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Development of implantable devices based on the principles of in vitro organogenesis has been hindered due to the prolonged time required to develop an implantable device. Herein we assessed the influence of serum concentration (0.5 % and 10 %), oxygen tension (0.5 %, 2 % and 20 %) and macromolecular crowding (75 μg/ml carrageenan) in extracellular matrix deposition in human corneal fibroblast culture (3, 7 and 14 days). The highest extracellular matrix deposition was observed after 14 days in culture at 0.5 % serum, 2 % oxygen tension and 75 μg/ml carrageenan. These data indicate that low oxygen tension coupled with macromolecular crowding significantly accelerate the development of scaffold-free tissue-like modules. This article is protected by copyright. All rights reserved.

The extracellular matrix (ECM) has unique biochemical, mechanical and organisational properties through which it provides a physical scaffolding for cells; a barrier that protects tissues; several signals that affect cell behaviour; and a reservoir for biologically active molecules. Considering the importance of ECM in regulating many fundamental cell processes, a myriad of strategies and materials has been developed to reproduce its properties. The first part of the chapter covers various approaches aiming to generate scaffolds whose fibre size, orientation and stiffness could mimic the ECM nanofibrous structure. In particular, the use of natural fibrous proteins, the application of electrospinning and freeze-drying and examples of tissue engineering applications are presented. The second part discusses strategies aiming to address the ECM ligand-binding function and to reproduce the dynamic, reciprocal, dialogue between cells and their microenvironment; examples of 3D scaffolds for controlled release of growth factors, drugs and genetic material are reported. Researchers have also used native ECM components to recapitulate the biochemical and biophysical properties of ECM. In the third part of the chapter, the use of fibrinogen and fibrin is presented as an example of natural scaffolds recapitulating ECM functions. Fibrinogen and fibrin can be used as provisional matrix in regenerating tissues; moreover, by varying the fabrication method and by blending them with other materials, it is possible to produce biodegradable scaffolds with reasonable control of degradation rate and drug release.

Self-assembling biomaterials offer an unprecedented chance of successfully facing most of the challenges of various biomedical fields, and, in particular, of tissue engineering. Nonetheless co-assembling peptides (CAPs), taking advantage of the theory and empirical findings developed for self-assembling peptides, could provide an even better control over cell cultures, drug delivery, and transplantation therapies. This study follows a “full” bottom-up approach to develop new CAPs for neural tissue engineering applications. After molecular aggregation analysis via coarse-grained simulations, LKLK12, LDLD12, and the functionalized KLPGWSG-LDLD12 CAPs are synthesized and characterized assessing their co-assembled secondary structures, the biomechanical properties of the obtained hydrogels, and the morphological features of the assembled nanofibers. The biological influence on viability and differentiation of human and murine neural stem cells are tested in vitro and neuroregenerative potentials in complete spinal cord transections are verified in vivo. Upon mixing of CAPs, the spontaneous formation of double layers of β-sheets with a high degree of integration of the two CAP species is demonstrated. The formation of entangled nanofibrous structures give rise to shear-thinning hydrogels. The in vitro results are comparable to a standard state-of-the-art cell culture substrate and nervous regeneration in vivo is enhanced.

Spinal cord injury presents a significant therapeutic challenge since the treatments available are mostly vain. The use of stem cells to treat this condition represents a promising new therapeutic strategy; therefore, a variety of stem cell treatments have been recently examined in animal models of CNS trauma. In this work, we analyzed the effects of third trimester amniotic fluid cells in a mouse model of spinal cord injury. Among the different cultures used for transplantation, some were able to induce a significant improvement in motor recovery (cultures #3.5, #3.6 and #7.30), evaluated by means of open field free locomotion. All effective cell cultures expressed the surface marker nerve/glial antigen 2, ortholog of the human chondroitin sulfate proteoglycan 4, which is present on several types of immature progenitor cells. The improved motor functional recovery was correlated with higher myelin preservation in the ventral horn white matter and an increased vascularization in the peri-lesion area. Real-Time PCR analysis showed higher expression levels of vascular endothelial growth factor and hypoxia-inducible factor-1α mRNA two days after cells transplantation compared to PBS-treated animals, indicating that an angiogenic pathway might have been activated by these cells, possibly through the production of hepatocyte growth factor. This cytokine appears to be produced mostly in filtering organs, such as the lung, of the transplanted animals and is likely released in the blood suggesting an endocrine role of hepatocyte growth factor in targeting the injury site.

