Three-Dimensional Humanized Model of the Periodontal Gingival Pocket to Study Oral Microbiome.

The oral cavity contains distinct microenvironments that serve as oral barriers, such as the non-shedding surface of the teeth (e.g., enamel), the epithelial mucosa and gingival tissue (attached gingiva) where microbial communities coexist. The interactions and balances between these communities are responsible for oral tissue homeostasis or dysbiosis, that ultimately dictate health or disease. Disruption of this equilibrium can lead to chronic inflammation and permanent tissue damage in the case of chronic periodontitis. There are currently no experimental tissue models able to mimic the structural, physical, and metabolic conditions present in the human oral gingival tissue to support the long-term investigation of host-pathogens imbalances. Herein, the authors report an in vitro 3D anatomical gingival tissue model, fabricated from silk biopolymer by casting a replica mold of an adult human mandibular gingiva to recreate a tooth-gum unit. The model is based on human primary cultures that recapitulate physiological tissue organization, as well as a native oxygen gradient within the gingival pocket to support human subgingival plaque microbiome with a physiologically relevant level of microbial diversity up to 24 h. The modulation of inflammatory markers in the presence of oral microbiome indicates the humanized functional response of this model and establishes a new set of tools to investigate host-pathogen imbalances in gingivitis and periodontal diseases.

Genetic inhibition of RIPK3 ameliorates functional outcome in controlled cortical impact independent of necroptosis.

Traumatic brain injury (TBI) is a leading cause of death and disability with no specific effective therapy, in part because disease driving mechanisms remain to be elucidated. Receptor interacting protein kinases (RIPKs) are serine/threonine kinases that assemble multi-molecular complexes that induce apoptosis, necroptosis, inflammasome and nuclear factor kappa B activation. Prior studies using pharmacological inhibitors implicated necroptosis in the pathogenesis of TBI and stroke, but these studies cannot be used to conclusively demonstrate a role for necroptosis because of the possibility of off target effects. Using a model of cerebral contusion and RIPK3 and mixed lineage kinase like knockout (MLKL-/-) mice, we found evidence for activation of RIPK3 and MLKL and assembly of a RIPK1-RIPK3-MLKL necrosome complex in pericontusional brain tissue. Phosphorylated forms of RIPK3 and MLKL were detected in endothelium, CD11b + immune cells, and neurons, and RIPK3 was upregulated and activated in three-dimensional human endothelial cell cultures subjected to CCI. RIPK3-/- and MLKL-/- mice had reduced blood-brain barrier damage at 24 h (p < 0.05), but no differences in neuronal death (6 h, p = ns in CA1, CA3 and DG), brain edema (24 h, p = ns), or lesion size (4 weeks, p = ns) after CCI. RIPK3-/-, but not MLKL-/- mice, were protected against postinjury motor and cognitive deficits at 1-4 weeks (RIPK3-/- vs WT: p < 0.05 for group in wire grip, Morris water maze hidden platform trials, p < 0.05 for novel object recognition test, p < 0.01 for rotarod test). RIPK3-/- mice had reduced infiltrating leukocytes (p < 0.05 vs WT in CD11b + cells, microglia and macrophages), HMGB1 release and interleukin-1 beta activation at 24-48 h (p < 0.01) after CCI. Our data indicate that RIPK3 contributes to functional outcome after cerebral contusion by mechanisms involving inflammation but independent of necroptosis.

Developing a self-organized tubulogenesis model of human renal proximal tubular epithelial cells in vitro.

