A Catalytic Approach for the Synthesis of Peptide-Oligonucleotides Conjugates in Aqueous Solution or On-Column.

Peptide-oligonucleotide conjugates (POCs) are covalent architectures composed of a DNA or RNA molecules linked to a peptide. These constructs have found widespread applications ranging from hybrid nanomaterials to gene-targeted therapies. Considering the important role of POCs, a new catalytic approach for their preparation is reported here, that could be applied either on solid support in anhydrous media, or post-synthetically in aqueous buffer. Single amino acids, peptides and cell penetrating peptides (CPPs) were conjugated to various oligo(ribo)nucleotides with high conversions and good isolated yields. The applicability of the method was demonstrated on more than 35 examples including an analogue of a commercial therapeutic oligonucleotide. Other conjugation partners, such as deoxycholic acid and biotin were also successfully conjugated to oligonucleotides. To highlight the potential of this catalytic approach, these conditions have been applied to iterative processes, which is of high interest for the development of DNA-Encoded Libraries.

The Hydrazine Moiety in the Synthesis of Modified Nucleosides and Nucleotides.

Synthetic nucleoside mimics are re-emerging as crucial contenders for antiviral and anticancer medications. While, Ribavirin stands out for its unique antiviral properties, predominantly associated with its distinctive triazole heterocycle as a nucleobase, the exploration of alternative nitrogen-based aromatic heterocycles hold great promises for the discovery of novel bioactive nucleoside mimics. Although nucleoside derivatives synthesized from hydrazine-ribose units have been in development for many decades, they have been little evaluated biologically and even less for their antiviral properties. With the aim of taking a closer look at these under-explored derivatives and investigating their synthetic pathways, this review provides an overview of the molecular design, the chemical synthesis, and the biological activity, when available, of these nucleoside analogues. Overall, the entire body of work already done motivates further exploration of these analogues and encourages us of formulating structurally novel nucleoside drug candidates featuring innovative mode of action.

Boron-containing carbonic anhydrases inhibitors.

Over the last decades, the medicinal chemistry of boron-based compounds has been extensively explored, designing valuable small molecule drugs to tackle diseases and conditions, such as cancer, infections, inflammatory and neurological disorders. Notably, boron has proven to also be a valuable element for the development of inhibitors of the metalloenzymes carbonic anhydrases (CAs), a class of drug targets with significant potential in medicinal chemistry. Incorporating boron into carbonic anhydrase inhibitors (CAIs) can modulate the ligand ability to recognize the target and/or influence selectivity towards different CA isoforms, using the tail approach and boron-based tails. The electron-deficient nature of boron and its associated properties have also led to the discovery of novel zinc-binding CAIs, such as boronic acids and the benzoxaboroles, capable of inhibiting the CAs upon a Lewis acid-base mechanism of action. The present manuscript reviews the state-of-the-art of boron-based CAIs. As research in the applications of boron compounds in medicinal chemistry continues, it is anticipated that new boron-based CAIs will soon expand the current array of such compounds. However, further research is imperative to fully unlock the potential of boron-based CAIs and to advance them towards clinical applications.

6-Substituted Triazolyl Benzoxaboroles as Selective Carbonic Anhydrase Inhibitors: In Silico Design, Synthesis, and X-ray Crystallography.

Benzoxaborole is currently a scaffold of great relevance in medicinal chemistry. In 2016, it was reported to be a new and valuable chemotype for designing carbonic anhydrase (CA) inhibitors. Herein, using an in silico design, we report the synthesis and characterization of substituted 6-(1H-1,2,3-triazol-1-yl)benzoxaboroles. 6-Azidobenzoxaborole was described for the first time as a molecular platform to prepare libraries of inhibitors by a copper(I)-catalyzed azide-alkyne cycloaddition via a click chemistry strategy. With inhibition constants below 30 nM, some derivatives, such as compound 20, showed efficacy as selective hCA VII and IX inhibitors. The design hypothesis was validated by crystallographic investigation on the hCA II/20 adduct, which provided explanations over the different inhibition behavior observed against the five evaluated hCA isoforms. Overall, this study identified 20 as a new promising lead compound to develop novel anticancer agents targeting the tumor-associated hCA IX but also potent neuropathic pain relievers targeting hCA VII.

