Toll-like receptor (TLR) agonists as a driving force behind next-generation vaccine adjuvants and cancer therapeutics.

Until recently, the development of new human adjuvants was held back by a poor understanding of their mechanisms of action. The field was revolutionized by the discovery of the toll-like receptors (TLRs), innate immune receptors that directly or indirectly are responsible for detecting pathogen-associated molecular patterns (PAMPs) and respond to them by activating innate and adaptive immune pathways. Hundreds of ligands targeting various TLRs have since been identified and characterized as vaccine adjuvants. This work has important implications not only for the development of vaccines against infectious diseases but also for immuno-therapies against cancer, allergy, Alzheimer's disease, drug addiction and other diseases. Each TLR has its own specific tissue localization and downstream gene signalling pathways, providing researchers the opportunity to precisely tailor adjuvants with specific immune effects. TLR agonists can be combined with other TLR or alternative adjuvants to create combination adjuvants with synergistic or modulatory effects. This review provides an introduction to the various classes of TLR adjuvants and their respective signalling pathways. It provides an overview of recent advancements in the TLR field in the past 2-3 years and discusses criteria for selecting specific TLR adjuvants based on considerations, such as disease mechanisms and correlates of protection, TLR immune biasing capabilities, route of administration, antigen compatibility, new vaccine technology platforms, and age- and species-specific effects.

Covax-19/Spikogen® vaccine based on recombinant spike protein extracellular domain with Advax-CpG55.2 adjuvant provides single dose protection against SARS-CoV-2 infection in hamsters.

COVID-19 presents an ongoing global health crisis. Protein-based COVID-19 vaccines that are well-tolerated, safe, highly-protective and convenient to manufacture remain of major interest. We therefore sought to compare the immunogenicity and protective efficacy of a number of recombinant SARS-CoV-2 spike protein candidates expressed in insect cells. By comparison to a full length (FL) spike protein detergent-extracted nanoparticle antigen, the soluble secreted spike protein extracellular domain (ECD) generated higher protein yields per liter of culture and when formulated with either Alum-CpG55.2 or Advax-CpG55.2 combination adjuvants elicited robust antigen-specific humoral and cellular immunity in mice. In hamsters, the spike ECD when formulated with either adjuvant induced high serum neutralizing antibody titers even after a single dose. When challenged with the homologous SARS-CoV-2 virus, hamsters immunized with the adjuvanted spike ECD exhibited reduced viral load in day 1-3 oropharyngeal swabs and day 3 nasal turbinate tissue and had no recoverable infectious virus in day 3 lung tissue. The reduction in lung viral load correlated with less weight loss and lower lung pathology scores. The formulations of spike ECD with Alum-CpG55.2 or Advax-CpG55.2 were protective even after just a single dose, although the 2-dose regimen performed better overall and required only half the total amount of antigen. Pre-challenge serum neutralizing antibody levels showed a strong correlation with lung protection, with a weaker correlation seen with nasal or oropharyngeal protection. This suggests that serum neutralizing antibody levels may correlate more closely with systemic, rather than mucosal, protection. The spike protein ECD with Advax-CpG55.2 formulation (Covax-19® vaccine) was selected for human clinical development.

Artificial Intelligence in Vaccine and Drug Design.

Knowledge in the fields of biochemistry, structural biology, immunological principles, microbiology, and genomics has all increased dramatically in recent years. There has also been tremendous growth in the fields of data science, informatics, and artificial intelligence needed to handle this immense data flow. At the intersection of wet lab and data science is the field of bioinformatics, which seeks to apply computational tools to better understanding of the biological sciences. Like so many other areas of biology, bioinformatics has transformed immunology research leading to the discipline of immunoinformatics. Within this field, many new databases and computational tools have been created that increasingly drive immunology research, in many cases drawing upon artificial intelligence and machine learning to predict complex immune system behaviors, for example, prediction of B cell and T cell epitopes. In this book chapter, we provide an overview of computational tools and artificial intelligence being used for protein modeling, drug screening, vaccine design, and highlight how these tools are being used to transform approaches to pandemic countermeasure development, by reference to the current COVID-19 pandemic.

