Revolutionizing Dental Implant Research: A Systematic Review on Three-Dimensional In Vitro Models.

Dental implants have been clinically used for almost five decades with high success rates. In vitro research models used in implant dentistry are limited to two-dimensional experiments, which are reproducible and well adapted to evaluate a single parameter but do not reproduce the complexity of clinical settings. On the contrary, the in vivo research models using animals offer similar histological and anatomical features to humans, and tissue healing can be close to a clinical situation, but those models are usually accompanied with ethical concerns, and their outcomes could not be extrapolated to humans because of interspecies variabilities. This makes the development of novel in vitro models that recapitulate physiological events occurring during dental implant placement of particular interest for current research in dentistry. Also, such models could be challenged by setting a pathological environment (peri-implantitis) to better understand the disease and eventually serve as a platform to evaluate novel treatment modalities. The aim of this systematic literature review was to cover all the in vitro three-dimensional (3D) complex models available for research in implant dentistry. To accomplish this, a comprehensive search of the literature present on Scopus and PubMed databases was done using specific keywords, as well as inclusion/exclusion criteria. Out of 1334 articles found, we have finally included 27 articles in this review with publication dates between 2001 and 2022. In those articles, the 3D models were designed to study tissue-implant interface behavior in bone or gingival tissue. The articles focused on simulating implant integration, evaluating the effect of different conditions on implant integration, or developing an infection model for the implant integration process. The methods used involved implant material and cells organized in a specific 3D structure. The 3D models developed were able to simulate the process of dental implant osseo- and soft tissue integration and lead to results comparable with conventional in vitro and in vivo models. A relatively limited number of articles were obtained, which indicates that this is an emerging field, highly dependent on progresses made in biotechnologies and tissue engineering, and that further investigation is needed to enhance these 3D in vitro models.

[Uses of 3D printing and Bioprinting for pre-implant bone reconstruction in Oral Surgery]. / Impression 3D et bioimpression pour la régénération osseuse en chirurgie orale.

Pre-implant bone surgery in oral surgery allows to reconstruct maxillary atrophies related to traumatic, infectious or tumoral processes. In this context, the ideal biomaterial remains autogenous bone, but biomaterials (of natural or synthetic origin) allow to limit the morbidity linked to bone harvesting, and to simplify these surgical procedures. In this article, we illustrate how 3D printing technologies can be used as an adjuvant to treat bone defects of complex shape or to create anatomical models used to plan interventions. Finally, some perspectives brought by tissue engineering and bioprinting (creation of complex in vitro models) are presented.

Title: Impression 3D et bioimpression pour la régénération osseuse en chirurgie orale. Abstract: La chirurgie osseuse pré-implantaire en chirurgie orale permet de reconstruire les atrophies des maxillaires en rapport avec des processus traumatiques, infectieux ou tumoraux. Dans ce contexte, le biomatériau idéal reste l'os autogène mais les biomatériaux (d'origine naturelle ou synthétique) permettent de limiter la morbidité liée aux prélèvements osseux et de simplifier ces interventions chirurgicales. Dans cet article, nous illustrons l'apport récent de l'impression 3D dans ce contexte pour traiter des défauts osseux de forme complexe ou pour créer des modèles anatomiques servant à planifier les interventions. Enfin, les perspectives apportées par l'ingénierie tissulaire et la bioimpression (création de modèles in vitro complexes) sont détaillées.

Development of Novel Polysaccharide Membranes for Guided Bone Regeneration: In Vitro and In Vivo Evaluations.

INTRODUCTION: Guided bone regeneration (GBR) procedures require selecting suitable membranes for oral surgery. Pullulan and/or dextran-based polysaccharide materials have shown encouraging results in bone regeneration as bone substitutes but have not been used to produce barrier membranes. The present study aimed to develop and characterize pullulan/dextran-derived membranes for GBR. MATERIALS AND METHODS: Two pullulan/dextran-based membranes, containing or not hydroxyapatite (HA) particles, were developed. In vitro, cytotoxicity evaluation was performed using human bone marrow mesenchymal stem cells (hBMSCs). Biocompatibility was assessed on rats in a subcutaneous model for up to 16 weeks. In vivo, rat femoral defects were created on 36 rats to compare the two pullulan/dextran-based membranes with a commercial collagen membrane (Bio-Gide®). Bone repair was assessed radiologically and histologically. RESULTS: Both polysaccharide membranes demonstrated cytocompatibility and biocompatibility. Micro-computed tomography (micro-CT) analyses at two weeks revealed that the HA-containing membrane promoted a significant increase in bone formation compared to Bio-Gide®. At one month, similar effects were observed among the three membranes in terms of bone regeneration. CONCLUSION: The developed pullulan/dextran-based membranes evidenced biocompatibility without interfering with bone regeneration and maturation. The HA-containing membrane, which facilitated early bone regeneration and offered adequate mechanical support, showed promising potential for GBR procedures.

Downregulation of Type 3 Deiodinase in the Hypothalamus During Inflammation.

Background: Inflammation is associated with marked changes in cellular thyroid hormone (TH) metabolism in triiodothyronine (T3) target organs. In the hypothalamus, type 2 deiodinase (D2), the main T3 producing enzyme, increases upon inflammation, leading to an increase in local T3 availability, which in turn decreases thyrotropin releasing hormone expression in the paraventricular nucleus. Type 3 deiodinase (D3), the T3 inactivating enzyme, decreases during inflammation, which might also contribute to the increased T3 availability in the hypothalamus. While it is known that D2 is regulated by nuclear factor κB (NF-κB) during inflammation, the underlying mechanisms of D3 regulation are unknown. Therefore, the aim of the present study was to investigate inflammation-induced D3 regulation using in vivo and in vitro models. Methods: Mice were injected with a sublethal dose of bacterial endotoxin (lipopolysaccharide [LPS]) to induce a systemic acute-phase response. A human neuroblastoma (SK-N-AS) cell line was used to test the involvement of the thyroid hormone receptor alpha 1 (TRα1) as well as the activator protein-1 (AP-1) and NF-κB inflammatory pathways in the inflammation-induced decrease of D3. Results: D3 expression in the hypothalamus was decreased 24 hours after LPS injection in mice. This decrease was similar in mice lacking the TRα. Incubation of SK-N-AS cells with LPS robustly decreased both D3 mRNA expression and activity. This led to increased intracellular T3 concentrations. The D3 decrease was prevented when NF-κB or AP-1 was inhibited. TRα1 mRNA expression decreased in SK-N-AS cells incubated with LPS, but knockdown of the TRα in SK-N-AS cells did not prevent the LPS-induced D3 decrease. Conclusions: We conclude that the inflammation-induced D3 decrease in the hypothalamus is mediated by the inflammatory pathways NF-κB and AP-1, but not TRα1. Furthermore, the observed decrease modulates intracellular T3 concentrations. Our results suggest a concerted action of inflammatory modulators to regulate both hypothalamic D2 and D3 activities to increase the local TH concentrations.

Human amniotic membrane for guided bone regeneration of calvarial defects in mice.

Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.

A new composite hydrogel combining the biological properties of collagen with the mechanical properties of a supramolecular scaffold for bone tissue engineering.

Tissue engineering is a promising alternative to autografts, allografts, or biomaterials to address the treatment of severe and large bone lesions. Classically, tissue engineering products associate a scaffold and cells and are implanted or injected into the lesion. These cells must be embedded in an appropriate biocompatible scaffold, which offers a favourable environment for their survival and differentiation. Here, we designed a composite hydrogel composed of collagen I, an extracellular matrix protein widely used in several therapeutic applications, which we associated with a physical hydrogel generated from a synthetic small amphiphilic molecule. This composite showed improved mechanical and biological characteristics as compared with gels obtained from each separate compound. Incorporation of the physical hydrogel prevented shrinkage of collagen and cell diffusion out of the gel and yielded a gel with a higher elastic modulus than those of gels obtained with each component alone. The composite hydrogel allowed cell adhesion and proliferation in vitro and long-term cell survival in vivo. Moreover, it promoted the differentiation of human adipose-derived stem cells in the absence of any osteogenic factors. In vivo, cells embedded in the composite gel and injected subcutaneously in immunodeficient mice produced lamellar osteoid tissue and differentiated into osteoblasts. This study points this new composite hydrogel as a promising scaffold for bone tissue engineering applications.

Prolonged delivery of BMP-2 by a non-polymer hydrogel for bone defect regeneration.

Bone morphogenetic protein 2 (BMP-2) is a potent inducer of bone formation that is currently used in a limited number of clinical indications to treat extensive bone loss. Extending the field of applications of this molecule requires design of the delivery system to protect the protein from early degradation and allow a slow long-term release. This study describes the use of a non-polymer hydrogel, based on the self-assembly of small amphiphilic glycosyl-nucleolipids into micellar structures, as a new type of delivery system for BMP-2. BMP-2 was readily encapsulated in glycosyl-nucleosyl-fluorinated (GNF)-based gels and slowly released in vitro, while maintaining its osteogenic activity. When hydrogel pieces containing fluorophore-tagged BMP-2 were deposited onto a calvaria defect in mouse, the signal detected in living mice gradually decreased and was still detectable at 3 weeks. Gel-embedded protein promoted significant calvarial bone defect regeneration at 8 weeks after surgery. In contrast, when a solution of BMP-2 without hydrogel was injected into the defects, the fluorescence signal decreased rapidly and no significant bone formation was observed. The unique property of the GNF-based hydrogel as an injectable delivery system for low doses of BMP-2 was revealed in a subcutaneous model of ectopic bone formation. Injected BMP-2-laden GNF hydrogel blocks elicited the formation of cancellous bone, showing all the typical features of remodeling bone that contains bone marrow. These results show that this GNF-based hydrogel is an easy-to-use, efficient delivery system for BMP-2 and osteogenic material to support bone regeneration.

Low molecular weight hydrogels derived from urea based-bolaamphiphiles as new injectable biomaterials.

There is a critical need for soft materials in the field of regenerative medicine and tissue engineering. However, designing injectable hydrogel scaffolds encompassing both adequate mechanical and biological properties remains a key challenge for in vivo applications. Here we use a bottom-up approach for synthesizing supramolecular gels to generate novel biomaterial candidates. We evaluated the low molecular weight gels candidates in vivo and identified one urea-containing molecule, compound 16, that avoid foreign body reactions in mice. The self-assembly of bolaamphiphiles creates a unique hydrogel supramolecular structures featuring fast gelation kinetics, high elastic moduli, thixotropic, and thermal reversibility properties. This soft material, which inhibits recognition by macrophages and fibrous deposition, exhibits long-term stability after in vivo injection.

Cellularizing hydrogel-based scaffolds to repair bone tissue: How to create a physiologically relevant micro-environment?

Tissue engineering is a promising alternative to autografts or allografts for the regeneration of large bone defects. Cell-free biomaterials with different degrees of sophistication can be used for several therapeutic indications, to stimulate bone repair by the host tissue. However, when osteoprogenitors are not available in the damaged tissue, exogenous cells with an osteoblast differentiation potential must be provided. These cells should have the capacity to colonize the defect and to participate in the building of new bone tissue. To achieve this goal, cells must survive, remain in the defect site, eventually proliferate, and differentiate into mature osteoblasts. A critical issue for these engrafted cells is to be fed by oxygen and nutrients: the transient absence of a vascular network upon implantation is a major challenge for cells to survive in the site of implantation, and different strategies can be followed to promote cell survival under poor oxygen and nutrient supply and to promote rapid vascularization of the defect area. These strategies involve the use of scaffolds designed to create the appropriate micro-environment for cells to survive, proliferate, and differentiate in vitro and in vivo. Hydrogels are an eclectic class of materials that can be easily cellularized and provide effective, minimally invasive approaches to fill bone defects and favor bone tissue regeneration. Furthermore, by playing on their composition and processing, it is possible to obtain biocompatible systems with adequate chemical, biological, and mechanical properties. However, only a good combination of scaffold and cells, possibly with the aid of incorporated growth factors, can lead to successful results in bone regeneration. This review presents the strategies used to design cellularized hydrogel-based systems for bone regeneration, identifying the key parameters of the many different micro-environments created within hydrogels.

Cation Tuning of Supramolecular Gel Properties: A New Paradigm for Sustained Drug Delivery.

Hydrogels formed by the self-assembly of low-molecular-weight gelators (LMWGs) are promising scaffolds for drug-delivery applications. A new biocompatible hydrogel, resulting from the self-assembly of nucleotide-lipid salts can be safely injected in vivo. The resulting hydrogel provides sustained-release of protein for more than a week.

Patterning of Endothelial Cells and Mesenchymal Stem Cells by Laser-Assisted Bioprinting to Study Cell Migration.

Tissue engineering of large organs is currently limited by the lack of potent vascularization in vitro. Tissue-engineered bone grafts can be prevascularized in vitro using endothelial cells (ECs). The microvascular network architecture could be controlled by printing ECs following a specific pattern. Using laser-assisted bioprinting, we investigated the effect of distance between printed cell islets and the influence of coprinted mesenchymal cells on migration. When printed alone, ECs spread out evenly on the collagen hydrogel, regardless of the distance between cell islets. However, when printed in coculture with mesenchymal cells by laser-assisted bioprinting, they remained in the printed area. Therefore, the presence of mesenchymal cell is mandatory in order to create a pattern that will be conserved over time. This work describes an interesting approach to study cell migration that could be reproduced to study the effect of trophic factors.

Control of stem-cell behavior by fine tuning the supramolecular assemblies of low-molecular-weight gelators.

Controlling the behavior of stem cells through the supramolecular architecture of the extracellular matrix remains an important challenge in the culture of stem cells. Herein, we report on a new generation of low-molecular-weight gelators (LMWG) for the culture of isolated stem cells. The bola-amphiphile structures derived from nucleolipids feature unique rheological and biological properties suitable for tissue engineering applications. The bola-amphiphile-based hydrogel scaffold exhibits the following essential properties: it is nontoxic, easy to handle, injectable, and features a biocompatible rheology. The reported glycosyl-nucleoside bola-amphiphiles (GNBA) are the first examples of LMWG that allow the culture of isolated stem cells in a gel matrix. The results (TEM observations and rheology) suggest that the supramolecular organizations of the matrix play a role in the behavior of stem cells in 3D environments.

Chondrocytes play a major role in the stimulation of bone growth by thyroid hormone.

Thyroid hormone (T3) is required for postnatal skeletal growth. It exerts its effect by binding to nuclear receptors, TRs including TRα1 and TRß1, which are present in most cell types. These cell types include chondrocytes and osteoblasts, the interactions of which are known to regulate endochondral bone formation. In order to analyze the respective functions of T3 stimulation in chondrocytes and osteoblasts during postnatal growth, we use Cre/loxP recombination to express a dominant-negative TRα1(L400R) mutant receptor in a cell-specific manner. Phenotype analysis revealed that inhibiting T3 response in chondrocytes is sufficient to reproduce the defects observed in hypothyroid mice, not only for cartilage maturation, but also for ossification and mineralization. TRα1(L400R) in chondrocytes also results in skull deformation. In the meantime, TRα1(L400R) expression in mature osteoblasts has no visible effect. Transcriptome analysis identifies a number of changes in gene expression induced by TRα1(L400R) in cartilage. These changes suggest that T3 normally cross talks with several other signaling pathways to promote chondrocytes proliferation, differentiation, and skeletal growth.

Repression of osteoblast maturation by ERRα accounts for bone loss induced by estrogen deficiency.

ERRα is an orphan member of the nuclear receptor family, the complete inactivation of which confers resistance to bone loss induced by ageing and estrogen withdrawal to female mice in correlation with increased bone formation in vivo. Furthermore ERRα negatively regulates the commitment of mesenchymal cells to the osteoblast lineage ex vivo as well as later steps of osteoblast maturation. We searched to determine whether the activities of ERRα on osteoblast maturation are responsible for one or both types of in vivo induced bone loss. To this end we have generated conditional knock out mice in which the receptor is normally present during early osteoblast differentiation but inactivated upon osteoblast maturation. Bone ageing in these animals was similar to that observed for control animals. In contrast conditional ERRαKO mice were completely resistant to bone loss induced by ovariectomy. We conclude that the late (maturation), but not early (commitment), negative effects of ERRα on the osteoblast lineage contribute to the reduced bone mineral density observed upon estrogen deficiency.

A thermosensitive low molecular weight hydrogel as scaffold for tissue engineering.

Hydrogels that are non-toxic, easy to use, cytocompatible, injectable and degradable are valuable biomaterials for tissue engineering and tissue repair. However, few compounds currently fulfil these requirements. In this study, we describe the biological properties of a new type of thermosensitive hydrogel based on low-molecular weight glycosyl-nucleosyl-fluorinated (GNF) compound. This gel forms within 25 min by self-assembly of monomers as temperature decreases. It degrades slowly in vitro and in vivo. It induces moderate chronic inflammation and is progressively invaded by host cells and vessels, suggesting good integration to the host environment. Although human adult mesenchymal stem cells derived from adipose tissue (ASC) cannot adhere on the gel surface or within a 3D gel scaffold, cell aggregates grow and differentiate normally when entrapped in the GNF-based gel. Moreover, this hydrogel stimulates osteoblast differentiation of ASC in the absence of osteogenic factors. When implanted in mice, gel-entrapped cell aggregates survive for several weeks in contrast with gel-free spheroids. They are maintained in their original site of implantation where they interact with the host tissue and adhere on the extracellular matrix. They can differentiate in situ into alkaline phosphatase positive osteoblasts, which deposit a calcium phosphate-rich matrix. When injected into subcutaneous sites, gel-encapsulated cells show similar biological properties as implanted gel-cells complexes. These data point GNF-based gels as a novel class of hydrogels with original properties, in particular osteogenic potential, susceptible of providing new therapeutic solutions especially for bone tissue engineering applications.

Layer-by-layer tissue microfabrication supports cell proliferation in vitro and in vivo.

Layer-by-layer biofabrication represents a novel strategy to create three-dimensional living structures with a controlled internal architecture, using cell micromanipulation technologies. Laser assisted bioprinting (LAB) is an effective printing method for patterning cells, biomolecules, and biomaterials in two dimensions. "Biopapers," made of thin polymer scaffolds, may be appropriate to achieve three-dimensional constructs and to reinforce mechanical properties of printed materials. The aim of this work was to evaluate the effect of the tridimensional organization of cells and biomaterials on cell proliferation in vitro and in vivo. The experimental LAB setup was comprised of an infrared laser, focused onto a glass ribbon coated with an absorbing layer of gold. The cell bioink was made of MG63 cells (50 millions cells/mL in culture medium and 1% alginate), transduced with Luciferase gene for tracking and quantification. The printing substrate was a 100-µm-thick polycaprolacton (PCL) electrospun scaffold. The building sequence comprised sequential layers of cells and PCL scaffolds stacked using two different tridimensional arrangements, which were compared in this study (layer-by-layer vs. seeding on a single locus of the scaffolds). Then the cell-seeded materials were cultured in vitro or implanted in vivo in NOD-SCID mice. The qualitative follow-up involved scanning electron microscopy (SEM) observations, live-dead assays, and histology. The cell amount was quantified by photon imager during 21 days in vitro and 2 months in vivo. Live- dead assay and SEM revealed that the cells survived after printing and spread onto PCL membranes. Circle-shaped patterns were maintained in vitro during the first week but they were no longer observable after 2 weeks, due to cell proliferation. Luciferase tracking displayed that the cell amount was increased in vitro and in vivo when the materials and the cells where stacked layer by layer. Histological sections of the in vivo samples revealed a thicker fibrous tissue in the layer-by-layer samples. We have demonstrated in this study that PCL electrospun biopapers can act as a shock-absorbing mattress for cell printing and could further support cell proliferation. The layer-by-layer printing provided an appropriate 3D environment for cell survival and enhanced cell proliferation in vitro and in vivo.

Glycosylated nucleoside lipid promotes the liposome internalization in stem cells.

We report new glycosyl-nucleoside-lipid based liposomes decorated with sugar moieties. The GNL-liposomes feature a suitable glycosylated surface for their internalization into ADSC stem cells.

Thyroid hormone receptor ß mediates thyroid hormone effects on bone remodeling and bone mass.

Excess thyroid hormone (TH) in adults causes osteoporosis and increases fracture risk. However, the mechanisms by which TH affects bone turnover are not elucidated. In particular, the roles of thyroid hormone receptor (TR) isotypes in the mediation of TH effects on osteoblast-mediated bone formation and osteoclast-mediated bone resorption are not established. In this study we have induced experimental hypothyroidism or hyperthyroidism in adult wild-type, TRα- or TRß-deficient mice and analyzed the effects of TH status on the structure and remodeling parameters of trabecular bone. In wild-type mice, excess TH decreased bone volume and mineralization. High TH concentrations were associated with a high bone-resorption activity, assessed by increased osteoclast surfaces and elevated concentrations of serum bone-resorption markers. Serum markers of bone formation also were higher in TH-treated mice. TRα deficiency did not prevent TH action on bone volume, bone mineralization, bone formation, or bone resorption. In contrast, TRß deficiency blocked all the early effects of excess TH observed in wild-type mice. However, prolonged exposure to low or high TH concentrations of TRß-deficient mice induced mild modifications of bone structure and remodeling parameters. Together our data suggest that TRß receptors mediate the acute effects produced by transient changes of TH concentrations on bone remodeling, whereas TRα receptors mediate long-term effects of chronic alterations of TH metabolism. These data shed new light on the respective roles of TRs in the control of bone metabolism by TH.

Antiproliferative effect on HepaRG cell cultures of new calix[4]arenes. Part II.

Cell cycle progression is dependent on the intracellular iron level and chelators can lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of some new synthetic calix[4]arene podands bearing diamino-tetraesters, diamino-tetraalcohols, diamino-tetraacid and tetraaryloxypentoxy groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670A (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in the human hepatocarcinoma HepaRG cell cultures using cell nuclei counting after staining with the DNA intercalating fluorescence dye, Hoechst 33342. Their cytotoxicity was evaluated by the extracellular LDH activity. Preliminary results indicated that their antiproliferative effect was mainly due to their cytotoxicity. The efficiency of these compounds, being comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that the new substituted calix[4]arenes could open the way to valuable new approaches for medicinal chemistry scaffolding.

Thyroid hormone receptor {alpha} modulates lipopolysaccharide-induced changes in peripheral thyroid hormone metabolism.

Acute inflammation is characterized by low serum T(3) and T(4) levels accompanied by changes in liver type 1 deiodinase (D1), liver D3, muscle D2, and muscle D3 expression. It is unknown at present whether thyroid hormone receptor alpha (TRalpha) plays a role in altered peripheral thyroid hormone metabolism during acute illness in vivo. We induced acute illness in TRalpha-deficient (TRalpha(0/0)) mice by administration of a sublethal dose of LPS. Compared with wild-type, TRalpha(0/0) mice have lower basal serum T(4) and lower liver D1 activity and muscle D3 mRNA expression, whereas liver D3 activity is higher. These changes are gender specific. The inflammatory response to LPS was similar in WT and TRalpha(0/0) mice. The decrease in serum thyroid hormones and liver D1 was attenuated in TRalpha(0/0) mice, whereas the LPS induced fall in liver D3 mRNA was more pronounced in TRalpha(0/0) mice. Muscle D2 mRNA increased similarly in both strains, whereas muscle D3 mRNA decreased less pronounced in TRalpha(0/0) mice. We conclude that alterations in peripheral thyroid hormone metabolism induced by LPS administration are partly regulated via TRalpha.