Liquid Transmission Electron Microscopy for Probing Collagen Biomineralization.

Collagen biomineralization is fundamental to hard tissue assembly. While studied extensively, collagen mineralization processes are not fully understood, with the majority of theories derived from electron microscopy (EM) under static, dehydrated, or frozen conditions, unlike the liquid phase environment where mineralization occurs. Herein, novel liquid transmission EM (TEM) strategies are presented, in which collagen mineralization was explored in liquid for the first time via TEM. Custom thin-film enclosures were employed to visualize the mineralization of reconstituted collagen fibrils in a calcium phosphate and polyaspartic acid solution to promote intrafibrillar mineralization. TEM highlighted that at early time points precursor mineral particles attached to collagen and progressed to crystalline mineral platelets aligned with fibrils at later time points. This aligns with observations from other techniques and validates the liquid TEM approach. This work provides a new liquid imaging approach for exploring collagen biomineralization, advancing toward understanding disease pathogenesis and remineralization strategies for hard tissues.

Screening of functionalized collagen membranes with a porcine periodontal regeneration model.

OBJECTIVES: Current methods for periodontal regeneration do not promote collagen fiber insertions into new bone and cementum. We used a pig wound model to screen different functionalized collagen membranes in promoting periodontal reattachment to root surfaces. METHODS: Treatment groups included (1) control with no membranes, (2) collagen-coated membranes, (3) membranes with insulin-like growth factor-1 (IGF-1), (4) membranes with amelotin, or (5) membranes attached with calcium phosphate cement (CPC), or with CPC combined with IGF-1. Flap procedures were performed on mandibular and maxillary premolars of each pig. RESULTS: Histomorphometric, micro-CT, and clinical measurements obtained at 4 and 12 weeks after surgery showed cementum formation on denuded roots and reformation of alveolar bone, indicating that the pig model can model healing responses in periodontal regeneration. Calcium phosphate cement simplified procedures by eliminating the need for sutures and improved regeneration of alveolar bone (p < 0.05) compared with other treatments. There was a reduction (p < 0.05) of PD only for the IGF group. Large observed variances between treatment groups indicated that a priori power analyses should be conducted to optimize statistical analysis. CONCLUSIONS: Pigs can model discrete elements of periodontal healing using collagen-based, functionalized membranes. Screening indicates that membrane anchorage with calcium phosphate cements improve regeneration of alveolar bone.

Glycosaminoglycans accelerate biomimetic collagen mineralization in a tissue-based in vitro model.

Mammalian teeth are attached to the jawbone through an exquisitely controlled mineralization process: unmineralized collagen fibers of the periodontal ligament anchor directly into the outer layer of adjoining mineralized tissues (cementum and bone). The sharp interface between mineralized and nonmineralized collagenous tissues makes this an excellent model to study the mechanisms by which extracellular matrix macromolecules control collagen mineralization. While acidic phosphoproteins, localized in the mineralized tissues, play key roles in control of mineralization, the role of glycosaminoglycans (GAGs) is less clear. As several proteoglycans are found only in the periodontal ligament, it has been hypothesized that these inhibit mineralization of collagen in this tissue. Here we used an in vitro model based on remineralization of mouse dental tissues to determine the role of matrix GAGs in control of mineralization. GAGs were selectively removed from demineralized mouse periodontal sections via enzymatic digestion. Proteomic analysis confirmed that enzymatic GAG removal does not significantly alter protein content. Analysis of remineralized tissue sections by transmission electron microscopy (TEM) shows that GAG removal reduced the rate of remineralization in mineralized tissues compared to the untreated control, while the ligament remained unmineralized. Protein removal with trypsin also reduced the rate of mineralization, but to a lesser extent than GAG removal, despite a much larger effect on protein content. These results indicate that GAGs promote mineralization in mineralized dental tissues rather than inhibiting mineral formation in the ligament, which may have broader implications for understanding control of collagen mineralization in connective tissues.

Polyaminoacids in Biomimetic Collagen Mineralization: Roles of Isomerization and Disorder in Polyaspartic and Polyglutamic Acids.

