Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model.

The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.

Phenomenological modelling and simulation of cell clusters in 3D cultures.

Cell clustering and aggregation are fundamental processes in the development of several tissues and the progression of many diseases. The formation of these aggregates also has a direct impact on the oxygen concentration in their surroundings due to cellular respiration and poor oxygen diffusion through clusters. In this work, we propose a mathematical model that is capable of simulating cell cluster formation in 3D cultures through combining a particle-based and a finite element approach to recreate complex experimental conditions. Cells are modelled considering cell proliferation, cell death and cell-cell mechanical interactions. Additionally, the oxygen concentration profile is calculated through finite element analysis using a reaction-diffusion model that considers cell oxygen consumption and diffusion through the extracellular matrix and the cell clusters. In our model, the local oxygen concentration in the medium determines both cell proliferation and cell death. Numerical predictions are also compared with experimental data from the literature. The simulation results indicate that our model can predict cell clustering, cluster growth and oxygen distribution in 3D cultures. We conclude that the initial cell distribution, cell death and cell proliferation dynamics determine the size and density of clusters. Moreover, these phenomena are directly affected by the oxygen transport in the 3D culture.

Proton pump inhibitors protect mice from acute systemic inflammation and induce long-term cross-tolerance.

Incidence of sepsis is increasing, representing a tremendous burden for health-care systems. Death in acute sepsis is attributed to hyperinflammatory responses, but the underlying mechanisms are still unclear. We report here that proton pump inhibitors (PPIs), which block gastric acid secretion, selectively inhibited tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) secretion by Toll-like receptor (TLR)-activated human monocytes in vitro, in the absence of toxic effects. Remarkably, the oversecretion of IL-1ß that represents a hallmark of monocytes from patients affected by cryopyrin-associated periodic syndrome is also blocked. Based on these propaedeutic experiments, we tested the effects of high doses of PPIs in vivo in the mouse model of endotoxic shock. Our data show that a single administration of PPI protected mice from death (60% survival versus 5% of untreated mice) and decreased TNF-α and IL-1ß systemic production. PPIs were efficacious even when administered after lipopolysaccharide (LPS) injection. PPI-treated mice that survived developed a long-term cross-tolerance, becoming resistant to LPS- and zymosan-induced sepsis. In vitro, their macrophages displayed impaired TNF-α and IL-1ß to different TLR ligands. PPIs also prevented sodium thioglycollate-induced peritoneal inflammation, indicating their efficacy also in a non-infectious setting independent of TLR stimulation. Lack of toxicity and therapeutic effectiveness make PPIs promising new drugs against sepsis and other severe inflammatory conditions.

Combining self-assembling peptide gels with three-dimensional elastomer scaffolds.

Some of the problems raised by the combination of porous scaffolds and self-assembling peptide (SAP) gels as constructs for tissue engineering applications are addressed for the first time. Scaffolds of poly(ethyl acrylate) and the SAP gel RAD16-I were employed. The in situ gelation of the SAP gel inside the pores of the scaffolds was studied. The scaffold-cum-gel constructs were characterized morphologically, physicochemically and mechanically. The possibility of incorporating an active molecule (bovine serum albumin, taken here as a model molecule for others) in the gel within the scaffold's pores was assessed, and the kinetics of its release in phosphate-buffered saline was followed. Cell seeding and colonization of these constructs were preliminarily studied with L929 fibroblasts and subsequently checked with sheep adipose-tissue-derived stem cells intended for further preclinical studies. Static (conventional) and dynamically assisted seedings were compared for bare scaffolds and the scaffold-cum-gel constructs. The SAP gel inside the pores of the scaffold significantly improved the uniformity and density of cell colonization of the three-dimensional (3-D) structure. These constructs could be of use in different advanced tissue engineering applications, where, apart from a cell-friendly extracellular matrix -like aqueous environment, a larger-scale 3-D structure able to keep the cells in a specific place, give mechanical support and/or conduct spatially the tissue growth could be required.

Towards on line monitoring the evolution of the myocardium infarction scar with an implantable electrical impedance spectrum monitoring system.