Three-dimensional tissue culture models which recapitulate the phenotype and function of human renal tissue have attracted significant interest as valuable tools for studying kidney development, disease pathophysiology, and nephrotoxicity. Here, a layer-by-layered three-dimensional (3D) co-culture technique was employed to bioengineer an improved human proximal tubule tissue model through incorporating human renal proximal tubule epithelial cells (RPTECs) with two types of interstitial cells on the layered extracellular matrix-like culture matrix. The resulting cultures were characterized by their growth profile, metabolic and proliferative activity, morphological characteristics as well as their functional gene expression. Our results found that the cultures were able to enable the self-organization of RPTECs and promote the tubule-like structure formation in vitro. A well-defined lumen structure and polarized expression of some key protein markers including actin, P-gp, Na+ -K+ -ATPase, and SGLT2 were also observed in the 3D co-cultures. Moreover, compared to the 3D monocultures, the tubule-like structures formed within the 3D co-cultures displayed more significant polarity and enhanced functional gene expression. This suggested the important role played by the renal stromal cells in supporting the tubulogenesis and differentiation of RPTECs. Thus, the 3D co-culture model reported here would benefit bioengineering approaches toward more physiologically relevant proximal tubule tissue in vitro, providing more robust tool not only for better understanding kidney development and pathophysiology but also for drug screening for nephrotoxicity.

Biodegradable silk catheters for the delivery of therapeutics across anatomical repair sites.

Biodegradable silk catheters for the delivery of therapeutics are designed with a focus on creating porous gradients that can direct the release of molecules away from the implantation site. Though suitable for a range of applications, these catheters are designed for drug delivery to transplanted adipose tissue in patients having undergone a fat grafting procedure. A common complication for fat grafts is the rapid reabsorption of large volume adipose transplants. In order to prolong volume retention, biodegradable catheters can be embedded into transplanted tissue to deliver nutrients, growth factors or therapeutics to improve adipocyte viability, proliferation, and ultimately extend volume retention. Two fabrication methods are developed: a silk gel-spinning technique, which uses a novel flash-freezing step to induce high porosity throughout the bulk of the tube, and a dip-coating process using silk protein solutions doped with a water soluble porogen. Increased porosity aids in the diffusion of drug through the silk tube in a controllable way. Additionally, we interface the porous tubes with ALZET osmotic pumps for implantation into a subcutaneous nude mouse model. The work described herein will discuss the processing parameters as well as the interfacing between pump and cargo therapeutic and the resulting release profiles. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 501-510, 2019.

Repetitive head injury in adolescent mice: A role for vascular inflammation.

Repetitive mild traumatic brain injury during adolescence can induce neurological dysfunction through undefined mechanisms. Interleukin-1 (IL-1) contributes to experimental adult diffuse and contusion TBI models, and IL-1 antagonists have entered clinical trials for severe TBI in adults; however, no such data exist for adolescent TBI. We developed an adolescent mouse repetitive closed head injury (rCHI) model to test the role of IL-1 family members in post-injury neurological outcome. Compared to one CHI, three daily injuries (3HD) produced acute and chronic learning deficits and emergence of hyperactivity, without detectable gliosis, neurodegeneration, brain atrophy, and white matter loss at one year. Mature IL-1ß and IL-18 were induced in brain endothelium in 3HD but not 1HD, three hit weekly, or sham animals. IL-1ß processing was induced cell-autonomously in three-dimensional human endothelial cell cultures subjected to in vitro concussive trauma. Mice deficient in IL-1 receptor-1 or caspase-1 had improved post-injury Morris water maze performance. Repetitive mild CHI in adolescent mice may induce behavioral deficits in the absence of significant histopathology. The endothelium is a potential source of IL-1ß and IL-18 in rCHI, and IL-1 family members may be therapeutic targets to reduce or prevent neurological dysfunction after repetitive mild TBI in adolescents.

Three-Dimensional Tissue Models for Studying Ex Vivo Megakaryocytopoiesis and Platelet Production.

Three-dimensional (3D) tissue cultures in vitro enable a more physiological reconstruction of native tissues and organs. The bone marrow environment, structure and composition regulate megakaryocyte function and platelet production. Here, we describe the use of silk fibroin protein biomaterials to assemble 3D scaffolds mimicking the bone marrow niche architecture and extracellular matrix composition to support platelet release from human megakaryocytes. Additionally, we also propose the use of hyaluronan hydrogels, functionalized with extracellular matrix components, to reproduce the 3D matrix structure of the bone marrow environment for studying human megakaryocyte function.

Multi-channel silk sponge mimicking bone marrow vascular niche for platelet production.

