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.

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.

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.

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.

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.

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.

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.

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.

The effect of polyaspartate chain length on mediating biomimetic remineralization of collagenous tissues.

Formation of hydroxyapatite (HAP) within collagen fibrils, as found in bone, dentine and cementum, is thought to be mediated by proteins rich in aspartate (Asp) and glutamate such as osteopontin and bone sialoprotein, respectively. Indeed polyaspartate (pAsp), a homopolymer analogue of such proteins, has been shown to induce intrafibrillar mineralization of collagen from solutions of calcium and phosphate that are supersaturated with respect to HAP. To elucidate the role of pAsp in mineralization of collagen, we explored the effect of pAsp chain length on in vitro HAP deposition in demineralized mouse periodontal tissue sections. Through characterization of both tissue sections and mineralizing solution, we show that chain length contributes to the effectiveness of pAsp in mediating intrafibrillar mineralization. This function appears to be associated with inhibition of otherwise kinetically favoured crystallization in the bulk solution, which allows for intrafibrillar crystallization, though this does not preclude the possibility of a more active role for pAsp in addition. Inhibition of crystallization in solution by pAsp occurs by slowing the growth of amorphous calcium phosphate and stabilization of this phase, rather than by sequestration of Ca2+ ions. These results suggest that the length of Asp-rich sequences of mineralizing proteins may be essential to their function, and could also be useful in optimization of mineralized tissue replacement synthesis.

Interplay of Geometric Cues and RGD/BMP-2 Crosstalk in Directing Stem Cell Fate.

Within the native microenvironment, extracellular matrix (ECM) components are thought to display a complex and heterogeneous distribution, spanning several length scales. Herein, the objective is to mimic, in vitro, the hierarchical organization of proteins and growth factors as well as their crosstalk. Photolithography technique was used to adjacently pattern geometrically defined regions of RGD and BMP-2 mimetic peptides onto glass substrates. These ECM-derived ligands are known to jointly regulate mesenchymal stem cells (MSCs) osteogenic differentiation. By manipulating the spatial distribution of dually grafted peptides, the extent of human MSCs osteogenic differentiation was significantly affected, depending on the shape of peptide micropatterns. Our data highlight the existence of a strong interplay between geometric cues and biochemical signals. Such in vitro systems provide a valuable tool to investigate mechanisms by which multiple ECM cues overlap to regulate stem cell fate, thereby contributing to the design of bioinspired biomaterials for bone tissue engineering applications.

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.

Inhibition of Notch Signaling During Mouse Incisor Renewal Leads to Enamel Defects.

The continuously growing rodent incisor is an emerging model for the study of renewal of mineralized tissues by adult stem cells. Although the Bmp, Fgf, Shh, and Wnt pathways have been studied in this organ previously, relatively little is known about the role of Notch signaling during incisor renewal. Notch signaling components are expressed in enamel-forming ameloblasts and the underlying stratum intermedium (SI), which suggested distinct roles in incisor renewal and enamel mineralization. Here, we injected adult mice with inhibitory antibodies against several components of the Notch pathway. This blockade led to defects in the interaction between ameloblasts and the SI cells, which ultimately affected enamel formation. Furthermore, Notch signaling inhibition led to the downregulation of desmosome-specific proteins such as PERP and desmoplakin, consistent with the importance of desmosomes in the integrity of ameloblast-SI attachment and enamel formation. Together, our data demonstrate that Notch signaling is critical for proper enamel formation during incisor renewal, in part by regulating desmosome-specific components, and that the mouse incisor provides a model system to dissect Jag-Notch signaling mechanisms in the context of mineralized tissue renewal.

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.

Structural changes in collagen fibrils across a mineralized interface revealed by cryo-TEM.

The structure of the mineralized collagen fibril, which is the basic building block of mineralized connective tissues, is critical to its function. We use cryo-TEM to study collagen structure at a well-defined hard-soft tissue interface, across which collagen fibrils are continuous, in order to evaluate changes to collagen upon mineralization. To establish a basis for the analysis of collagen banding, we compared cryo-TEM images of rat-tail tendon collagen to a model based on the X-ray structure. While there is close correspondence of periodicity, differences in band intensity indicate fibril regions with high density but lacking order, providing new insight into collagen fibrillar structure. Across a mineralized interface, we show that mineralization results in an axial contraction of the fibril, concomitant with lateral expansion, and that this contraction occurs only in the more flexible gap region of the fibril. Nevertheless, the major features of the banding pattern are not significantly changed, indicating that the axial arrangement of molecules remains largely intact. These results suggest a mechanism by which collagen fibrils are able to accommodate large amounts of mineral without significant disruption of their molecular packing, leading to synergy of mechanical properties.

Novel proteins identified in the insoluble byssal matrix of the freshwater zebra mussel.

The freshwater zebra mussel, Dreissena polymorpha, is an invasive, biofouling species that adheres to a variety of substrates underwater, using a proteinaceous anchor called the byssus. The byssus consists of a number of threads with adhesive plaques at the tips. It contains the unusual amino acid 3, 4-dihydroxyphenylalanine (DOPA), which is believed to play an important role in adhesion, in addition to providing structural integrity to the byssus through cross-linking. Extensive DOPA cross-linking, however, renders the zebra mussel byssus highly resistant to protein extraction, and therefore limits byssal protein identification. We report here on the identification of seven novel byssal proteins in the insoluble byssal matrix following protein extraction from induced, freshly secreted byssal threads with minimal cross-linking. These proteins were identified by LC-MS/MS analysis of tryptic digests of the matrix proteins by spectrum matching against a zebra mussel cDNA library of genes unique to the mussel foot, the organ that secretes the byssus. All seven proteins were present in both the plaque and thread. Comparisons of the protein sequences revealed common features of zebra mussel byssal proteins, and several recurring sequence motifs. Although their sequences are unique, many of the proteins display similarities to marine mussel byssal proteins, as well as to adhesive and structural proteins from other species. The large expansion of the byssal proteome reported here represents an important step towards understanding zebra mussel adhesion.