The human heart tissue has a limited capacity for regeneration. Tissue and cellular therapies based on the use of stem cells may be useful alternatives to limit the size of myocardial infarction. In this paper, the preliminary results from an experimental campaign for on-line monitoring of myocardium scar infarction are presented. This study has been carried out under a research project that has as main objective the development and application of a bioactive patch implant for regeneration of myocardial infarction. Electrical Impedance Spectroscopy (EIS) has been chosen as a tissue state monitoring technique. What is presented in this communication is the first results of an implantable EIS measurement system which has been implanted in a subset of the animals corresponding to the control group, along one month. In all the animals, the myocardial infarction was induced by the ligation of the first circumflex marginal artery. In the animal group presented, the bioactive patch scaffold and the electrodes were implanted without the stem cells load. The scaffold is a piece of decellularized human pericardium, lyophilized and rehydrated with hydrogel RAD16-I. Nanogold particles were also placed near the electrodes to improve the electrode area conductivity. The results presented correspond to the subset of animals (n = 5), which had implanted the bioimpedance system monitoring the electrical impedance spectrum in vivo during 1 month. Two electrodes were connected to the bioactive patch implant. A total of 14 logarithmically spaced frequencies were measured every 5 minutes, from 100 Hz to 200 kHz. Results show a convergence of low-frequency and high frequency impedance magnitudes along the measurement period, which is coherent with the scar formation.

Influence of electrical stimulation on 3D-cultures of adipose tissue derived progenitor cells (ATDPCs) behavior.

Tissue engineering has a fundamental role in regenerative medicine. Still today, the major motivation for cardiac regeneration is to design a platform that enables the complete tissue structure and physiological function regeneration of injured myocardium areas. Although tissue engineering approaches have been generally developed for two-dimensional (2D) culture systems, three-dimensional (3D) systems are being spotlighted as the means to mimic better in vivo cellular conditions. This manuscript examines the influence of electrical stimulation on 3D cultures of adipose tissue-derived progenitor cells (ATDPCs). ATDPCs cells were encapsulated into a self-assembling peptide nanoscaffold (RAD16-I) and continuously electro stimulated during 14-20 days with 2-ms pulses of 50mV/cm at a frequency of 1 Hz. Good cellular network formation and construct diameter reduction was observed in electro stimulated samples. Importantly, the process of electro stimulation does not disrupt cell viability or connectivity. As a future outlook, differentiation studies to cardiomyocytes-like cells will be performed analyzing gene profile and protein expression.

Self-assembling peptides: from bio-inspired materials to bone regeneration.

In recent years, the development of new biomaterials with specifications for tissue and organ functional requirements-such as proper biological, structural, and biomechanical properties as well as designed control for biodegradation and therapeutic drug-release capacity-is the main aim of many academic and industrial programs. Hence, the concept of molecular self-assembly is the driving force for the development of new biomaterials that support the growth and functional differentiation of cells and tissues in a controlled manner. The discovery, properties, and development of self-assembling peptides to be used as three-dimensional (3D) scaffolds based on their similarity (in structure and mechanical features) to extracellular matrices are described. Self-assembling peptides can be used for in vitro applications for cell 3D culture as well as in vivo for tissue regeneration such as bone and optical nerve repair, as well as for drug delivery of mediators to improve therapy, as in the case of myocardial infarction. Finally, the use of self-assembling materials in combination with a bioengineering platform is proposed to assist functional bone regeneration in cases of larger bone defects, including exposed fractures due to trauma and spinal disorders dealing with high loadings, as well as replacement of big bone structures due to tumors.

Primary sequence of ionic self-assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis.