The extracellular matrix of hard connective tissues is composed primarily of mineralized collagen fibrils. Acidic noncollagenous proteins play important roles in mediating mineralization of collagen. Polyaspartate, a homopolymer substitute for such proteins, has been used extensively in in vitro models to produce biomimetic mineralized collagen. Polyglutamate behaves differently in mineralization models, despite its chemical similarity. We show that polyaspartate is a 350 times more effective inhibitor of solution precipitation of hydroxyapatite than polyglutamate. Supersaturated CaP solutions stabilized with polyaspartic acid produce collagen with aligned intrafibrillar mineral, while solutions containing polyglutamate lead to the formation of unaligned mineral clusters on the fibril surface. Molecular analysis showed that the commercial polyaspartic acid contains substantial isomerization, unlike polyglutamic acid. Hence, the secondary structure of polyaspartic acid is more disordered than that of polyglutamic acid. The increased flexibility of the polyaspartic acid chain may explain its potency as an inhibitor of solution crystallization and a mediator of intrafibrillar collagen mineralization.

In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Otoliths are one of the biominerals whose formation is highly controlled by proteins. The first protein discovered to be involved in otolith biomineralization in zebrafish was starmaker (Stm). Previously, Stm was shown to be responsible for the preferential formation of aragonite, a polymorph of calcium carbonate, in otoliths. In this work, proteomic analysis of adult zebrafish otoliths was performed. Stm is the only highly phosphorylated protein found in our studies. Besides previously studied otolith proteins, we discovered several dozens of unknown proteins that reveal the likely mechanism of biomineralization. A comparison of aragonite and vaterite otoliths showed similarities in protein composition. We observed the presence of Stm in both types of otoliths. In vitro studies of 2 characteristic Stm fragments indicated that the DS-rich region has a special biomineralization activity, especially after phosphorylation.-Kalka, M., Markiewicz, N., Ptak, M., Sone, E. D., Ozyhar, A., Dobryszycki, P., Wojtas, M. In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Spatial distribution of proteins in the quagga mussel adhesive apparatus.

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious 'anchor' (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.

A Top-down Approach to Elucidate the Role of Matrix-Bound Phosphoproteins in Control of Collagen Biomineralization.

The periodontium is the set of tissues responsible for tooth anchorage, and consists of interconnected layers of mineralized and unmineralized tissues (bone, ligament and cementum). The ligament-cementum interface is a particularly elegant example of biological control of mineralization and the controlling factors are poorly understood. Here we use a tissue-based in vitro model of mineralization, in which sections of demineralized mouse jaw remineralize with the same selectivity as found in vivo, to probe the molecular mechanism of control over collagen mineralization in the periodontium. Removal or enzymatic cleavage of noncollagenous proteins have very similar effects: a reduction in the rate of remineralization that is much more drastic in cementum than in dentin. The periodontal ligament does not mineralize within experimental parameters even after protein removal/digestion. Dephosphorylation results in a slight reduction in mineralization in dentin and cementum. Understanding the mechanisms controlling selective mineralization in the periodontium will help elucidate the molecular factors controlling collagen biomienralization, and provide inspiration for the development of scaffolds for regeneration of hard-soft tissue interfaces.

BoneTape: A novel osteosynthetic device for the stabilization of zygomatic fractures.

BACKGROUND: The study aims to assess the safety and effectiveness of BoneTape™, a new resorbable bone fixation device, using a zygomatic fracture model in rabbits. METHODS: The study followed BoneTape™ samples and control (sham) groups over 2-, 6-, and 12-week periods post-zygomaticomaxillary (ZM) osteotomy and zygomaticofrontal (ZF) disarticulation. The osteotomized segments were analyzed for bone healing, inflammatory response, and tissue healing. µCT imaging and histological analysis were used to examine the axial alignment, offset, and quality of new bone formation. RESULTS: BoneTape™ samples demonstrated enhanced maintenance of the initial intraoperative positioning, reduced axial offset, and better alignment when compared with the control group, enabling stable bone healing under physiological loading conditions. Complete union was observed at 12-weeks in both groups. The BoneTape™ group experienced minimal immune and tissue reactions, classically associated with wound healing, and showed an increased number of giant cells at 6 and 12-weeks. CONCLUSION: BoneTape™ represents a promising advancement in osteosynthesis, demonstrating efficacy in maintaining stable zygomatic reconstruction and eliciting minimal immune response in a rabbit model. This study introduces BoneTape™ as a disruptive solution specifically designed for clinical application in cranio-maxillofacial fracture fixation, with the potential to eliminate the use of over-engineered solutions while offering benefits such as ease of application and fewer biologically disruptive steps.

In vitro models of collagen biomineralization.