In the bone marrow, the interaction of progenitor cells with the vasculature is fundamental for the release of blood cells into circulation. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising protein biomaterial for bone marrow tissue engineering, because of its tunable architecture and mechanical properties, the capacity to incorporate labile compounds without loss of bioactivity and the demonstrated ability to support blood cell formation without premature activation. In this study, we fabricated a custom perfusion chamber to contain a multi-channel lyophilized silk sponge mimicking the vascular network in the bone marrow niche. The perfusion system consisted in an inlet and an outlet and 2 splitters that allowed funneling flow in each single channel of the silk sponge. Computational Fluid Dynamic analysis demonstrated that this design permitted confined flow inside the vascular channels. The silk channeled sponge supported efficient platelet release from megakaryocytes (Mks). After seeding, the Mks localized along SDF-1α functionalized vascular channels in the sponge. Perfusion of the channels allowed the recovery of functional platelets as demonstrated by increased PAC-1 binding upon thrombin stimulation. Further, increasing the number of channels in the silk sponge resulted in a proportional increase in the numbers of platelets recovered, suggesting applicability to scale-up for platelet production. In conclusion, we have developed a scalable system consisting of a multi-channeled silk sponge incorporated in a perfusion chamber that can provide useful technology for functional platelet production ex vivo.

High-Strength, Durable All-Silk Fibroin Hydrogels with Versatile Processability toward Multifunctional Applications.

Hydrogels have been the focus of extensive research due to their potential use in fields including biomedical, pharmaceutical, biosensors, and cosmetics. However, the general weak mechanical properties of hydrogels limit their utility. Here, we generate pristine silk fibroin (SF) hydrogels with excellent mechanical properties via a binary solvent induced conformation transition (BSICT) strategy. In this method, the conformational transition of SF is regulated by moderate binary solvent diffusion and SF/solvent interactions. ß-sheet formation serves as the physical crosslinks that connect disparate protein chains to form continuous 3D hydrogel networks, avoiding complex chemical and/or physical treatments. The Young's modulus of these new BSICT-silk fibroin hydrogels can reach up to 6.5±0.2 MPa, tens to hundreds of times higher than that of conventional hydrogels (0.01-0.1 MPa). These new materials filled the "empty soft materials space" in the elastic modulus/strain Ashby plot. More remarkably, the BSICT-SF hydrogels can be processed into different constructions through different polymer and/or metal based processing techniques, such as molding, laser cutting, and machining. Thus, these new hydrogel systems exhibit potential utility in many biomedical and engineering fields.

Label free monitoring of megakaryocytic development and proplatelet formation in vitro.

Megakaryopoiesis and platelet production are complex biological processes that require tight regulation of successive lineage commitment steps and are ultimately responsible for maintaining and renewing the pool of circulating platelets in the blood. Despite major advancements in the understanding of megakaryocytic biology, the detailed mechanisms driving megakaryocytic differentiation have yet to be elucidated. Here we show that automated image analysis algorithms applied to two-photon excited fluorescence (TPEF) images can non-invasively monitor structural and metabolic megakaryocyte behavior changes occurring during differentiation and platelet formation in vitro. Our results demonstrate that high-contrast, label-free two photon imaging holds great potential in studying the underlying physiological processes controlling the intricate process of platelet production.

Modular flow chamber for engineering bone marrow architecture and function.

The bone marrow is a soft, spongy, gelatinous tissue found in the hollow cavities of flat and long bones that support hematopoiesis in order to maintain the physiologic turnover of all blood cells. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising biomaterial for bone marrow engineering, because of its tunable architecture and mechanical properties, the capacity of incorporating labile compounds without loss of bioactivity and demonstrated ability to support blood cell formation. In this study, we developed a bone marrow scaffold consisting of a modular flow chamber made of polydimethylsiloxane, holding a silk sponge, prepared with salt leaching methods and functionalized with extracellular matrix components. The silk sponge was able to support efficient platelet formation when megakaryocytes were seeded in the system. Perfusion of the chamber allowed the recovery of functional platelets based on multiple activation tests. Further, inhibition of AKT signaling molecule, which has been shown to be crucial in regulating physiologic platelet formation, significantly reduced the number of collected platelets, suggesting the applicability of this tissue model for evaluation of the effects of bone marrow exposure to compounds that may affect platelet formation. In conclusion, we have bioengineered a novel modular system that, along with multi-porous silk sponges, can provide a useful technology for reproducing a simplified bone marrow scaffold for blood cell production ex vivo.