Appropriate choice of biomaterial supports is critical for the study of capillary morphogenesis in vitro as well as to support vascularization of engineered tissues in vivo. Self-assembling peptides are a class of synthetic, ionic, oligopeptides that spontaneously assemble into gels with an ECM-like microarchitecture when exposed to salt. In this paper, the ability of four different self-assembling peptide gels to promote endothelial cell adhesion and capillary morphogenesis is explored. Human umbilical vein endothelial cells (HUVECs) were cultured within ionic self-assembling peptide family members, RAD16-I ((RADA)(4)), RAD16-II ((RARADADA)(2)), KFE-8 ((FKFE)(2)), or KLD-12 ((KLDL)(3)). HUVECs suspended in RAD16-I or RAD16-II gels elongated and formed interconnected capillary-like networks resembling in vivo capillaries, while they remained round and formed clusters within KFE-8 or KLD-12 gels. As HUVECs attach to RAD16-I and RAD16-II significantly better than the other peptides, these differences appear to be explained by differences in cell adhesion. Although adhesion likely occurs via bound adhesion proteins, there appears to be no difference in protein binding to the peptides investigated. Results indicate that, although these oligopeptides have similar mechanisms of self- assembly, their primary sequence can greatly affect cell adhesion. Additionally, a subset of these biomimetic ECM-like materials support capillary morphogenesis and thus may be useful for supporting vascularization.

Fabrication of a three-dimensional nanostructured biomaterial for tissue engineering of bone.

A plasma process for the surface modification of HA powders has been developed. Acrylic acid and acrylic acid/octadiene plasma deposited films onto HA particles have demonstrated to interact with SBF allowing the calcium dissolution-precipitation mechanism. Therefore, a nanostructured composite between HA and a self-assembling peptide scaffold (RAD16-I) has been developed. The differentiation of mESC in this scaffold has been studied, in order to test the osteogenic capacity of the new composite material. We have observed that the mESC can be induced to produce Ca salts (mineralization) in a 3D-microenvironment and moreover, this activity can be enhanced by the presence of HA particles into the nanofiber scaffold.

Somatostatin receptor distribution and function in immune system.

Somatostatin and cortistatin, a recently discovered endogenous neuropeptide relative of somatostatin, have multiple modulatory effects on the immune system. The specific somatostatin receptor distribution might in part explain the heterogeneity of effects of somatostatin or its analogs on immunocytes. In fact, somatostatin receptor subtypes are differentially expressed on specific cell subsets within the organs of the immune system and the expression is dynamically regulated and seems to depend on the traffic of these cells through and within lymphoid structure and homing in tissues. Somatostatin effects on immune cells are mainly based on autocrine and paracrine modes of action. In fact, activated cells producing somatostatin (or cortistatin) may interact with other cells expressing the receptors. Here, we review the postulated modes of action of somatostatin and somatostatin-like peptides, including the currently available synthetic somatostatin analogs, in cells of the immune system. We also discuss the wide distribution of somatostatin and its specific five receptor subtypes in immune cell lines, as well as throughout animal and human lymphoid organs, in both normal and pathological conditions.

Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair.

Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix (ECM) accumulation in a three-dimensional geometry. A major challenge in choosing an appropriate scaffold for cartilage repair is the identification of a material that can simultaneously stimulate high rates of cell division and high rates of cell synthesis of phenotypically specific ECM macromolecules until repair evolves into steady-state tissue maintenance. We have devised a self-assembling peptide hydrogel scaffold for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time-dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically functional neo-tissue. Taken together, these results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like ECM within a three-dimensional cell culture for cartilage tissue repair.

A defective expression of ICAM-1 (CD54) on secretory endometrial cells is associated with endometriosis.

A defect in the cytolytic activity against autologous endometrial cells refluxed in the peritoneal cavity has been hypothesized as being involved in the etiology of endometriosis, although its causes have not been definitively identified. Cell adhesion molecules are necessary not only for cell-to-cell contact but also for the binding of immune effectors to their targets. In this study, the expression of CD54, CD58 and CD106, three adhesion molecules with a crucial role in cytotoxic mechanisms, was quantitatively studied on fresh endometrial cells by immunofluorescence and flow cytometry. Samples were collected from endometriosis patients (n=10) and controls (n=12), either during the follicular or luteal phase of the cycle. While no significant differences were observed for CD58 and CD106, a significantly reduced expression of CD54 in the secretory endometrial cells of women with endometriosis was observed (-75% with respect to apparently healthy controls). These findings could account for an apparently cyclic defect in the expression of CD54 that could result in poor binding of immune effectors to secretory endometrial cells in vivo. The defective recognition and removal of refluxed endometrial cells could, at least in part, be involved in the pathogenesis of endometriosis.