An Advax-Adjuvanted Inactivated Cell-Culture Derived Japanese Encephalitis Vaccine Induces Broadly Neutralising Anti-Flavivirus Antibodies, Robust Cellular Immunity and Provides Single Dose Protection.

ccJE+Advax is an inactivated cell culture Japanese encephalitis (JE) vaccine formulated with Advax, a novel polysaccharide adjuvant based on delta inulin. This vaccine has previously shown promise in murine and equine studies and the current study sought to better understand its mechanism of action and assess the feasibility of single dose vaccine protection. Mice immunised with ccJE+Advax had higher serum neutralisation titres than those immunised with ccJE alone or with alum adjuvant. ccJE+Advax induced extraordinarily broad cross-neutralising antibodies against multiple flaviviruses including West Nile virus (WNV), Murray Valley encephalitis virus (MVEV), St Louis encephalitis virus (SLEV) and Dengue virus-1 and -2 (DENV-1 and -2). Notably, the DENV-2 cross-neutralising antibodies from ccJE+Advax immunised mice uniquely had no DENV-2 antibody-dependent infection enhancement (ADIE) activity, in contrast to high ADIE activity seen with DENV-1 cross-reactive antibodies induced by mbJE or ccJE alone or with alum adjuvant. JEV-stimulated splenocytes from ccJE+Advax immunised mice showed increased IL-17 and IFN-γ production, consistent with a mixed Th1 and Th17 response, whereas ccJE-alum was associated with production of mainly Th2 cytokines. In a mouse lethal challenge study against highly virulent JaTH160 JEV strain, ccJE+Advax conferred complete protection in a two-dose schedule with 50 ng of vaccine antigen and near complete protection after a single 200 ng dose of vaccine antigen. There is an ongoing lack of human vaccines against particular flaviviruses, including WNV, SLEV and MVEV. Given its ability to provide single-dose JEV protection and induce broadly neutralising antibodies devoid of ADIE activity, ccJE+Advax vaccine could be useful in situations where rapid protection is desirable, e.g., during a local outbreak or for use in travellers or armies requiring rapid deployment to JEV endemic regions.

Rapid development of analytical methods for evaluating pandemic vaccines: a COVID-19 perspective.

Vaccines are key in charting a path out of the COVID-19 pandemic. However, development of new vaccines is highly dependent on availability of analytical methods for their design and evaluation. This paper highlights the challenges presented in having to rapidly develop vaccine analytical tools during an ongoing pandemic, including the need to address progressive virus mutation and adaptation which can render initial assays unreliable or redundant. It also discusses the potential of new computational modeling techniques to model and analyze key viral proteins and their attributes to assist vaccine production and assay design. It then reviews the current range of analytical tools available for COVID-19 vaccine application, ranging from in vitro assays for immunogen characterization to assays to measure vaccine responses in vivo. Finally, it provides a future perspective for COVID-19 vaccine analytical tools and attempts to predict how the field might evolve over the next 5-10 years.

A humanized orthotopic tumor microenvironment alters the bone metastatic tropism of prostate cancer cells.

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, and bone is the most frequent site of metastasis. The tumor microenvironment (TME) impacts tumor growth and metastasis, yet the role of the TME in PCa metastasis to bone is not fully understood. We used a tissue-engineered xenograft approach in NOD-scid IL2Rγnull (NSG) mice to incorporate two levels of humanization; the primary tumor and TME, and the secondary metastatic bone organ. Bioluminescent imaging, histology, and immunohistochemistry were used to study metastasis of human PC-3 and LNCaP PCa cells from the prostate to tissue-engineered bone. Here we show pre-seeding scaffolds with human osteoblasts increases the human cellular and extracellular matrix content of bone constructs, compared to unseeded scaffolds. The humanized prostate TME showed a trend to decrease metastasis of PC-3 PCa cells to the tissue-engineered bone, but did not affect the metastatic potential of PCa cells to the endogenous murine bones or organs. On the other hand, the humanized TME enhanced LNCaP tumor growth and metastasis to humanized and murine bone. Together this demonstrates the importance of the TME in PCa bone tropism, although further investigations are needed to delineate specific roles of the TME components in this context.