Hyaluronan based hydrogels provide an improved model to study megakaryocyte-matrix interactions.

Hyaluronan (HA) is a glycosamminoglican involved in cell biology as well as a relevant polymer for tissue engineering and regenerative medicine. Megakaryocytes (Mks) are immersed in a mesh of extracellular matrix (ECM) components that regulate their maturation in the bone marrow (BM) and the release of platelets into the bloodstream. While fibrous ECMs such as collagens and fibronectin have been demonstrated to differently regulate Mk function and platelet release, the role of HA, that fills the majority of the BM extracellular interstitial space, has not been investigated so far. Here we demonstrated that, although human Mks express HA receptors, they are not affected by HA in terms of in vitro differentiation, maturation and platelet formation. Importantly, chemical properties of HA were exploited to generate hydrogels with entrapped ECMs that represent a useful model to more closely mimic the tridimensional characteristics of the BM environment for studying Mk function. In conclusion, in this work we demonstrated that HA is an ideal candidate for a 3D ex vivo model of human BM ECM component environment.

The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control.

Megakaryocytes are rare cells found in the bone marrow, responsible for the everyday production and release of millions of platelets into the bloodstream. Since the discovery and cloning, in 1994, of their principal humoral factor, thrombopoietin, and its receptor c-Mpl, many efforts have been directed to define the mechanisms underlying an efficient platelet production. However, more recently different studies have pointed out new roles for megakaryocytes as regulators of bone marrow homeostasis and physiology. In this review we discuss the interaction and the reciprocal regulation of megakaryocytes with the different cellular and extracellular components of the bone marrow environment. Finally, we provide evidence that these processes may concur to the reconstitution of the bone marrow environment after injury and their deregulation may lead to the development of a series of inherited or acquired pathologies.

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies.

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.

Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms.

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.

mTOR inhibitors alone and in combination with JAK2 inhibitors effectively inhibit cells of myeloproliferative neoplasms.

BACKGROUND: Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN), usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells. FINDINGS: Mouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001) and an ATP-competitive (PP242) mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib). mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with polycythemia vera. CONCLUSIONS/SIGNIFICANCE: These findings support mTOR inhibitors as novel potential drugs for the treatment of MPN and advocate for clinical trials exploiting the combination of mTOR and JAK2 inhibitor.

Spleen endothelial cells from patients with myelofibrosis harbor the JAK2V617F mutation.

Increased microvessel density contributes to abnormal BM and spleen microenvironment in myelofibrosis (MF). Taking advantage of the JAK2V617F mutation as a marker of malignancy, in the present study, we investigated whether splenic endothelial cells (ECs) obtained from capillaries by laser microdissection or from fresh spleen tissue by cell culture or cell sorting harbored such mutation in patients bearing the mutation in their granulocytes and undergoing splenectomy for therapeutical reasons. To extend the analysis to the ECs of large vessels, endothelial tissue from the splenic vein was also studied. We found JAK2V617F(+) ECs in 12 of 18 patients also bearing the mutation in their granulocytes. In 3 patients, the mutation was found in at least 2 different EC samples obtained by laser microdissection, cell culture, or cell sorting. The mutation was detected in the splenic vein ECs of 1 of 6 patients investigated. In conclusion, we provide evidence that some ECs from the spleen and splenic veins of patients with MF bear the JAK2V617F mutation. We suggest that splenic ECs are involved in the process of malignant transformation in MF.

Safety and efficacy of everolimus, a mTOR inhibitor, as single agent in a phase 1/2 study in patients with myelofibrosis.