Analysis of HLA-class-I specific natural killer cell receptors expressed on T lymphocytes infiltrating non-small-cell lung cancer.

HLA-class I-specific natural killer cell receptors (HNKR) have been described to significantly interfere with both specific and non-specific functional activities of T lymphocytes. Despite the clear evidences obtained in T cells derived from peripheral blood, little is known about the activity of HNKR expressed in tumor infiltrating lymphocytes. For this reason, we have studied T lymphocytes derived from advanced non small cell lung cancers (NSCLC). The population of T cells expressing the HNKR(+) phenotype was rare both in NSCLC-associated lymphocytes and in the peripheral blood. The two populations were clearly oligoclonal, as shown by the analysis of T cell receptor repertoire. Interestingly, while HNKR(+) T cells derived from the peripheral blood belonged to the CD45R0 phenotype, the large majority of HNKR(+) T cells in TIL were CD45RA. Functionally, all HNKR(+) T cells displayed a cytolytic activity against allogeneic NSCLC. Autologous NSCLC, tested in a single patient, was lysed efficiently by HNKR(+) T cells, thus suggesting that at least in this model, the presence of HNKR did not significantly interfere with the functional capacity of effector cells.

Intensified chemotherapy supported by DMSO-free peripheral blood progenitor cells in breast cancer patients.

BACKGROUND: The majority of high-dose chemotherapy (HDC)-related complications results from bone marrow aplasia, but the graft infusion per se may cause adverse reactions due to the injection of both dimethyl sulfoxide (DMSO) and cell lysis products. We evaluated the feasibility of a two-step chemotherapy regimen with peripheral blood progenitor cell (PBPC) support in association with a novel procedure to remove DMSO and products of cell lysis from the cryopreserved cells. PATIENTS AND METHODS: Stage III and IV breast cancer patients received induction chemotherapy with three cycles of CEF (cyclophosphamide 600 mg/m2, epirubicin 100 mg/m2, 5-fluorouracil 600 mg/m2) followed by three cycles of HDC consisting of escalating doses of cyclophosphamide (dose range 1200 3000 mg/m2) and carboplatin (dose range 600-1000 mg/m2), supported by DMSO-free PBPC reinfusion. DMSO was removed by a washing/enzymatic digestion procedure. RESULTS: Twenty patients received induction chemotherapy and eighteen completed the entire chemotherapy program; a total of fifty-four cycles of HDC were administered. Dose limiting toxicity of HDC was long-lasting grade 4 neutropenia associated with documented infection. The maximum tolerated dose (MTD) was cyclophosphamide 3000 mg/m2 and carboplatin 600 mg/m2. No side effects related to PBPC reinfusion were observed. CONCLUSIONS: The proposed two-step chemotherapy regimen, associated with a novel washing/enzymatic digestion procedure, is feasible in advanced breast cancer patients in the absence of complications related to the specific toxicity of PBPC reinfusion.

HLA class I molecule expression is up-regulated during maturation of dendritic cells, protecting them from natural killer cell-mediated lysis.

It has been widely demonstrated that natural killer (NK) cells are able to discriminate between normal and abnormal cells on the basis of the interaction of their NKRs with the MHC molecules expressed on the target cells. Recent studies showed that also normal dendritic cells are susceptible to NK cell-mediated lysis. In this work, the potential relationships between the expression of different surface molecules on both immature and mature DC and susceptibility to lysis have been investigated. The reduced density of HLA class I on the surface of immature DC resulted in the only permissive signal to NK cell mediated killing. On the contrary, the remarkable increase in HLA class I molecules detected during DC maturation was strictly related to the resistance to NK cell. Contemporary, no clear evidences of a role for other surface molecules modulated during DC maturation (CD80, CD83, CD86 and CD40), were obtained.

