cells. Bio-implants were transplanted into the upper first molar region of a tooth-extraction mouse model. Within 8 weeks the bio-implant generated fibrous connective tissue, a localised blood vessel network and new bone growth fused into the alveolar bone socket. The study presents a bio-implant engineered with human cells, specialised for the root connection, and resulted in the partial reconstruction of a naturalised tooth attachment complex (periodontium), consisting of all the principal tissue types, cementum, PDL and alveolar bone.

Insect Analogue to the Lotus Leaf: A Planthopper Wing Membrane Incorporating a Low-Adhesion, Nonwetting, Superhydrophobic, Bactericidal, and Biocompatible Surface.

Nature has produced many intriguing and spectacular surfaces at the micro- and nanoscales. These small surface decorations act for a singular or, in most cases, a range of functions. The minute landscape found on the lotus leaf is one such example, displaying antiwetting behavior and low adhesion with foreign particulate matter. Indeed the lotus leaf has often been considered the "benchmark" for such properties. One could expect that there are animal counterparts of this self-drying and self-cleaning surface system. In this study, we show that the planthopper insect wing (Desudaba danae) exhibits a remarkable architectural similarity to the lotus leaf surface. Not only does the wing demonstrate a topographical likeness, but some surface properties are also expressed, such as nonwetting behavior and low adhering forces with contaminants. In addition, the insect-wing cuticle exhibits an antibacterial property in which Gram-negative bacteria (Porphyromonas gingivalis) are killed over many consecutive waves of attacks over 7 days. In contrast, eukaryote cell associations, upon contact with the insect membrane, lead to a formation of integrated cell sheets (e.g., among human stem cells (SHED-MSC) and human dermal fibroblasts (HDF)). The multifunctional features of the insect membrane provide a potential natural template for man-made applications in which specific control of liquid, solid, and biological contacts is desired and required. Moreover, the planthopper wing cuticle provides a "new" natural surface with which numerous interfacial properties can be explored for a range of comparative studies with both natural and man-made materials.

Strategies to promote chondrogenesis and osteogenesis from human bone marrow cells and articular chondrocytes encapsulated in polysaccharide templates.

The aim of this study was to synthesize functional in vitro and in vivo 3-dimensional (3D) constructs using a mix of human mesenchymal populations and articular chondrocytes encapsulated in biomineralized polysaccharide templates. Single-cell-type populations or mixtures of both cell types were encapsulated in alginate/chitosan and cultured within a rotating-bioreactor, perfused bioreactor system, or static conditions for 28 days. Within single cell-type populations, type II collagen immunopositive cells were present within lacunae in rotating-bioreactor capsules, with an increased proportion of metabolically active cells compared with perfused and static constructs. Biochemical analysis indicated significantly increased ( p < 0.05) DNA and protein in rotating-bioreactor conditions compared with perfused or static. However, in coculture samples, DNA and protein was significantly increased in static cultures owing to the formation of large regions of partially mineralized osteoid. This osteoid was found only in static cultures and when the ratio of human bone marrow cells to chondrocytes was 2:1 or, to a lesser extent, 5:1 ratio capsules. Subcutaneous implantation of capsules into immunocompromised mice also showed optimal osteoid formation when the ratio was 2:1. The current studies demonstrate the pivotal role of robust 3D biomimetic microenvironments and indicate the potential to harness the interactions between different cell types to create specific tissues.

Small-Scale Fabrication of Biomimetic Structures for Periodontal Regeneration.