A truncated glycoprotein G vaccine formulated with Advax-CpG adjuvant provides protection of mice against genital herpes simplex virus 2 infection.

Herpes simplex virus type 2 (HSV-2) is a common sexually transmitted disease that affects approximately 500 million individuals globally. There is currently no approved vaccine to prevent HSV-2 infection. EXCT4 is a truncated form of the mature glycoprotein G-2 (mgG-2) that unlike full mature form is secreted by expressing cells enabling it to be rapidly scaled up for production. The current study examined whether EXCT4 immunity in mice could be further enhanced through use of adjuvants. EXCT4 formulated with Advax-CpG adjuvant induced a strong Th1-type immune response characterized by interferon gamma (IFN-γ) and protected animals against a lethal genital challenge with HSV-2. This response was associated with reduced viral load in vaginal washes, spinal cord, and dorsal root ganglia. Together the results provide proof of concept that EXCT4 formulated with Advax-CpG adjuvant is a promising HSV-2 vaccine candidate warranting further investigation.

A M2 protein-based universal influenza vaccine containing Advax-SM adjuvant provides newborn protection via maternal or neonatal immunization.

BACKGROUND: Despite newborns being at increased risk of serious influenza infection, influenza vaccines are currently not recommended for use in infants under 6 months of age. We therefore sought to evaluate the protective efficacy in mice of an M2-based influenza vaccine (CapM2e) formulated with Advax-SM adjuvant. Vaccine protection was assessed via both passive maternal immunization and direct neonatal immunization. METHODS: For maternal transfer studies, female mice were immunized 1 week before and after mating. Blood was collected from both mother and offspring during weaning and pups were challenged when they reached 3 weeks of age with lethal doses of H1N1 and homologous reassortment influenza strain H3N2 with conserved M2. For direct immunization studies, newborns were immunized at 1 and 3 weeks of age and blood was collected prior to challenge at 4 weeks of age. RESULTS: Maternal immunization with CapM2e + Advax-SM vaccine induced high maternal M2e antibody levels that were passively transferred to their offspring and provided them with protection against both H1N1 and H3N2 influenza strains when challenged at 3 weeks of age. When used for direct immunization of neonatal mice, CapM2e + Advax-SM vaccine similarly induced high serum M2e antibody levels and protected against H1N1 and H3N2 influenza challenges with protection associated with inhibition of virus replication with a significant reduction in lung virus load in immunized pups. CONCLUSION: CapM2e + Advax-SM vaccine could be useful for protecting newborns against diverse influenza A strains, with opportunities to achieve protection by passive maternal immunization or active neonatal immunization. This data supports further development of this promising M2e-based vaccine candidate.

Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection.

The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. Covax-19 vaccine induced high spike protein binding antibody levels that neutralised the original lineage B.1.319 virus from which the vaccine spike protein was derived, as well as the variant B.1.1.7 lineage virus. Covax-19 vaccine also induced a high frequency of spike-specific CD4 + and CD8 + memory T-cells with a dominant Th1 phenotype associated with the ability to kill spike-labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus given two weeks after the last immunisation. Notably, ferrets that received the two higher doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting that in addition to lung protection, Covax-19 vaccine may have the potential to reduce virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.

Advax-CpG Adjuvant Provides Antigen Dose-Sparing and Enhanced Immunogenicity for Inactivated Poliomyelitis Virus Vaccines.