In addition to dysregulated JAK/STAT signaling, activation of the AKT/mTOR pathway occurs in myelofibrosis, a myeloproliferative neoplasm with no approved therapies. We conducted a phase 1/2 study with everolimus, an mTOR inhibitor, in 39 high- or intermediate-risk primary or postpolycythemia vera/postessential thrombocythemia myelofibrosis subjects. Responses were evaluated in 30 patients of phase 2. No dose-limiting toxicity was observed in phase 1 up to 10 mg/d. When this dose was used in phase 2, grade ≥ 3 toxicities were infrequent; the commonest toxicity was grade 1-2 stomatitis. Rapid and sustained splenomegaly reduction of > 50% and > 30% occurred in 20% and 44% of subjects, respectively. A total of 69% and 80% experienced complete resolution of systemic symptoms and pruritus. Response in leukocytosis, anemia, and thrombocytosis occurred in 15%-25%. Clinical responses were not associated with reduced JAK2V617F burden, circulating CD34(+) cells, or cytokine levels, whereas CCDN1 mRNA and phospho-p70S6K level, known targets of mTOR, and WT1 mRNA were identified as possible biomarkers associated with response. Response rate was 60% when European Network for Myelofibrosis criteria were used (8 major, 7 moderate, 3 minor responses) or 23% when IWG-MRT criteria (1 partial response, 6 clinical improvements) were used. These results provide proof-of-concept that targeting mTOR pathway in myelofibrosis may be clinically relevant.

Overexpression of microRNA-16-2 contributes to the abnormal erythropoiesis in polycythemia vera.

Deregulated expression of microRNAs is associated with neoplasia. Here, we show that mature miR-16 levels are abnormally increased in CD34(+) cells of patients with polycythemia vera as a consequence of preferential expression of miR-16-2 on chromosome 3 rather than of miR-16-1 on chromosome 13. Forced expression of miRNA-16 in normal CD34(+) cells stimulated erythroid cell proliferation and maturation. Conversely, exposure of polycythemia vera CD34(+) cells to small interfering RNA against pre-miR-16-2 reduced erythroid colonies and largely prevented formation of erythropoietin-independent colonies; myeloid progenitors remained unaffected. Experiments with knock down of JAK2 indicated that overexpression of miR-16 was independent of JAK/STAT pathway activation. Mice injected with an miR-16 antagomir showed a blunted erythroid response to exogenous erythropoietin, which indicates a role of miR-16 in normal erythropoiesis. These data suggest that deregulation of miR-16-2 contributes to abnormal expansion of erythroid lineage in polycythemia vera. However, the mechanisms for miR-16-2 overexpression remain to be elucidated, because no genetic abnormalities at the miR-16-2 locus were discovered.

Influence of JAK2V617F allele burden on phenotype in essential thrombocythemia.

BACKGROUND: Fifty to sixty percent of patients with essential thrombocythemia harbor the JAK2(V617F) mutation. The impact of this mutation on clinical phenotype is still debated. The aim of this study was to evaluate possible correlations between JAK2(V617F) mutant allele burden and both clinical presentation and hematologic abnormalities in essential thrombocythemia patients. DESIGN AND METHODS: In this single-center retrospective study, JAK2(V617F) allele load was measured by sensitive quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the granulocytes of 260 patients diagnosed as having essential thrombocythemia according to WHO criteria. RESULTS: Median V617F allele burden in patients with the mutation (n=165, 63.4%) was 24%, ranging from 1% to 87%; an allele burden greater than 51% was found in 5% of the patients. Older patients presented progressively higher percentages of the V617F allele. Signs of stimulated erythropoiesis and myelopoiesis, as well as higher PRV-1 levels, were found in patients with the mutation, but no linear correlation with load of mutant allele could be ascertained; on the other hand, the frequency of patients with erythropoietin-independent erythroid colonies progressively increased depending on mutant allele load. Splenomegaly and microvessel symptoms were significantly more represented among patients with greater than 50% and 25% JAK2(V617F) allele burden, respectively. Increasing mutant allele load correlated with higher frequency of arterial thrombosis at diagnosis, as confirmed also in multivariate analysis; the relative risk was 3.0 (95% CI 1.3-6.8; p=0.01) in patients having a greater than 25% mutant allele burden. CONCLUSIONS: The JAK2(V617F) mutant allele burden contributes to determining the clinical phenotype in patients with essential thrombocythemia.