Hanson Fong

Hsp70 and Hsp40 attenuate formation of spherical and annular polyglutamine oligomers by partitioning monomer

Protein conformational changes that result in misfolding, aggregation and amyloid fibril formation are a common feature of many neurodegenerative disorders. Studies with β-amyloid (Aβ), α-synuclein and other amyloid-forming proteins indicate that the assembly of misfolded protein conformers into fibrils is a complex process that may involve the population of metastable spherical and/or annular oligomeric assemblies. Here, we show by atomic force microscopy that a mutant huntingtin fragment with an expanded polyglutamine repeat forms spherical and annular oligomeric structures reminiscent of those formed by Aβ and α-synuclein. Notably, the molecular chaperones Hsp70 and Hsp40, which are protective in animal models of neurodegeneration, modulate polyglutamine aggregation reactions by partitioning monomeric conformations and disfavoring the accretion of spherical and annular oligomers.

Biological Organization of Hydroxyapatite Crystallites into a Fibrous Continuum Toughens and Controls Anisotropy in Human Enamel

Enamel forms the outer surface of teeth, which are of complex shape and are loaded in a multitude of ways during function. Enamel has previously been assumed to be formed from discrete rods and to be markedly aniostropic, but marked anisotropy might be expected to lead to frequent fracture. Since frequent fracture is not observed, we measured enamel organization using histology, imaging, and fracture mechanics modalities, and compared enamel with crystalline hydroxyapatite (Hap), its major component. Enamel was approximately three times tougher than geologic Hap, demonstrating the critical importance of biological manufacturing. Only modest levels of enamel anisotropy were discerned; rather, our measurements suggest that enamel is a composite ceramic with the crystallites oriented in a complex three-dimensional continuum. Geologic apatite crystals are much harder than enamel, suggesting that inclusion of biological contaminants, such as protein, influences the properties of enamel. Based on our findings, we propose a new structural model.

Nano-mechanical properties profiles across dentin–enamel junction of human incisor teeth

Abstract Understanding how load is transferred from enamel to dentin and how the two tissues function as a single mechanical unit during mastication requires studies of micromechanics in relation to microstructure of the dentin–enamel junction (DEJ) zone. In this investigation, nano-hardness and elastic modulus of human incisor teeth were studied across the DEJ. It was found that, over a length scale of about 20 μm, there were decreasing trends in both hardness and elastic modulus across the DEJ zone profiling from enamel to dentin. Images obtained using atomic force microscopy from polished surfaces of cross-sectioned dental samples showed an interpenetrated microstructure of enamel and dentin at the DEJ zone. This result suggests that the nano-mechanical property profiles across the DEJ were due to a continuous variation in the ratios of relative amount of enamel and dentin. These characteristics of the DEJ zone could be significant for describing the structural and mechanical coupling of the two tissues. By increasing the contact area across the interface between the two hard tissues the stresses are dissipated reducing interfacial stress concentrations at the DEJ, thereby promoting effective load transfer from the hard (brittle) enamel to soft (tough) dentin.

Advances in defining regulators of cementum development and periodontal regeneration.

Substantial advancements have been made in defining the cells and molecular signals that guide tooth crown morphogenesis and development. As a result, very encouraging progress has been made in regenerating crown tissues by using dental stem cells and recombining epithelial and mesenchymal tissues of specific developmental ages. To date, attempts to regenerate a complete tooth, including the critical periodontal tissues of the tooth root, have not been successful. This may be in part due to a lesser degree of understanding of the events leading to the initiation and development of root and periodontal tissues. Controversies still exist regarding the formation of periodontal tissues, including the origins and contributions of cells, the cues that direct root development, and the potential of these factors to direct regeneration of periodontal tissues when they are lost to disease. In recent years, great strides have been made in beginning to identify and characterize factors contributing to formation of the root and surrounding tissues, that is, cementum, periodontal ligament, and alveolar bone. This review focuses on the most exciting and important developments over the last 5 years toward defining the regulators of tooth root and periodontal tissue development, with special focus on cementogenesis and the potential for applying this knowledge toward developing regenerative therapies. Cells, genes, and proteins regulating root development are reviewed in a question-answer format in order to highlight areas of progress as well as areas of remaining uncertainty that warrant further study.

Regulation of in vitro calcium phosphate mineralization by combinatorially selected hydroxyapatite-binding peptides.