Self-assembling peptides are promising biomaterials for spinal cord repair as they can easily be injected into the lesion site and can provide physical support to regrowing nervous tissue. However, to improve upon the design of synthetic scaffolds for spinal cord injury, characteristics of the scaffold/host relationship need to be further investigated. In the current study we aimed to evaluate both the mechanical properties and the therapeutic effect of two self-assembling peptides B24 and biotin-LDLK12 in spinal cord injury. Atomic force microscopy and rheology were used to characterise various concentrations of the two peptides in terms of the propensity to form nanostructures and the viscoelastic properties. Concurrently, these peptide solutions were injected into the contused spinal cord of rats to evaluate both diffusibility within the tissue, and scaffold formation in vivo. After selection of the best concentration for delivery in vivo, the two self-assembling peptides were tested in the contused spinal cord of rats for their influence on hematoma and cyst formation, biocompatibility and permissiveness for axonal growth. The results suggest that rheology can provide a useful indication to predict the hydrogel formation and diffusibility of the self-assembling peptides in vivo. Moreover at three days post-injury both self-assembling peptides had a good hemostatic effect and at 28 days they improved axon regrowth. In summary, the injectable self-assembling hydrogels could attenuate hematoma and provide a therapeutic effect in a spinal cord injury model.

Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes.

A key challenge of in vitro organogenesis is the development in timely manner tissue equivalents. Herein, we assessed the simultaneous effect of oxygen tension (0.5%, 2% and 20%), foetal bovine serum concentration (0.5% and 10%) and macromolecular crowding (75 μg/ml carrageenan) in human dermal fibroblast culture. Our data demonstrate that cells cultured at 2% oxygen tension, in the presence of carrageenan and at 0.5% serum concentration deposited within 3 days in culture more extracellular matrix than cells grown for 14 days, at 20% oxygen tension, 10% serum concentration and in the absence of carrageenan. These data suggest that optimal oxygen tension coupled with macromolecular crowding are important in vitro microenvironment modulators for accelerated development of tissue-like modules in vitro. Statement of Significance To enable clinical translation and commercialisation of in vitro organogenesis therapies, we cultured human dermal fibroblast at 2% oxygen tension, under macromolecular crowding conditions (75 μg/ml carrageenan) and at low foetal bovine serum concentration (0.5%). Within 3 days in culture, more extracellular matrix was deposited under these conditions than cells grown for 14 days, at 20% oxygen tension, 10% FBS concentration and in the absence of crowding agents. These data bring us closer to the development of more clinically relevant tissue-like modules.

Development of implantable devices based on the principles of in vitro organogenesis has been hindered due to the prolonged time required to develop an implantable device. Herein we assessed the influence of serum concentration (0.5% and 10%), oxygen tension (0.5%, 2% and 20%) and macromolecular crowding (75 μg/ml carrageenan) in extracellular matrix deposition in human corneal fibroblast culture (3, 7 and 14 days). The highest extracellular matrix deposition was observed after 14 days in culture at 0.5% serum, 2% oxygen tension and 75 μg/ml carrageenan. These data indicate that low oxygen tension coupled with macromolecular crowding significantly accelerate the development of scaffold-free tissue-like modules. Copyright © 2016 John WileySons, Ltd.