Global immunization campaigns have resulted in a major decline in the global incidence of polio cases, with wild-type poliovirus remaining endemic in only two countries. Live oral polio vaccine (OPV) played a role in the reduction in polio case numbers; however, the risk of OPV developing into circulating vaccine-derived poliovirus makes it unsuitable for eradication programs. Trivalent inactivated polio virus (TIPV) vaccines which contain formalin-inactivated antigens produced from virulent types 1, 2 and 3 reference polio strains grown in Vero monkey kidney cells have been advocated as a replacement for OPV; however, TIPVs have weak immunogenicity and multiple boosts are required before peak neutralizing titers are reached. This study examined whether the incorporation of the novel polysaccharide adjuvant, Advax-CpG, could boost the immunogenicity of two TIPV vaccines, (i) a commercially available polio vaccine (IPOL®, Sanofi Pasteur) and (ii) a new TIPV formulation developed by Statens Serum Institut (SSI). Mice were immunized intramuscularly based on recommended vaccine dosage schedules and serum antibody titers were followed for 12 months post-immunization. Advax-CpG significantly enhanced the long-term immunogenicity of both TIPV vaccines and had at least a 10-fold antigen dose-sparing effect. An exception was the poor ability of the SSI TIPV to induce serotype type 1 neutralizing antibodies. Immunization with monovalent IPVs suggested that the low type 1 response to TIPV may be due to antigen competition when the type 1 antigen was co-formulated with the type 2 and 3 antigens. This study provides valuable insights into the complexity of the formulation of multivalent polio vaccines and supports the further development of adjuvanted antigen-sparing TIPV vaccines in the fight to eradicate polio.

3D Quantification of Vascular-Like Structures in z Stack Confocal Images.

Optical slice microscopy is commonly used to characterize the morphometric features of 3D cellular cultures, such as in vitro vascularization. However, the quantitative analysis of those structures is often performed on a single 2D maximum intensity projection image, limiting the accuracy of data obtained from 3D cultures. Here, we present a protocol for the quantitative analysis of z stack images, utilizing Fiji, Amira, and WinFiber3D. This protocol facilitates the in-depth examination of vascular-like structures within 3D cell culture models. For complete details on the use and execution of this protocol, please refer to Koch et al. (2020).

Investigation of Sustained BMP Delivery in the Prevention of Medication-Related Osteonecrosis of the Jaw (MRONJ) in a Rat Model.

Medication-related osteonecrosis of the jaw (MRONJ) poses an ongoing challenge for clinicians and researchers. Currently, there is a lack of preventative measures available for at-risk patients undergoing tooth extractions, especially those with prior bisphosphonate treatment due to osteoporosis or bone metastasis diagnoses. Here, these issues are addressed using a preventative tissue engineering strategy against MRONJ development. This study evaluates the efficacy of a poly(ethylene glycol)-heparin hydrogel as a tool for the delivery of arginylglycylaspartic acid (RGD) and recombinant human bone morphogenic protein-2 (rhBMP-2). Three groups of skeletally mature rats each receive two doses of intravenous zoledronic acid prior to surgery and undergo extraction of the right first mandibular molar with gingival closure. Experimental groups either have the sockets left empty, filled with hydrogel minus rhBMP-2, or filled with hydrogel plus rhBMP-2. Eight weeks postoperatively specimens are analyzed using radiological, histological, and scanning electron microscopy (SEM) techniques. µCT analysis shows increased bone formation with hydrogel/rhBMP-2 delivery compared to the empty socket. Hydrogel-treated groups display increased presence of osteocytes and increased osteoclastic action compared to the empty sockets. These results represent the first step toward improved delivery of rhBMP-2 and a potential MRONJ preventative for patients undergoing bisphosphonate treatment.

Microenvironment engineering of osteoblastic bone metastases reveals osteomimicry of patient-derived prostate cancer xenografts.

Representative in vitro models that mimic the native bone tumor microenvironment are warranted to support the development of more successful treatments for bone metastases. Here, we have developed a primary cell 3D model consisting of a human osteoblast-derived tissue-engineered construct (hOTEC) indirectly co-cultured with patient-derived prostate cancer xenografts (PDXs), in order to study molecular interactions in a patient-derived microenvironment context. The engineered biomimetic microenvironment had high mineralization and embedded osteocytes, and supported a high degree of cancer cell osteomimicry at the gene, protein and mineralization levels when co-cultured with prostate cancer PDXs from a lymph node metastasis (LuCaP35) and bone metastasis (BM18) from patients with primary prostate cancer. This fully patient-derived model is a promising tool for the assessment of new molecular mechanisms and as a personalized pre-clinical platform for therapy testing for patients with prostate cancer bone metastases.

Additively Manufactured Multiphasic Bone-Ligament-Bone Scaffold for Scapholunate Interosseous Ligament Reconstruction.