We report selection and characterization of hydroxyapatite-binding heptapeptides from a peptide-phage library and demonstrate the effects of two peptides, with different binding affinities and structural properties, on the mineralization of calcium phosphate mineral. In vitro mineralization studies carried out using one strong- and one weak-binding peptide, HABP1 and HABP2, respectively, revealed that the former exhibited a drastic outcome on mineralization kinetics and particle morphology. Strong-binding peptide yielded significantly larger crystals, as observed by electron microscopy, in comparison to those formed in the presence of a weak-binding peptide or in the negative control. Molecular structural studies carried out by circular dichroism revealed that HABP1 and HABP2 differed in their secondary structure and conformational stability. The results indicate that sequence, structure, and molecular stability strongly influence the mineralization activity of these peptides. The implication of the research is that the combinatorially selected short-sequence peptides may be used in the restoration or regeneration of hard tissues through their control over of the formation of calcium phosphate biominerals.

Biological response on a titanium implant-grade surface functionalized with modular peptides.

Titanium (Ti) and its alloys are among the most successful implantable materials for dental and orthopedic applications. The combination of excellent mechanical and corrosion resistance properties makes them highly desirable as endosseous implants that can withstand a demanding biomechanical environment. Yet, the success of the implant depends on its osteointegration, which is modulated by the biological reactions occurring at the interface of the implant. A recent development for improving biological responses on the Ti-implant surface has been the realization that bifunctional peptides can impart material binding specificity not only because of their molecular recognition of the inorganic material surface, but also through their self-assembly and ease of biological conjugation properties. To assess peptide-based functionalization on bioactivity, the present authors generated a set of peptides for implant-grade Ti, using cell surface display methods. Out of 60 unique peptides selected by this method, two of the strongest titanium binding peptides, TiBP1 and TiBP2, were further characterized for molecular structure and adsorption properties. These two peptides demonstrated unique, but similar molecular conformations different from that of a weak binder peptide, TiBP60. Adsorption measurements on a Ti surface revealed that their disassociation constants were 15-fold less than TiBP60. Their flexible and modular use in biological surface functionalization were demonstrated by conjugating them with an integrin recognizing peptide motif, RGDS. The functionalization of the Ti surface by the selected peptides significantly enhanced the bioactivity of osteoblast and fibroblast cells on implant-grade materials.

Tooth-forming potential in embryonic and postnatal tooth bud cells

Humans are genetically programmed to replace their teeth once during childhood. Therefore, when adult teeth are lost or damaged, they cannot be regenerated or regrown. However, with the advancement of stem cell biology and tissue engineering, regenerating the whole tooth has become a realistic and attractive option to replace a lost or damaged tooth, and therefore has strongly attracted attention in the field of dental research. During the past several years, significant progress has been made in this research endeavor, providing greater understanding of the production of an entire biological tooth by tissue engineering using stem cells. There are several ways to reproduce an entire biological tooth. Approaches are categorized according to the cell sources that have the potential to produce teeth. One source is the embryonic tooth bud, and the other is the postnatal tooth bud. The results from embryonic and postnatal tooth buds differ considerably. In particular, the potential to regulate the shape of the tooth crown from embryonic tooth bud is higher than from postnatal tooth bud. This article describes the achievements to date in production of biological teeth, mostly from our laboratory. In particular, we describe the potential to produce teeth from embryonic and postnatal tooth buds.

Enzyme replacement prevents enamel defects in hypophosphatasia mice

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity, leading to rickets or osteomalacia and to dental defects. HPP occurs from loss‐of‐function mutations within the gene that encodes the tissue‐nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl−/−, aka Akp2−/−) mice closely phenocopy infantile HPP, including the rickets, vitamin B6‐responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl−/− mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral‐targeted TNAP (ENB‐0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage‐specific expression of TNAP in ameloblasts. In the Alpl−/− mice, histological, µCT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter‐rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral‐targeting, human TNAP at 8.2 mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization and demonstrate the efficacy of mineral‐targeted TNAP to prevent enamel defects in HPP.

Enamel structure properties controlled by engineered proteins in transgenic mice

Amelogenin protein has regulatory effects on enamel biofabrication in mammalian tooth. Using teeth obtained from transgenic mice that express two separate protein‐engineered versions of amelogenins, we made structure‐nanomechanical properties correlations and showed 21% hardness and 24% elastic modulus degradation compared with the age‐matched wildtype littermates. We attribute the inferior properties to the disorganization of the protein matrix resulting in defective mineral formation.

Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

ABSTRACT Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo.