Luiz E. Bertassoni

Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs

Vascularization remains a critical challenge in tissue engineering. The development of vascular networks within densely populated and metabolically functional tissues facilitate transport of nutrients and removal of waste products, thus preserving cellular viability over a long period of time. Despite tremendous progress in fabricating complex tissue constructs in the past few years, approaches for controlled vascularization within hydrogel based engineered tissue constructs have remained limited. Here, we report a three dimensional (3D) micromolding technique utilizing bioprinted agarose template fibers to fabricate microchannel networks with various architectural features within photocrosslinkable hydrogel constructs. Using the proposed approach, we were able to successfully embed functional and perfusable microchannels inside methacrylated gelatin (GelMA), star poly(ethylene glycol-co-lactide) acrylate (SPELA), poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(ethylene glycol) diacrylate (PEGDA) hydrogels at different concentrations. In particular, GelMA hydrogels were used as a model to demonstrate the functionality of the fabricated vascular networks in improving mass transport, cellular viability and differentiation within the cell-laden tissue constructs. In addition, successful formation of endothelial monolayers within the fabricated channels was confirmed. Overall, our proposed strategy represents an effective technique for vascularization of hydrogel constructs with useful applications in tissue engineering and organs on a chip.

Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels.

Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least eight days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms.

Biomechanical perspective on the remineralization of dentin

The objective of this article is to critically evaluate the methods that are used to assess outcomes of remineralization of dentin. Currently, the most used assessment methods fall either into quantitative analysis of the mineral content of the remineralized structures or dry measurements of their mechanical properties. Properties obtained from the dehydrated organic dentin matrix may not reflect the true mechanical behavior of the remineralized tissue under physiological and hydrated conditions. Here we seek to clarify the biomechanical aspects of remineralization of dentin, pointing out the effects of hydration and dehydration on the mechanical properties of treated tissues. We also emphasize that a more appropriate endpoint to evaluate the effectiveness of remineralization in dentin should be associated with the recovery of the mechanical properties of the hydrated tissue, which is presumed to correlate well with its overall functionality.

The dentin organic matrix – limitations of restorative dentistry hidden on the nanometer scale

The prevention and treatment of dental caries are major challenges occurring in dentistry. The foundations for modern management of this dental disease, estimated to affect 90% of adults in Western countries, rest upon the dependence of ultrafine interactions between synthetic polymeric biomaterials and nanostructured supramolecular assemblies that compose the tooth organic substrate. Research has shown, however, that this interaction imposes less than desirable long-term prospects for current resin-based dental restorations. Here we review progress in the identification of the nanostructural organization of the organic matrix of dentin, the largest component of the tooth structure, and highlight aspects relevant to understating the interaction of restorative biomaterials with the dentin substrate. We offer novel insights into the influence of the hierarchically assembled supramolecular structure of dentin collagen fibrils and their structural dependence on water molecules. Secondly, we review recent evidence for the participation of proteoglycans in composing the dentin organic network. Finally, we discuss the relation of these complexly assembled nanostructures with the protease degradative processes driving the low durability of current resin-based dental restorations. We argue in favour of the structural limitations that these complexly organized and inherently hydrated organic structures may impose on the clinical prospects of current hydrophobic and hydrolyzable dental polymers that establish ultrafine contact with the tooth substrate.

Mechanical Recovery of Dentin Following Remineralization In Vitro – an Indentation Study

This study sought to gain insights into the steps leading to remineralization and mechanical recovery of hydrated dentin. Mechanical recovery in water was hypothesized to result from effective mineral matrix binding and to occur from the innermost regions outwards due to an increase in the number of nucleation sites. Partially demineralized (0.05 M acetate, pH=5.0, 8h) dentin was remineralized using calcium and phosphate solutions of 10.1 or 9.8 degree of saturation (DS) for hydroxyapatite (pH=7.4) for 4, 8 or 24h. Remineralization used a constant solution composition approach, which allowed for a continuous mineral growth with relatively constant thermodynamic driving forces. Crystal growth rates (R) were calculated using concentrations of calcium and phosphate. Before and after de- and re-mineralization, specimens had their surface and cross-section elastic moduli measured using AFM-nanoindentation in water. DS=10.1 provided higher R and higher mechanical recovery at the surface (p<0.0001). Cross-sectional measurements showed that subsurface mechanical recovery occurred from the innermost demineralized areas gradually outwards for both groups with no statistical differences at different DS, thus suggesting that remineralization is driven by mineral growth within nucleation sites with preserved collagen fibrils. Further, mechanical recovery appeared to initially obey a heterogeneous pattern, which vanished with time. This study provides evidence of mechanical recovery of hydrated dentin after remineralization and novel insights into the steps leading to mechanical recovery of carious dentin.

Three-Dimensional Bioprinting for Regenerative Dentistry and Craniofacial Tissue Engineering

Craniofacial tissues are organized with complex 3-dimensional (3D) architectures. Mimicking such 3D complexity and the multicellular interactions naturally occurring in craniofacial structures represents one of the greatest challenges in regenerative dentistry. Three-dimensional bioprinting of tissues and biological structures has been proposed as a promising alternative to address some of these key challenges. It enables precise manufacture of various biomaterials with complex 3D architectures, while being compatible with multiple cell sources and being customizable to patient-specific needs. This review describes different 3D bioprinting methods and summarizes how different classes of biomaterials (polymer hydrogels, ceramics, composites, and cell aggregates) may be used for 3D biomanufacturing of scaffolds, as well as craniofacial tissue analogs. While the fabrication of scaffolds upon which cells attach, migrate, and proliferate is already in use, printing of all the components that form a tissue (living cells and matrix materials together) to produce tissue constructs is still in its early stages. In summary, this review seeks to highlight some of the key advantages of 3D bioprinting technology for the regeneration of craniofacial structures. Additionally, it stimulates progress on the development of strategies that will promote the translation of craniofacial tissue engineering from the laboratory bench to the chair side.

Evaluation of Surface Structural and Mechanical Changes Following Remineralization of Dentin

This study sought to gain insights into the surface structural and mechanical changes leading to remineralization of dentin. Remineralization was compared between a continuous remineralization approach and a nonbuffered static approach using solutions of the same initial composition. Artificial carious lesions were treated for 5 days and analyzed every 24 h using nanoindentation in water, SEM, and AFM. The continuous approach yielded a recovery of mechanical properties of up to 60% of normal dentin, whereas the static approach led to recovery of only 10%. Image analysis revealed that the static approach yielded the formation of areas suggestive of an apatite precipitate on the surface of the dentin matrix. In contrast, surface precipitate was absent using the continuous approach, suggesting that mineral formed within the lesion and re-associated with the collagenous matrix. This study provided evidence that mechanical recovery of dentin in near physiological conditions is attainable through the continuous delivery of calcium and phosphate ions.

Insights into the structure and composition of the peritubular dentin organic matrix and the lamina limitans.

Dentin is a mineralized dental tissue underlying the outer enamel that has a peculiar micro morphology. It is composed of micrometer sized tubules that are surrounded by a highly mineralized structure, called peritubular dentin (PTD), and embedded in a collagen-rich matrix, named intertubular dentin. The PTD has been thought to be composed of a highly mineralized collagen-free organic matrix with unknown composition. Here we tested the hypothesis that proteoglycans and glycosaminoglycans, two important organic structural features found in dentin, are key participants in the microstructure and composition of the PTD. To test this hypothesis dentin blocks were demineralized with 10 vol% citric acid for 2 min and either digested with 1mg/ml TPCK-treated trypsin with 0.2 ammonium bicarbonate at pH 7.9 (TRY) or 0.1 U/mL C-ABC with 50mM Tris, 60mM sodium acetate and 0.02% bovine serum albumin at pH 8.0 (C-ABC). TRY is known to cleave the protein core of dentin proteoglycans, whereas C-ABC is expected to selectively remove glycosaminoglycans. All specimens were digested for 48 h in 37°C, dehydrated in ascending grades of acetone, immersed in HMDS, platinum coated and imaged using an FE-SEM. Images of demineralized dentin revealed a meshwork of noncollagenous fibrils protruding towards the tubule lumen following removal of the peritubular mineral and confirmed the lack of collagen in the peritubular matrix. Further, images revealed that the peritubular organic network originates from a sheet-like membrane covering the entire visible length of tubule, called lamina limitans. Confirming our initial hypothesis, after the digestion with C-ABC the organic network appeared to vanish, while the lamina limitans was preserved. This suggests that glycosaminoglycans are the main component of the PTD organic network. Following digestion with TRY, both the organic network and the lamina limitans disappeared, thus suggesting that the lamina limitans may be primarily composed of proteoglycan protein cores. In summary, our results provide novel evidence that (1) PTD lacks collagen fibrils, (2) PTD contains an organic scaffold embedded with mineral and (3) the PTD organic matrix is manly composed of glycosaminoglycans, whereas the lamina limitans is primarily made of proteoglycans protein cores.

Nanotechnology in Dental Sciences: Moving towards a Finer Way of Doing Dentistry

Nanotechnologies are predicted to revolutionize: (a) the control over materials properties at ultrafine scales; and (b) the sensitivity of tools and devices applied in various scientific and technological fields. In this short review, we argue that dentistry will be no exception to this trend. Here, we present a dynamic view of dental tissues, an adoption of which may lead to finer, more effective and minimally invasive reparation approaches. By doing so, we aim at providing insights into some of the breakthroughs relevant to understanding the genesis of dental tissues at the nanostructural level or generating dental materials with nanoscale critical boundaries. The lineage of the progress of dental science, including the projected path along the presumed nanotechnological direction of research and clinical application is mentioned too. We conclude by claiming that dentistry should follow the trend of probing matter at nanoscale that currently dominates both materials and biological sciences in order to improve on the research strategies and clinical techniques that have traditionally rested on mechanistic assumptions.

Effect of pre- and postpolymerization on flexural strength and elastic modulus of impregnated, fiber-reinforced denture base acrylic resins

STATEMENT OF PROBLEM Impregnated fibers require light polymerization; however, little information exists about how different protocols might affect the mechanical properties of reinforced denture base materials. PURPOSE The purpose of this study was to compare the effects of pre- or postpolymerization of preimpregnated fibers on the flexural strength and elastic modulus of a reinforced autopolymerized and a heat-polymerized acrylic resin. MATERIAL AND METHODS Seventy-two specimens were divided into 12 treatment groups (n=6), according to type of acrylic resin (autopolymerized or heat polymerized), type of reinforcement, and its pre- or postpolymerization. Impregnated glass fibers (Fibrex-Lab), unimpregnated glass fibers (Fibrante), and ribs made from a restorative composite resin (Z250) were used as reinforcements. The reinforcements were light polymerized either before or after incorporation and processing of the acrylic resins. Specimens were tested in 3-point load and the data were analyzed using 2-way ANOVA and Tukey post hoc test (alpha=.05). Specimens were further examined using light microscopy, atomic force microscopy, and scanning electron microscopy. RESULTS Elastic modulus was significantly higher for heat-polymerized acrylic resins than for autopolymerized acrylic resins (P<.001). Prepolymerized fibers increased both flexural strength and elastic modulus of autopolymerized acrylic resins significantly more than postpolymerized fibers (P<.001); however, postpolymerized fibers yielded a higher elastic modulus than prepolymerized fibers for the heat-polymerized material (P<.001). CONCLUSIONS Prepolymerized fibers improved the overall mechanical properties of reinforced autopolymerized acrylic resins more than postpolymerized fibers. However, postpolymerization of fibers yielded higher elastic modulus for reinforced heat-polymerized acrylics.