Aim: We carried out an association study between T-cell receptor beta polymorphism (TCRB) and endometriosis to investigate the difference in allelic frequency. Polymorphisms in T-cell receptor genes can provide important information for the study of the immune response and autoimmune diseases; indeed, rs1800907, a very common single nucleotide polymorphism (SNP) of the TCRB, has been extensively studied in autoimmune diseases in the 1990s using Southern blot analysis and more recently polymerase chain reaction (PCR) and sequencing. An autoimmune etiology for endometriosis has been strongly suggested for the presence of antibodies against endometrium, high rates of autoimmune disorders and associated atopic diseases. Material and Methods: We investigated 70 patients with endometriosis and 120 controls. DNA of patients and controls was studied by PCR followed by restriction digestion and sequencing to determine genotype and presence of linkage disequilibrium (LD). Statistical analysis was carried out using STATA Routine GENHW (StataCorp, College Station, TX, USA) for estimation of Hardy–Weinberg equilibrium and test power calculation. The difference of allele distribution between patients and controls was calculated according to Pearson's and Fisher's tests. Test power for the estimation of linkage disequilibrium is low (0.16). Results: We performed an association study of the SNP rs1800907 of TCRB between 70 patients with endometriosis and 120 controls, and did not find any significant difference (χ2 = 0.27 and P = 0.87). Fisher's test confirmed a P-value of 0.872. Conclusion: Our study does not suggest an evidential and major involvement of TCRB in the pathogenesis of endometriosis in an Italian population in a small case control study.

Tissue engineering by self-assembly hypothesises that optimal repair and regeneration can be achieved best by using the cells’ inherent ability to create organs with proficiency still unmatched by currently available scaffold fabrication technologies. However, the prolonged culture time required to develop an implantable device jeopardises clinical translation and commercialisation of such techniques. Herein, we report that macromolecular crowding, a biophysical in vitro microenvironment modulator, dramatically accelerates extracellular matrix deposition in cultured human corneal, lung and dermal fibroblasts and human bone marrow mesenchymal stem cells. In fact, an almost 5 to 30 fold increase in collagen type I deposition was recorded as early as 48 hours in culture, without any negative effect in cell phenotype and function.

Traditional ex vivo culture setups fail to imitate the native tissue niche, leading to cellular senescence, phenotypic drift, growth arrest and loss of stem cell multipotency. Growing evidence suggests that surface topography, substrate stiffness, mechanical stimulation, oxygen tension and localised density influence cellular functions and longevity, enhance tissue-specific extracellular matrix deposition and direct stem cell differentiation. In this review, we discuss how these cues will facilitate engineering of physiological in vitro microenvironments to enable clinical translation of cell based therapies and development of in vitro models for drug discovery applications.

Nanostructured scaffolds recently showed great promise in tissue engineering: nanomaterials can be tailored at the molecular level and scaffold morphology may more closely resemble features of extracellular matrix components in terms of porosity, framing and biofunctionalities. As a consequence, both biomechanical properties of scaffold microenvironments and biomaterial–protein interactions can be tuned, allowing for improved transplanted cell engraftment and better controlled diffusion of drugs. Easier said than done, a nanotech-based regenerative approach encompasses different fields of know-how, ranging from in silico simulations, nanomaterial synthesis and characterization at the nano-, micro- and mesoscales to random library screening methods (e.g. phage display), in vitro cellular-based experiments and validation in animal models of the target injury. All of these steps of the “assembly line” of nanostructured scaffolds are tightly interconnected both in their standard analysis techniques and in their most recent breakthroughs: indeed their efforts have to jointly provide the deepest possible analyses of the diverse facets of the challenging field of neural tissue engineering. The purpose of this review is therefore to provide a critical overview of the recent advances in and drawbacks and potential of each mentioned field, contributing to the realization of effective nanotech-based therapies for the regeneration of peripheral nerve transections, spinal cord injuries and brain traumatic injuries. Far from being the ultimate overview of such a number of topics, the reader will acknowledge the intrinsic complexity of the goal of nanotech tissue engineering for a conscious approach to the development of a regenerative therapy and, by deciphering the thread connecting all steps of the research, will gain the necessary view of its tremendous potential if each piece of stone is correctly placed to work synergically in this impressive mosaic.