Dendritic cells efficiently cross-prime HLA class I-restricted cytolytic T lymphocytes when pulsed with both apoptotic and necrotic cells but not with soluble cell-derived lysates.

Dendritic cells (DC) have been shown to efficiently present antigen to CD8(+) cytolytic T cells (CTL) when pulsed with apoptotic cells as a source of cell-derived antigen. Such cross-priming could not be detected by the use of necrotic cells, while conflicting results have been reported for cell-derived soluble lysates. In this study, we reinvestigated this issue by using autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) as a source of antigen to pulse monocyte-derived DC. Both autologous and HLA-mismatched allogeneic LCL have been used either in the form of apoptotic or of necrotic cells or of soluble cell lysates. At day 7, DC were co-cultured with the autologous CD8(+) lymphocyte fraction for an additional 9 days, in the presence of exogenous IL-2 (added after 48 h). At the end of the culture period, CD8(+) CTL efficiently lysed autologous LCL only when they had been co-cultured with DC pulsed with necrotic or apoptotic cells. That an efficient cross-priming of autologous CD8(+) cells could be induced by DC pulsed with apoptotic or necrotic LCL but not with cell lysates was further demonstrated in assays of IFN-gamma production in response to short-term re-stimulation of CD8(+) cells with LCL. In addition, LCL-specific CD8(+) cells could specifically lyse autologous DC that had been pulsed with LCL-derived antigens, further suggesting that DC presented exogenous antigens on HLA class I molecules.

Natural killer cells lyse autologous herpes simplex virus infected targets using cytolytic mechanisms distributed clonotypically.

Natural killer (NK) cells have the capability of lysing targets that have down-regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV-infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti-p70, anti-p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV-infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV-infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer-cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV-infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA-A and HLA-B and did not modify the expression of HLA-C, but also that NK cell clones expressing the "activating" form of the anti HLA-C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV-infected autologous target cells.

Cetirizine-induced downregulation of airway fibroblast proliferation and function: a rationale for a different approach to allergy treatment?

Recently, airway fibroblasts captured the attention of both allergists and basic scientists since they are no longer considered as mere bystanders, as far as allergic airway diseases are concerned. The aim of the present study was to assess the effects of different Cetirizine (Cet) concentrations (0.01, 0.05, 0.1 mg/ml) on human airway fibroblast proliferation and on CD54 expression. By means of flow cytometry analysis, we evaluated CD54 expression by airway fibroblasts in basal conditions or after gammaIFN stimulation in the presence of Cetirizine; we also evaluated the effect of the drug on cell proliferation by a [3H]thymidine incorporation assay. All of the tested doses of Cetirizine were able to significantly reduce CD54 upregulation induced by gammaIFN; concerning the fibroblast proliferation, we observed a dose-dependent inhibition of [3H]thymidine incorporation. These results show that Cetirizine exerts a biologic effect directly on human airway fibroblasts, suggesting a new rationale in the use of this compound.

Chitin oligosaccharides as candidate patterning agents in zebrafish embryogenesis.

In this work we investigate the possible function of N-acetyl-chitooligosaccharides (NACOs) produced during zebrafish (Danio rerio) development. First, we show that NACOs are synthesized in vivo during early embryogenesis in the zebrafish. Second, we demonstrate that injection of a pure bacterial chitinase into one-cell stage embryos elicits developmental defects in which the posterior trunk and tail of developing embryo are severely affected. In addition, an endogenous chitinase activity detected both intra- and extracellularly is described, suggesting that cells may secrete it into the extracellular space. Moreover, this compartmentalization appears to be functionally relevant as inhibition of the extracellular, but not the intracellular, endogenous chitinase activity causes morphological defects similar to those seen in embryos injected with chitinase 63. Finally, analysis of the expression of the zebrafish ZDG42 gene, which has been suggested to be involved in synthesis of NACOs, is described. Transcripts are detected from late blastula stage, during gastrulation, and move as an anterior-posterior wave of expression in adaxial mesoderm during somitogenesis.