The scapholunate interosseous ligament (SLIL) is a frequently torn wrist ligament, and current surgical options for SLIL tears are suboptimal. This research aims to develop a novel multiphasic bone-ligament-bone scaffold (BLB) with a porous interface using 3D-printing and cell sheet technology for the reconstruction of the dorsal scapholunate interosseous ligament. The BLB comprises two bone compartments bridged by aligned polycaprolactone fibers mimicking the architecture of the native tissue. Mechanical testing of the BLBs shows their ability to withstand physiological forces. Combination of the BLB with human bone marrow mesenchymal stem cell sheet demonstrates that the harvesting did not compromise cell viability, while allowing homogeneous distribution in the ligament compartment. The BLBs are loaded with cell sheets and bone morphogenetic protein-2 in the ligament and bone compartment respectively prior to ectopic implantation into athymic rats. The histology demonstrates rapid tissue infiltration, high vascularization, and more importantly the maintenance of the compartmentalization as bone formation remains localized to the bone compartment despite the porous interface. The cells in the ligament compartment become preferentially aligned, and this proof-of-concept study demonstrates that the BLB can provide sufficient compartmentalization and fiber guiding properties necessary for the regeneration of the dorsal SLIL.

A 3D tumor microenvironment regulates cell proliferation, peritoneal growth and expression patterns.

Peritoneal invasion through the mesothelial cell layer is a hallmark of ovarian cancer metastasis. Using tissue engineering technologies, we recreated an ovarian tumor microenvironment replicating this aspect of disease progression. Ovarian cancer cell-laden hydrogels were combined with mesothelial cell-layered melt electrospun written scaffolds and characterized with proliferation and transcriptomic analyses and used as intraperitoneal xenografts. Here we show increased cancer cell proliferation in these 3D co-cultures, which we validated using patient-derived cells and linked to peritoneal tumor growth in vivo. Transcriptome-wide expression analysis identified IGFBP7, PTGS2, VEGFC and FGF2 as bidirectional factors deregulated in 3D co-cultures compared to 3D mono-cultures, which we confirmed by immunohistochemistry of xenograft and patient-derived tumor tissues and correlated with overall and progression-free survival. These factors were further increased upon expression of kallikrein-related proteases. This clinically predictive model allows us to mimic the complexity and processes of the metastatic disease that may lead to therapies that protect from peritoneal invasion or delay the development of metastasis.

Humanization of bone and bone marrow in an orthotopic site reveals new potential therapeutic targets in osteosarcoma.

BACKGROUND: Existing preclinical murine models often fail to predict effects of anti-cancer drugs. In order to minimize interspecies-differences between murine hosts and human bone tumors of in vivo xenograft platforms, we tissue-engineered a novel orthotopic humanized bone model. METHODS: Orthotopic humanized tissue engineered bone constructs (ohTEBC) were fabricated by 3D printing of medical-grade polycaprolactone scaffolds, which were seeded with human osteoblasts and embedded within polyethylene glycol-based hydrogels containing human umbilical vein endothelial cells (HUVECs). Constructs were then implanted at the femur of NOD-scid and NSG mice. NSG mice were then bone marrow transplanted with human CD34 + cells. Human osteosarcoma (OS) growth was induced within the ohTEBCs by direct injection of Luc-SAOS-2 cells. Tissues were harvested for bone matrix and marrow morphology analysis as well as tumor biology investigations. Tumor marker expression was analyzed in the humanized OS and correlated with the expression in 68 OS patients utilizing tissue micro arrays (TMA). RESULTS: After harvesting the femurs micro computed tomography and immunohistochemical staining showed an organ, which had all features of human bone. Around the original mouse femur new bone trabeculae have formed surrounded by a bone cortex. Staining for human specific (hs) collagen type-I (hs Col-I) showed human extracellular bone matrix production. The presence of nuclei staining positive for human nuclear mitotic apparatus protein 1 (hs NuMa) proved the osteocytes residing within the bone matrix were of human origin. Flow cytometry verified the presence of human hematopoietic cells. After injection of Luc-SAOS-2 cells a primary tumor and lung metastasis developed. After euthanization histological analysis showed pathognomic features of osteoblastic OS. Furthermore, the tumor utilized the previously implanted HUVECS for angiogenesis. Tumor marker expression was similar to human patients. Moreover, the recently discovered musculoskeletal gene C12orf29 was expressed in the most common subtypes of OS patient samples. CONCLUSION: OhTEBCs represent a suitable orthotopic microenvironment for humanized OS growth and offers a new translational direction, as the femur is the most common location of OS. The newly developed and validated preclinical model allows controlled and predictive marker studies of primary bone tumors and other bone malignancies.