Facile access to 4'-(N-acylsulfonamide) modified nucleosides and evaluation of their inhibitory activity against SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

N-Acylsulfonamides possess an additional carbonyl function compared to their sulfonamide analogues. Due to their unique physico-chemical properties, interest in molecules containing the N-acylsulfonamide moiety and especially nucleoside derivatives is growing in the field of medicinal chemistry. The recent renewal of interest in antiviral drugs derived from nucleosides containing a sulfonamide function has led us to evaluate the therapeutic potential of N-acylsulfonamide analogues. While these compounds are usually obtained by a difficult acylation of sulfonamides, we report here the easy and efficient synthesis of 20 4'-(N-acylsulfonamide) adenosine derivatives via the sulfo-click reaction. The target compounds were obtained from thioacid and sulfonyl azide synthons in excellent yields and were evaluated as potential inhibitors of the SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

Activation of NRF2 and ATF4 Signaling by the Pro-Glutathione Molecule I-152, a Co-Drug of N-Acetyl-Cysteine and Cysteamine.

I-152 combines two pro-glutathione (GSH) molecules, namely N-acetyl-cysteine (NAC) and cysteamine (MEA), to improve their potency. The co-drug efficiently increases/replenishes GSH levels in vitro and in vivo; little is known about its mechanism of action. Here we demonstrate that I-152 not only supplies GSH precursors, but also activates the antioxidant kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (KEAP1/NRF2) pathway. The mechanism involves disulfide bond formation between KEAP1 cysteine residues, NRF2 stabilization and enhanced expression of the γ-glutamil cysteine ligase regulatory subunit. Accordingly, a significant increase in GSH levels, not reproduced by treatment with NAC or MEA alone, was found. Compared to its parent compounds, I-152 delivered NAC more efficiently within cells and displayed increased reactivity to KEAP1 compared to MEA. While at all the concentrations tested, I-152 activated the NRF2 pathway; high doses caused co-activation of activating transcription factor 4 (ATF4) and ATF4-dependent gene expression through a mechanism involving Atf4 transcriptional activation rather than preferential mRNA translation. In this case, GSH levels tended to decrease over time, and a reduction in cell proliferation/survival was observed, highlighting that there is a concentration threshold which determines the transition from advantageous to adverse effects. This body of evidence provides a molecular framework for the pro-GSH activity and dose-dependent effects of I-152 and shows how synergism and cross reactivity between different thiol species could be exploited to develop more potent drugs.

Redox homeostasis as a target for new antimycobacterial agents.

Despite early treatment with antimycobacterial combination therapy, drug resistance continues to emerge. Maintenance of redox homeostasis is essential for Mycobacterium avium (M. avium) survival and growth. The aim of the present study was to investigate the antimycobacterial activity of two pro-glutathione (pro-GSH) drugs that are able to induce redox stress in M. avium and to modulate cytokine production by macrophages. Hence, we investigated two molecules shown to possess antiviral and immunomodulatory properties: C4-GSH, an N-butanoyl GSH derivative; and I-152, a prodrug of N-acetyl-cysteine (NAC) and ß-mercaptoethylamine (MEA). Both molecules showed activity against replicating M. avium, both in the cell-free model and inside macrophages. Moreover, they were even more effective in reducing the viability of bacteria that had been kept in water for 7 days, proving to be active both against replicating and non-replicating bacteria. By regulating the macrophage redox state, I-152 modulated cytokine production. In particular, higher levels of interferon-gamma (IFN-γ), interleukin 1 beta (IL-1ß), IL-18 and IL-12, which are known to be crucial for the control of intracellular pathogens, were found after I-152 treatment. Our results show that C4-GSH and I-152, by inducing perturbation of redox equilibrium, exert bacteriostatic and bactericidal activity against M. avium. Moreover, I-152 can boost the host response by inducing the production of cytokines that serve as key regulators of the Th1 response.

I-152, a supplier of N-acetyl-cysteine and cysteamine, inhibits immunoglobulin secretion and plasma cell maturation in LP-BM5 murine leukemia retrovirus-infected mice by affecting the unfolded protein response.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 µmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

Splitting aptamers and nucleic acid enzymes for the development of advanced biosensors.

In analogy to split-protein systems, which rely on the appropriate fragmentation of protein domains, split aptamers made of two or more short nucleic acid strands have emerged as novel tools in biosensor set-ups. The concept relies on dissecting an aptamer into a series of two or more independent fragments, able to assemble in the presence of a specific target. The stability of the assembled structure can further be enhanced by functionalities that upon folding would lead to covalent end-joining of the fragments. To date, only a few aptamers have been split successfully, and application of split aptamers in biosensing approaches remains as promising as it is challenging. Further improving the stability of split aptamer target complexes and with that the sensitivity as well as efficient working modes are important tasks. Here we review functional nucleic acid assemblies that are derived from aptamers and ribozymes/DNAzymes. We focus on the thrombin, the adenosine/ATP and the cocaine split aptamers as the three most studied DNA split systems and on split DNAzyme assemblies. Furthermore, we extend the subject into split light up RNA aptamers used as mimics of the green fluorescent protein (GFP), and split ribozymes.

Bis-benzoxaboroles: Design, Synthesis, and Biological Evaluation as Carbonic Anhydrase Inhibitors.

The synthesis, characterization, and biological evaluation of a series of compounds incorporating two or three benzoxaborole moieties is reported. Three different synthetic strategies were used to explore within this series as much chemical space as possible, all starting from the 6-aminobenzoxaborole reagent: amide coupling, imine bond formation, and squarate coupling. Eleven new compounds were isolated in pure form, and single crystals were obtained for two of them. These compounds were then evaluated as carbonic anhydrase inhibitors against the cytosolic hCA I and II and the transmembrane hCA IV, IX, and XII isoforms. While the benzoxaborole scaffold has been recently introduced as a new chemotype for carbonic anhydrase inhibition, these new multivalent derivatives exhibited superior inhibitory activity against the tumor-associated isoform hCA IX. In particular, compared to monovalent 6-aminobenzoxaborole (K I = 813 nM) and 6-carboxybenzoxaborole (K I = 400 nM), derivative 2h characterized by a glutamic acid structural core and two benzoxaborole moieties was found to be more potent (K I = 64 nM) and more selective over human hCA II.

Boosting GSH Using the Co-Drug Approach: I-152, a Conjugate of N-acetyl-cysteine and ß-mercaptoethylamine.

Glutathione (GSH) has poor pharmacokinetic properties; thus, several derivatives and biosynthetic precursors have been proposed as GSH-boosting drugs. I-152 is a conjugate of N-acetyl-cysteine (NAC) and S-acetyl-ß-mercaptoethylamine (SMEA) designed to release the parent drugs (i.e., NAC and ß-mercaptoethylamine or cysteamine, MEA). NAC is a precursor of L-cysteine, while MEA is an aminothiol able to increase GSH content; thus, I-152 represents the very first attempt to combine two pro-GSH molecules. In this review, the in-vitro and in-vivo metabolism, pro-GSH activity and antiviral and immunomodulatory properties of I-152 are discussed. Under physiological GSH conditions, low I-152 doses increase cellular GSH content; by contrast, high doses cause GSH depletion but yield a high content of NAC, MEA and I-152, which can be used to resynthesize GSH. Preliminary in-vivo studies suggest that the molecule reaches mouse organs, including the brain, where its metabolites, NAC and MEA, are detected. In cell cultures, I-152 replenishes experimentally depleted GSH levels. Moreover, administration of I-152 to C57BL/6 mice infected with the retroviral complex LP-BM5 is effective in contrasting virus-induced GSH depletion, exerting at the same time antiviral and immunomodulatory functions. I-152 acts as a pro-GSH agent; however, GSH derivatives and NAC cannot completely replicate its effects. The co-delivery of different thiol species may lead to unpredictable outcomes, which warrant further investigation.

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics.

UNLABELLED: Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152), an N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice. IMPORTANCE: The first report of an association between Th2 polarization and alteration of the redox state in LP-BM5 infection is presented. Moreover, it provides evidence that LP-BM5 infection causes a decrease in the thiol content of peritoneal macrophages, which can influence IL-12 production. The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection, as it reestablishes the Th1/Th2 balance. Immunotherapy based on the use of pro-GSH molecules would permit LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and a Th1/Th2 imbalance to be more effectively combated.

Synthesis of new lipoic acid conjugates and evaluation of their free radical scavenging and neuroprotective activities.

A series of new lipoic acid derivatives were designed and synthesized as multitarget ligands against Alzheimer's disease. In particular, analogues combining both lipoic acid and cysteine core structures were synthesized. The antioxidant properties of these compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS(•+) ) radical cation scavenging assays and ferrous ion chelation. The antioxidant potential of the synthesized compounds was also evaluated in a cellular context and compared to α-lipoic acid and its reduced form, dihydrolipoic acid. The antioxidant effects observed for these compounds in vitro confirmed the importance of free thiol functions for effective antioxidant capacities. However, these promising in vitro results were not mirrored by the antioxidant activity in T67 cell line. This suggests that multiple factors are at stake and warrant further investigations.

Molecules altering the intracellular thiol content modulate NF-kB and STAT-1/IRF-1 signalling pathways and IL-12 p40 and IL-27 p28 production in murine macrophages.

BACKGROUND: The aim of this study was to investigate the molecular mechanisms involved in the production of Th1 cytokines, namely IL-12 and IL-27, when the intra-macrophage redox state was altered by different chemical entities such as GSH-C4, which is reduced glutathione carrying an aliphatic chain, or I-152, a pro-drug of N-acetyl-cysteine (NAC) and beta-mercaptoethylamine. We had already demonstrated that GSH-C4 and I-152 could shift the immune response towards Th1 in Ovalbumin-immunized mice as well as enhance Th1 response in HIV-1 Tat-immunized mice. METHODOLOGY/PRINCIPAL FINDINGS: By a new high performance liquid chromatography method, we found that 20 mM GSH-C4 provided a number of thiol species in the form of GSH, while 20 mM I-152 decreased GSH and increased the thiols in the form of NAC and I-152. Under these experimental conditions, GSH-C4 and I-152 enhanced and suppressed respectively the mRNA expression levels of IL-12 p40 induced by LPS/IFN-γ as assessed by Real-Time PCR. The protein production of IL-12 p40 was increased by GSH-C4 and decreased by I-152 as determined by Enzyme-linked immunosorbent assay. Western immunoblot and electrophoretic mobility shift assays revealed that Nuclear Factor -kB (NF-kB) activation was inhibited by I-152 and prolonged by GSH-C4. Twenty mM I-152 stimulated IL-27 p28 gene expression and sustained Signal Transducer and Activator of Transcription (STAT)-mediated interferon regulator factor 1 (IRF-1) de novo synthesis. By contrast, 20 mM GSH-C4 did not exert any effect on IL-27 p28 gene expression. CONCLUSIONS AND SIGNIFICANCE: an increase in the intra-macrophage redox state by GSH-C4 and I-152 enhances Th1 cytokine production although the chemical structure and the intra-cellular metabolism influence differently signalling pathways involved in IL-27 or IL-12 production. GSH-C4 and I-152 may be used as Th1 immunomodulators in some pathologies and in ageing where GSH depletion may contribute to the Th1/Th2 imbalance, and in new immunization strategies.

Borononucleotides as substrates/binders for human NMP kinases: enzymatic and spectroscopic evaluation.

Borononucleotides are a family of natural nucleotide monophosphate analogues with a 5'-boronic acid function. As B-O-P linkages are known to be unstable in solution, we evaluated the ability of borononucleotides to be recognized by nucleoside monophosphate kinases and eventually foil the phosphorylation process. In this context, and with the idea of probing the influence of their size, shape, and flexibility, a library of borononucleotides were synthetized starting from the borononucleotide analogue of thymidine, which was shown to behave as a slow substrate of human TMP kinase. This study thus constitutes a good starting point for the development of new monophosphate mimics as potential substrates or ligands for NMP kinases.

Straightforward synthesis of triazoloacyclonucleotide phosphonates as potential HCV inhibitors.

Preparation of several triazoloacyclic nucleoside phosphonates is described. The key step of the synthesis involves a copper(I)-catalysed azide-alkyne 1,3-dipolar cycloaddition between azidoalkylphosphonates and propargylated nucleobases. The antiviral properties of these new analogues have been evaluated and revealed interesting potencies.

The increase in intra-macrophage thiols induced by new pro-GSH molecules directs the Th1 skewing in ovalbumin immunized mice.

In the present work, the capacity of new pro-GSH molecules to increase the intra-macrophage thiol content in vitro and in vivo as well as to shift the immune response to Th1 in ovalbumin (Ova)-sensitized mice were examined. The molecules were the N-butanoyl GSH derivative, GSH-C4, and a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA), I-152. In vitro, 2h-incubation with both molecules was found to increase intra-macrophage thiol content; in vivo, Ova-sensitized mice pre-treated by intraperitoneal administration of the pro-GSH molecules showed an increase in plasma anti-Ova IgG2a and IgG2b, characterizing Th1 immune response, and a decrease in IgG1, typical of the Th2 response. Such findings were connected to a shift to a Th1 response also involving splenocyte IFN-γ production as revealed by ELISPOT assay and higher levels of IL-12 in circulation. Although immune responses are in vivo mediated both by dendritic cells and macrophages, the data reported in this paper corroborate the suggestion that the pro-GSH molecules, increasing the intra-cellular thiol pool, modulate the Th1/Th2 balance favouring Th1-type responses and may be employed as Th1-directing adjuvants in new vaccination protocols and as immunomodulators in those diseases where Th1 response patterns are compromised in favour of Th2.

Morphological and functional characteristics of three-dimensional engineered bone-ligament-bone constructs following implantation.

The incidence of ligament injury has recently been estimated at 400,000/year. The preferred treatment is reconstruction using an allograft, but outcomes are limited by donor availability, biomechanical incompatibility, and immune rejection. The creation of an engineered ligament in vitro solely from patient bone marrow stromal cells (has the potential to greatly enhance outcomes in knee reconstructions. Our laboratory has developed a scaffoldless method to engineer three-dimensional (3D) ligament and bone constructs from rat bone marrow stem cells in vitro. Coculture of these two engineered constructs results in a 3D bone-ligament-bone (BLB) construct with viable entheses, which was successfully used for medial collateral ligament (MCL) replacement in a rat model. 1 month and 2 month implantations were applied to the engineered BLBs. Implantation of 3D BLBs in a MCL replacement application demonstrated that our in vitro engineered tissues grew and remodeled quickly in vivo to an advanced phenotype and partially restored function of the knee. The explanted 3D BLB ligament region stained positively for type I collagen and elastin and was well vascularized after 1 and 2 months in vivo. Tangent moduli of the ligament portion of the 3D BLB 1 month explants increased by a factor of 2.4 over in vitro controls, to a value equivalent to those observed in 14-day-old neonatal rat MCLs. The 3D BLB 1 month explants also exhibited a functionally graded response that closely matched native MCL inhomogeneity, indicating the constructs functionally adapted in vivo.

Expanding the borononucleotide family: synthesis of borono-analogues of dCMP, dGMP and dAMP.

We previously reported the synthesis of a borononucleotide analogue of thymidine monophosphate and its association towards the formation of a new borono-linked dinucleotide. Here we describe the completion of the set of four 2'-deoxyborononucleotide analogues of natural nucleotide monophosphates, namely the previously unknown dCbn, dGbn and dAbn. These analogues were all prepared from the respective 5'-aldehydic nucleosides through a homologation/reduction sequence. The borononucleotides were subsequently obtained by either borylation (dCbn and dGbn) or cross-metathesis (CM) in the presence of the Hoveyda-Grubbs catalyst (dAbn). The reversible formation of the corresponding dinucleotides between these new analogues and uridine was studied by (1)H NMR, and semi-empirical calculations were carried out to provide bond length and electrostatic information that assess the structural similarities existing between these bioisosteres and their natural counterparts.

The effect of implantation on scaffoldless three-dimensional engineered bone constructs.

Our laboratory has previously developed scaffoldless engineered bone constructs (EBC). Bone marrow stromal cells (BMSC) were harvested from rat femur and cultured in medium that induced osteogenic differentiation. After reaching confluence, the monolayer of cells contracted around two constraint points forming a cylinder. EBCs were placed in small diameter (0.5905 x 0.0625 in.) or large diameter (0.5905 x 0.125 in.) silicone tubing and implanted intramuscularly in the hind limb of a rat. Bone mineral content (BMC) of the EBC was analyzed before implantation and at 1 and 2 mo following implantation and compared to that of native femur bone at different stages of development. Negligible BMC was observed in E-20 femur or EBCs prior to implantation. One-month implantation in both small and large tubing increased BMC in the EBC. BMC of EBC from large tubing was greater than in 14 d rat neonatal femurs, but was 2% and 3% of BMC content in adult bone after 1 and 2 mo of implantation, respectively. Alizarine Red and osteopontin staining of the EBCs before and after implantation confirmed increased bone mineralization in the implanted EBCs. Implanted EBCs also had extensive vascularization. Our data suggest that BMSC can be successfully used for the generation of scaffoldless EBC, and this model can be potentially used for the generation of autologous bone transplants in humans.