Over the last several years, significant progress has been made toward understanding the mechanisms involved in the mineralization of hard collagenous tissues, such as bone and dentin. Particularly notable are the identification of transient mineral phases that are precursors to carbonated hydroxyapatite, the identification and characterization of non-collagenous proteins that are involved in controlling mineralization, and significant improvements in our understanding of the structure of collagen. These advances not only represent a paradigm shift in the way collagen mineralization is viewed and understood, but have also brought new challenges to light. In this review, we discuss how recent in vitro models have addressed critical questions regarding the role of the non-collagenous proteins in controlling mineralization, the nature of the interactions between amorphous calcium phosphate and collagen during the early stages of mineralization, and the role of collagen in the mineralization process. We discuss the significance of these findings in expanding our understanding of collagen biomineralization, while addressing some of the limitations that are inherent to in vitro systems.

PERP regulates enamel formation via effects on cell-cell adhesion and gene expression.

Little is known about the role of cell-cell adhesion in the development of mineralized tissues. Here we report that PERP, a tetraspan membrane protein essential for epithelial integrity, regulates enamel formation. PERP is necessary for proper cell attachment and gene expression during tooth development, and its expression is controlled by P63, a master regulator of stratified epithelial development. During enamel formation, PERP is localized to the interface between the enamel-producing ameloblasts and the stratum intermedium (SI), a layer of cells subjacent to the ameloblasts. Perp-null mice display dramatic enamel defects, which are caused, in part, by the detachment of ameloblasts from the SI. Microarray analysis comparing gene expression in teeth of wild-type and Perp-null mice identified several differentially expressed genes during enamel formation. Analysis of these genes in ameloblast-derived LS8 cells upon knockdown of PERP confirmed the role for PERP in the regulation of gene expression. Together, our data show that PERP is necessary for the integrity of the ameloblast-SI interface and that a lack of Perp causes downregulation of genes that are required for proper enamel formation.

Byssal proteins of the freshwater zebra mussel, Dreissena polymorpha.

The freshwater zebra mussel (Dreissena polymorpha) is a notorious biofouling organism. It adheres to a variety of substrata underwater by means of a proteinaceous structure called the byssus, which consists of a number of threads with adhesive plaques at the tips. The byssal proteins are difficult to characterize due to extensive cross-linking of 3,4-dihydroxyphenylalanine (DOPA), which renders the mature structure largely resistant to protein extraction and immunolocalization. By inducing secretion of fresh threads and plaques in which cross-linking is minimized, three novel zebra mussel byssal proteins were identified following extraction and separation by gel electrophoresis. Peptide fragment fingerprinting was used to match tryptic digests of several gel bands against a cDNA library of genes expressed uniquely in the mussel foot, the organ which secretes the byssus. This allowed identification of a more complete sequence of Dpfp2 (D. polymorpha foot protein 2), a known DOPA-containing byssal protein, and a partial sequence of Dpfp5, a novel protein with several typical characteristics of mussel adhesive proteins.

Zebra mussel adhesion: structure of the byssal adhesive apparatus in the freshwater mussel, Dreissena polymorpha.

The freshwater zebra mussel (Dreissena polymorpha) owes a large part of its success as an invasive species to its ability to attach to a wide variety of substrates. As in marine mussels, this attachment is achieved by a proteinaceous byssus, a series of threads joined at a stem that connect the mussel to adhesive plaques secreted onto the substrate. Although the zebra mussel byssus is superficially similar to marine mussels, significant structural and compositional differences suggest that further investigation of the adhesion mechanisms in this freshwater species is warranted. Here we present an ultrastructural examination of the zebra mussel byssus, with emphasis on interfaces that are critical to its adhesive function. By examining the attached plaques, we show that adhesion is mediated by a uniform electron dense layer on the underside of the plaque. This layer is only 10-20 nm thick and makes direct and continuous contact with the substrate. The plaque itself is fibrous, and curiously can exhibit either a dense or porous morphology. In zebra mussels, a graded interface between the animal and the substrate mussels is achieved by interdigitation of uniform threads with the stem, in contrast to marine mussels, where the threads themselves are non-uniform. Our observations of several novel aspects of zebra mussel byssal ultrastructure may have important implications not only for preventing biofouling by the zebra mussel, but for the development of new bioadhesives as well.

Mineralized vectors for gene therapy.