The periodontium is the supporting tissues for the tooth organ and is vulnerable to destruction, arising from overpopulating pathogenic bacteria and spirochaetes. The presence of microbes together with host responses can destroy large parts of the periodontium sometimes leading tooth loss. Permanent tissue replacements are made possible with tissue engineering techniques. However, existing periodontal biomaterials cannot promote proper tissue architectures, necessary tissue volumes within the periodontal pocket and a "water-tight" barrier, to become clinically acceptable. New kinds of small-scale engineered biomaterials, with increasing biological complexity are needed to guide proper biomimetic regeneration of periodontal tissues. So the ability to make compound structures with small modules, filled with tissue components, is a promising design strategy for simulating the anatomical complexity of the periodotium attachment complexes along the tooth root and the abutment with the tooth collar. Anatomical structures such as, intima, adventitia, and special compartments such as the epithelial cell rests of Malassez or a stellate reticulum niche need to be engineered from the start of regeneration to produce proper periodontium replacement. It is our contention that the positioning of tissue components at the origin is also necessary to promote self-organizing cell-cell connections, cell-matrix connections. This leads to accelerated, synchronized and well-formed tissue architectures and anatomies. This strategy is a highly effective preparation for tackling periodontitis, periodontium tissue resorption, and to ultimately prevent tooth loss. Furthermore, such biomimetic tissue replacements will tackle problems associated with dental implant support and perimimplantitis.

A First Step in De Novo Synthesis of a Living Pulp Tissue Replacement Using Dental Pulp MSCs and Tissue Growth Factors, Encapsulated within a Bioinspired Alginate Hydrogel.

INTRODUCTION: A living, self-supporting pulp tissue replacement in vitro and for transplantation is an attractive yet unmet bioengineering challenge. Our aim is to create 3-dimensional alginate-based microenvironments that replicate the shape of gutta-percha and comprise key elements for the proliferation of progenitor cells and the release of growth factors. METHODS: An RGD-bearing alginate framework was used to encapsulate dental pulp stem cells and human umbilical vein endothelial cells in a ratio of 1:1. The alginate hydrogel also retained and delivered 2 key growth factors, vascular endothelial growth factor-121 and fibroblast growth factor, in a sufficient amount to induce proliferation. A method was then devised to replicate the shape of gutta-percha using RGD alginate within a custom-made mold of thermoresponsive N-isopropylacrylamide. Plugs of alginate containing different permutations of growth factor-based encapsulates were tested and evaluated for viability, proliferation, and release kinetics between 1 and 14 days. RESULTS: According to scanning electron microscopic and confocal microscopic observations, the encapsulated human endothelial cells and dental pulp stem cell distribution were frequent and extensive throughout the length of the construct. There were also high levels of viability in all test environments. Furthermore, cell proliferation was higher in the growth factor-based groups. Growth factor release kinetics also showed significant differences between them. Interestingly, the combination of vascular endothelial growth factor and fibroblast growth factor synergize to significantly up-regulate cell proliferation. CONCLUSIONS: RGD-alginate scaffolds can be fabricated into shapes to fill the pulp space by simple templating. The addition of dual growth factors to cocultures of stem cells within RGD-alginate scaffolds led to the creation of microenvironments that significantly enhance the proliferation of dental pulp stem cell/human umbilical vein endothelial cell combinations.

Marine Structural Biomaterials in Medical Biomimicry.

Marine biomaterials display properties, behaviors, and functions that have not been artificially matched in relation to their hierarchical construction, crack-stopping properties, growth adaptation, and energy efficiency. The discovery and understanding of such features that are characteristic of natural biomaterials can be used to manufacture more energy-efficient and lightweight materials. However, a more detailed understanding of the design of natural biomaterials with good performance and the mechanism of their design is required. Far-reaching biomolecular characterization of biomaterials and biostructures from the ocean world is possible with sophisticated analytical methods, such as whole-genome RNA-seq, and de novo transcriptome sequencing and mass spectrophotometry-based sequencing. In combination with detailed material characterization, the elements in newly discovered biomaterials and their properties can be reconstituted into biomimetic or bio-inspired materials. A major aim of harnessing marine biomaterials is their translation into biomimetic counterparts. To achieve full translation, the genome, proteome, and hierarchical material characteristics, and their profiles in space and time, have to be associated to allow for smooth biomimetic translation. In this article, we highlight the novel science of marine biomimicry from a materials perspective. We focus on areas of material design and fabrication that have excelled in marine biological models, such as embedded interfaces, chiral organization, and the use of specialized composite material-on-material designs. Our emphasis is primarily on key materials with high value in healthcare in which we evaluate their future prospects. Marine biomaterials are among the most exquisite and powerful aspects in materials science today.

Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.

The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems.

A gecko skin micro/nano structure - A low adhesion, superhydrophobic, anti-wetting, self-cleaning, biocompatible, antibacterial surface.

Geckos, and specifically their feet, have attracted significant attention in recent times with the focus centred around their remarkable adhesional properties. Little attention however has been dedicated to the other remaining regions of the lizard body. In this paper we present preliminary investigations into a number of notable interfacial properties of the gecko skin focusing on solid and aqueous interactions. We show that the skin of the box-patterned gecko (Lucasium sp.) consists of dome shaped scales arranged in a hexagonal patterning. The scales comprise of spinules (hairs), from several hundred nanometres to several microns in length, with a sub-micron spacing and a small radius of curvature typically from 10 to 20 nm. This micro and nano structure of the skin exhibited ultralow adhesion with contaminating particles. The topography also provides a superhydrophobic, anti-wetting barrier which can self clean by the action of low velocity rolling or impacting droplets of various size ranges from microns to several millimetres. Water droplets which are sufficiently small (10-100 µm) can easily access valleys between the scales for efficient self-cleaning and due to their dimensions can self-propel off the surface enhancing their mobility and cleaning effect. In addition, we demonstrate that the gecko skin has an antibacterial action where Gram-negative bacteria (Porphyromonas gingivalis) are killed when exposed to the surface however eukaryotic cell compatibility (with human stem cells) is demonstrated. The multifunctional features of the gecko skin provide a potential natural template for man-made applications where specific control of liquid, solid and biological contacts is required.

Linear poly(ethylenimine) cross-linked by methyl-ß-cyclodextrin for gene delivery.

Poly(ethylenimine) (PEI) is a cationic polymer extensively exploited for non-viral gene delivery; however, its wide application has been impeded by its cytotoxicity. PEI can assume either a branched or linear configuration. Whereas branched PEI (bPEI) is more chemically reactive and can form smaller complexes with DNA under salt-containing conditions, lPEI is generally less toxic and exhibits higher transfection efficiency. In this study, we cross-linked low-molecularweight lPEI with methyl ß-cyclodextrin (MßCD) to form MßCD-lPEI (MLP). The structure of MLP was successfully characterized by NMR, FT-IR, MALDI-TOF and elemental analysis. In the standard serum-free transfection environment, MLP could effectively transfect glioblastoma, melanoma and hepatocarcinoma cells. A high transfection efficiency was maintained in the presence of serum. Apart from its high transfection efficiency, MLP was found to have negligible cytotoxicity over a wide range of concentrations and to exhibit a low membrane disruptive capacity ex vivo. MLP warrants further development as a promising gene delivery system for future research.

Calcifying tissue regeneration via biomimetic materials chemistry.

Materials chemistry is making a fundamental impact in regenerative sciences providing many platforms for tissue development. However, there is a surprising paucity of replacements that accurately mimic the structure and function of the structural fabric of tissues or promote faithful tissue reconstruction. Methodologies in biomimetic materials chemistry have shown promise in replicating morphologies, architectures and functional building blocks of acellular mineralized tissues dentine, enamel and bone or that can be used to fully regenerate them with integrated cell populations. Biomimetic materials chemistry encompasses the two processes of crystal formation and mineralization of crystals into inorganic formations on organic templates. This review will revisit the successes of biomimetics materials chemistry in regenerative medicine, including coccolithophore simulants able to promote in vivo bone formation. In-depth knowledge of biomineralization throughout evolution informs the biomimetic materials chemist of the most effective techniques for regenerative framework construction exemplified via exploitation of liquid crystals (LCs) and complex self-organizing media. Therefore, a new innovative direction would be to create chemical environments that perform reaction-diffusion exchanges as the basis for building complex biomimetic inorganic structures. This has evolved widely in biology, as have LCs, serving as self-organizing templates in pattern formation of structural biomaterials. For instance, a study is highlighted in which artificially fabricated chiral LCs, made from bacteriophages are transformed into a faithful copy of enamel. While chemical-based strategies are highly promising at creating new biomimetic structures there are limits to the degree of complexity that can be generated. Thus, there may be good reason to implement living or artificial cells in 'morphosynthesis' of complex inorganic constructs. In the future, cellular construction is probably key to instruct building of ultimate biomimetic hierarchies with a totality of functions.

In situ continuous growth formation of synthetic biominerals.

Continuous self-assembled growth of both the organic and inorganic components of materials with nacre-like structure is achieved upon mineralisation of chitin and chitosan scaffolds using a combined soaking method and the inclusion of poly(acrylic acid) and chitosan oligomers as additives.

Biomimetic mineralisation of polymeric scaffolds using a combined soaking and Kitano approach.

Chitosan hydrogels are of considerable interest in synthetic biomimetic mineralisation strategies due to their favourable characteristics such as the presentation of a large surface area for crystal nucleation within a structured yet responsive scaffold. Chitosan hydrogels were prepared and subsequently calcium carbonate mineralisation was initiated using a method which combines alternate soaking of the films with precursor solutions followed by treatment with Kitano solution. This combined approach allows for increased extent of mineralisation, inducement of mineralisation uniformly throughout the hydrogel rather than only at the peripheral surface and ready scalability and shape manipulation. The base synthetic system is readily modified through the introduction of additives that manipulate the nucleation and growth of the calcium carbonate. Addition of poly(acrylic acid) inhibits nucleation and induces tangential crystal growth along the internal and external interfaces of the hydrogel. The resulting composite is comprised of stacked overlapping plates of calcium carbonate intercalated with carbohydrate. The method is applicable in combination with a variety of hydrogels including macroporous chitosan, chitosan-alginate bilayers and pure alginate hydrogels. The composite materials were analysed by SEM, XRD, microRaman spectroscopy and mechanical strength testing.

Augmentation of skeletal tissue formation in impaction bone grafting using vaterite microsphere biocomposites.

The development of particulate bone void fillers with added biological function to augment skeletal tissue formation will lead to improved efficacy in bone replacement surgery. We demonstrate the potential for vaterite microsphere biocomposites to augment bone matrix formation within an in vivo model for impaction bone grafting seeded with human bone marrow stromal cells. In vitro tests demonstrate the significance of vaterite microspheres in the activation and promotion of 3D skeletal tissue formation. Further in vitro experiments using functionalized microspheres with surface integrated RGD peptide activate co-cultured skeletal populations in pellets and promote secretion of extracellular matrix collagens and human osteocalcin. Specific temporal release of entrapped RNase A was successfully demonstrated using these specialized microspheres with integrated magnetic beads, which physically disrupted the inorganic macrostructure. These studies demonstrate that bio-inspired calcium carbonate microspheres augment in vivo bone formation in impaction bone grafting. Such microspheres with added biological functionality offer innovative therapeutic approaches to activate skeletal populations and enhance bone formation with reparative implications for hard tissues.

Tissue bionics: examples in biomimetic tissue engineering.

Many important lessons can be learnt from the study of biological form and the functional design of organisms as design criteria for the development of tissue engineering products. This merging of biomimetics and regenerative medicine is termed 'tissue bionics'. Clinically useful analogues can be generated by appropriating, modifying and mimicking structures from a diversity of natural biomatrices ranging from marine plankton shells to sea urchin spines. Methods in biomimetic materials chemistry can also be used to fabricate tissue engineering scaffolds with added functional utility that promise human tissues fit for the clinic.