A Novel 3D Cultured Model for Studying Early Changes in Age-Related Macular Degeneration.

Various in vitro culture systems have been used to investigate the pathogenesis of age-related macular degeneration (AMD). However, many still rely on oversimplified monolayer culture models. AMD is a complex disease, associated with the pathological changes to multiple structural components such as the Bruch's membrane, retinal pigment epithelium (RPE), and choroidal endothelial cells. This study aims to construct a novel 3D coculture model using the polycaprolactone (PCL)-gelatin electrospun scaffold, with human RPE cells (hRPE) and primate choroidal cells (RF-6A). Results from this study show that PCL-gelatin scaffolds have a highly porous ultrastructure that supports the attachment, proliferation, differentiation, and migration of the hRPEs and choroidal endothelial cells. It is also demonstrated that the PCL-gelatin 3D coculture model may be useful in exploring the molecular interplay between the hPRE and the choroidal endothelial cells, and their effects on growth factor modulation, which may be important in the pathogenesis of AMD.

Fetal Bone Marrow-Derived Mesenchymal Stem/Stromal Cells Enhance Humanization and Bone Formation of BMP7 Loaded Scaffolds.

Tissue engineered constructs built with human cells capable of generating a bone-like organ within the mouse have attracted considerable interest over the past decade. Here, we aimed to compare the utility of human mesenchymal stem/stromal cells (MSC) isolated from fetal term placenta (fPL-MSC) and fetal first trimester bone marrow (fBM-MSC) in a polycaprolactone scaffold/BMP7-based model in nude mice. Furthermore, fPL-MSC were co-seeded with fetal placenta-derived endothelial colony forming cells (ECFC) to assess the impact of ECFC on fPL-MSC osteogenesis. X-ray radiography and micro computed tomography analyses showed enhanced bone formation in all BMP7 groups; however there was no difference after 2 months in bone formation between scaffolds seeded with fPL-MSC alone or combination of ECFC and fPL-MSC. Of interest, fBM-MSC showed the highest level of bone formation. Additionally, endochondral ossification contributed in generation of bone in fBM-MSC. Histological analysis showed the primary role of BMP in generation of cortical and trabecular bone, and the recruitment of hematopoietic cells to the scaffolds. Current in vivo engineered bone organs can potentially be used for drug screening or as models to study bone tissue development in combination with haematopoiesis.

Comparison of Different Decalcification Methods Using Rat Mandibles as a Model.

Selection of decalcification agents is an essential consideration when processing mineralized tissues because the integrity and immunohistochemical characteristics of the tissues may be affected. Here, we report results obtained from the decalcification of rat mandibles using 10% ethylenediaminetetraacetic acid (EDTA) at room temperature (RT), 10% EDTA at 37C, 5% nitric acid, and 10% formic acid at RT. Decalcification endpoints were determined by microcomputed tomography. Morphological preservation and antigenicity were evaluated by hematoxylin and eosin staining and immunohistochemistry. Decalcification of the anterior and posterior portions of the mandible took 220 and 191 hr in 10% EDTA RT, 102 and 73 hr in 10% EDTA 37C, 13.5 and 4.3 hr in 5% nitric acid, and 140 and 36 hr in 10% formic acid, respectively. Decalcification in 10% EDTA at 37C was accelerated, but 10% EDTA at RT provided optimal results for immunohistochemistry and cellular and structural details. Decalcification using 5% nitric acid was accomplished in the shortest time and exhibited good cellular and architectural morphology, whereas 10% formic acid was suboptimal with respect to tissue and cellular morphology. Despite being the slowest method, EDTA at RT is still the recommended method for decalcifying mineralized tissues; however, if rapid decalcification is needed, 5% nitric acid is the best option, yielding acceptable tissue integrity and speed.