There is an intense interest in developing materials for safe and effective delivery of polynucleotides using non-viral vectors. Mineralization of organic templates has long been used to produce complex materials with outstanding biocompatibility. However, a lack of control over mineral growth has limited the applicability of mineralized materials to a few in vitro applications. With better control over mineral growth and surface functionalization, mineralized vectors have advanced significantly in recent years. Here, we review the recent progress in chemical synthesis, physicochemical properties, and applications of mineralized materials in gene therapy, focusing on structure-function relationships. We contrast the classical understanding of the mineralization mechanism with recent ideas of mineralization. A brief introduction to gene delivery is summarized, followed by a detailed survey of current mineralized vectors. The vectors derived from calcium phosphate are articulated and compared to other minerals with unique features. Advanced mineral vectors derived from templated mineralization and specialty coatings are critically analyzed. Mineral systems beyond the co-precipitation are explored as more complex multicomponent systems. Finally, we conclude with a perspective on the future of mineralized vectors by carefully demarcating the boundaries of our knowledge and highlighting ambiguous areas in mineralized vectors. STATEMENT OF SIGNIFICANCE: Therapy by gene-based medicines is increasingly utilized to cure diseases that are not alleviated by conventional drug therapy. Gene medicines, however, rely on macromolecular nucleic acids that are too large and too hydrophilic for cellular uptake. Without tailored materials, they are not functional for therapy. One emerging class of nucleic acid delivery system is mineral-based materials. The fact that they can undergo controlled dissolution with minimal footprint in biological systems are making them attractive for clinical use, where safety is utmost importance. In this submission, we will review the emerging synthesis technology and the range of new generation minerals for use in gene medicines.

The genome of the zebra mussel, Dreissena polymorpha: a resource for comparative genomics, invasion genetics, and biocontrol.

The zebra mussel, Dreissena polymorpha, continues to spread from its native range in Eurasia to Europe and North America, causing billions of dollars in damage and dramatically altering invaded aquatic ecosystems. Despite these impacts, there are few genomic resources for Dreissena or related bivalves. Although the D. polymorpha genome is highly repetitive, we have used a combination of long-read sequencing and Hi-C-based scaffolding to generate a high-quality chromosome-scale genome assembly. Through comparative analysis and transcriptomics experiments, we have gained insights into processes that likely control the invasive success of zebra mussels, including shell formation, synthesis of byssal threads, and thermal tolerance. We identified multiple intact steamer-like elements, a retrotransposon that has been linked to transmissible cancer in marine clams. We also found that D. polymorpha have an unusual 67 kb mitochondrial genome containing numerous tandem repeats, making it the largest observed in Eumetazoa. Together these findings create a rich resource for invasive species research and control efforts.

Attachment of zebra and quagga mussel adhesive plaques to diverse substrates.

Like marine mussels, freshwater zebra and quagga mussels adhere via the byssus, a proteinaceous attachment apparatus. Attachment to various surfaces allows these invasive mussels to rapidly spread, however the adhesion mechanism is not fully understood. While marine mussel adhesion mechanics has been studied at the individual byssal-strand level, freshwater mussel adhesion has only been characterized through whole-mussel detachment, without direct interspecies comparisons on different substrates. Here, adhesive strength of individual quagga and zebra mussel byssal plaques were measured on smooth substrates with varying hydrophobicity-glass, PVC, and PDMS. With increased hydrophobicity of substrates, adhesive failures occurred more frequently, and mussel adhesion strength decreased. A new failure mode termed 'footprint failure' was identified, where failure appeared to be adhesive macroscopically, but a microscopic residue remained on the surface. Zebra mussels adhered stronger and more frequently on PDMS than quagga mussels. While their adhesion strengths were similar on PVC, there were differences in the failure mode and the plaque-substrate interface ultrastructure. Comparisons with previous marine mussel studies demonstrated that freshwater mussels adhere with comparable strength despite known differences in protein composition. An improved understanding of freshwater mussel adhesion mechanics may help explain spreading dynamics and will be important in developing effective antifouling surfaces.

Identification and characterization of aortic valve mesenchymal progenitor cells with robust osteogenic calcification potential.

Advanced valvular lesions often contain ectopic mesenchymal tissues, which may be elaborated by an unidentified multipotent progenitor subpopulation within the valve interstitium. The identity, frequency, and differentiation potential of the putative progenitor subpopulation are unknown. The objectives of this study were to determine whether valve interstitial cells (VICs) contain a subpopulation of multipotent mesenchymal progenitor cells, to measure the frequencies of the mesenchymal progenitors and osteoprogenitors, and to characterize the osteoprogenitor subpopulation because of its potential role in calcific aortic valve disease. The multilineage potential of freshly isolated and subcultured porcine aortic VICs was tested in vitro. Progenitor frequencies and self-renewal capacity were determined by limiting dilution and colony-forming unit assays. VICs were inducible to osteogenic, adipogenic, chondrogenic, and myofibrogenic lineages. Osteogenic differentiation was also observed in situ in sclerotic porcine leaflets. Primary VICs had strikingly high frequencies of mesenchymal progenitors (48.0 +/- 5.7%) and osteoprogenitors (44.1 +/- 12.0%). High frequencies were maintained for up to six population doublings, but decreased after nine population doublings to 28.2 +/- 9.9% and 5.8 +/- 1.3%, for mesenchymal progenitors and osteoprogenitors, respectively. We further identified the putative osteoprogenitor subpopulation as morphologically distinct cells that occur at high frequency, self-renew, and elaborate bone matrix from single cells. These findings demonstrate that the aortic valve is rich in a mesenchyma l progenitor cell population that has strong potential to contribute to valve calcification.

The byssus of the zebra mussel (Dreissena polymorpha): spatial variations in protein composition.

The notorious biofouling organism Dreissena polymorpha (the zebra mussel) attaches to a variety of surfaces using a byssus, a series of protein threads that connect the animal to adhesive plaques secreted onto hard substrata. Here, the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize the composition of different regions of the byssus is reported. All parts of the byssus show mass peaks corresponding to small proteins in the range of 3.7-7 kDa, with distinctive differences between different regions. Indeed, spectra from thread and plaques are almost completely non-overlapping. In addition, several peaks were identified that are unique to the interfacial region of the plaque, and therefore likely represent specialized adhesive proteins. These results indicate a high level of control over the distribution of proteins, presumably with different functions, in the byssus of this freshwater species.

Fingerprinting of Proteins that Mediate Quagga Mussel Adhesion using a De Novo Assembled Foot Transcriptome.

The European freshwater mollusk Dreissena bugensis (quagga mussel), an invasive species to North America, adheres to surfaces underwater via the byssus: a non-living protein 'anchor'. In spite of its importance as a biofouling species, the sequence of the majority of byssal proteins responsible for adhesion are not known, and little genomic data is available. To determine protein sequence information, we utilized next-generation RNA sequencing and de novo assembly to construct a cDNA library of the quagga mussel foot transcriptome, which contains over 200,000 transcripts. Quagga mussel byssal proteins were extracted from freshly induced secretions and analyzed using LC-MS/MS; peptide spectra were matched to the transcriptome to fingerprint the entire protein primary sequences. We present the full sequences of fourteen novel quagga mussel byssal proteins, named Dreissena bugensis foot proteins 4 to 17 (Dbfp4-Dbfp17), and new sequence data for two previously observed byssal proteins Dbfp1 and Dbfp2. Theoretical masses of the newly discovered proteins range from 4.3 kDa to 21.6 kDa. These protein sequences are unique but contain features similar to glue proteins from other species, including a high degree of polymorphism, proteins with repeated peptide motifs, disordered protein structure, and block structures.

Oil-Infused Silicone Prevents Zebra Mussel Adhesion.

The remarkable underwater adhesion capacity of the invasive freshwater mussel species Dreissena polymorpha (zebra mussel) causes extensive damage each year. The adhesive interface between the substrate surface and the mussels' adhesive plaques plays a key role in zebra mussel biofouling. Silicone-oil-infused polydimethylsiloxane (iPDMS), an omniphobic material in the class of liquid-infused slippery surfaces, has been shown to develop a uniform, microscale, antifouling surface oil layer, which we hypothesized would be effective against zebra mussel fouling. iPDMS substrates with varying levels of oil saturation were tested for their ability to disrupt mussel adhesion by characterizing zebra mussel reattachment in a simulated freshwater environment for 3 days. On fully saturated iPDMS samples or those near full saturation, zebra mussels showed no reattachment, compared to 41% reattachment on PDMS controls (no oil infusion). For lower saturation levels, the frequency of reattachment was decreased relative to PDMS controls. Mussel detachment forces decreased in iPDMS as compared to PDMS, and adhesive failures occurred more frequently with higher iPDMS saturations. Surface analysis of the subsaturated iPDMS substrates showed incomplete coverage of the surface oil layer. After 3 days of immersion in artificial freshwater, subsaturated iPDMS substrates showed a decrease in slipperiness (measured by water slide angle), whereas in fully saturated iPDMS, the slipperiness was unchanged, despite no observed oil loss in either group. The decrease in slipperiness is attributed to microfouling of the subsaturated substrates, consistent with incomplete surface oil layer coverage, and supports the notion that full oil layer coverage is required for effective antifouling properties. Employing iPDMS as an antifouling coating shows promise against freshwater mussel adhesion, and this work further aids in understanding the antifouling mechanism of iPDMS and the role of the plaque-substrate interface in freshwater mussel adhesion.