Developing functionalized biomaterials for enhancing transplanted cell engraftment in vivo and stimulating the regeneration of injured tissues requires a multi-disciplinary approach customized for the tissue to be regenerated. In particular, nervous tissue engineering may take a great advantage from the discovery of novel functional motifs fostering transplanted stem cell engraftment and nervous fiber regeneration. Using phage display technology we have discovered new peptide sequences that bind to murine neural stem cell (NSC)-derived neural precursor cells (NPCs), and promote their viability and differentiation in vitro when linked to LDLK12 self-assembling peptide (SAPeptide). We characterized the newly functionalized LDLK12 SAPeptides via atomic force microscopy, circular dichroism and rheology, obtaining nanostructured hydrogels that support human and murine NSC proliferation and differentiation in vitro. One functionalized SAPeptide (Ac-FAQ), showing the highest stem cell viability and neural differentiation in vitro, was finally tested in acute contusive spinal cord injury in rats, where it fostered nervous tissue regrowth and improved locomotor recovery. Interestingly, animals treated with the non-functionalized LDLK12 had an axon sprouting/regeneration intermediate between Ac-FAQ-treated animals and controls. These results suggest that hydrogels functionalized with phage-derived peptides may constitute promising biomimetic scaffolds for in vitro NSC differentiation, as well as regenerative therapy of the injured nervous system. Moreover, this multi-disciplinary approach can be used to customize SAPeptides for other specific tissue engineering applications. © 2012 The Royal Society of Chemistry.

PURPOSE Our aim was the search for new sources of cells potentially useful for central nervous system regenerative medicine. Extra-embryonic tissues are promising sources of pluripotent stem cells. Among these, human second-trimester amniotic fluid (AF) contains cell populations exhibiting self-renewal capacity, multipotency and the expression of embryonic cell markers. METHODS Here we report the properties of the easily available third-trimester AF cells (AFCs). Different cell types from 6 of 9 AF samples were separated, expanded, and characterized by assessing their morphological, proliferative, and differentiative properties. RESULTS All isolated cultures presented CD105, CD90 and CD73 mesenchymal markers, whereas they differed among themselves in CD117, CD146, CD31, NG2 and CD133 expression. Their doubling time and telomere length were conserved throughout many passages. Importantly, immunofluorescence and Real-time PCR showed that, during their proliferative state and differentiation, several cultures expressed neuronal and glial markers such as nestin, GFAP, β-tubulin III and neurofilament H indicating their potential attitude towards a neural fate. Indeed, these cells showed a rather poor capacity to differentiate in adipogenic and osteogenic lineages. CONCLUSIONS In this work we report that cells with neural differentiation capability can be isolated from third-trimester AF, such properties could be useful for neuro-regenerative purposes.

The purpose of this study was to determine the fate and the effects of undifferentiated embryonic stem cells (ESCs) in mice after contusive lesion of the spinal cord (SCI). Reproducible traumatic lesion to the cord was performed at T8 level by means of the Infinite Horizon Device, and was followed by intravenous injection of one million of undifferentiated ESCs through the tail vein within 2 h from the lesion. The ESCs-treated animals showed a significant improvement of the recovery of motor function 28 days after lesion, with an average score of 4.61 ± 0.13 points of the Basso Mouse Scale (n = 14), when compared to the average score of vehicle treated mice, 3.58 ± 0.23 (n = 10). The number of identified ESCs found at the lesion site was 0.6% of the injected cells at 1 week after transplantation, and further reduced to 0.04% at 1 month. It is, thus, apparent that the promoted hind-limb recovery cannot be correlated to a substitution of the lost tissue performed by the exogenous ESC. The extensive evaluation of production of several neuroprotective and inflammatory cytokines did not reveal any effect by ESC-treatment, but unexpectedly the number of invading macrophages and neutrophils was greatly reduced. This may explain the improved preservation of lesion site ventral myelin, at both 1 week (29 ± 11%) and 1 month (106 ± 14%) after injury. No teratoma formation was observed, although an inappropriate colonization of the sacral cord by differentiated nestin- and β-tubulin III-positive ESCs was detected.

We searched for a difference in allele distribution between males and females of a single nucleotide polymorphism located in the human beta T-cell receptor, in 500 subjects (200 males and 300 females). Genotype analysis gave the following results: among the males, 114 (57%) were heterozygous for the T/C polymorphism, 52 (26%) were homozygous (T/T), and 34 (17%) were homozygous (C/C). Among the females, 142 (47.3%) were heterozygous, 73 (24.3%) were homozygous (T/T), and 85 (28.3%) were homozygous (C/C). The allele frequency was significantly different between sexes (chi2 = 8.799, P = 0.012).

We studied monosomy and deletions of chromosome 7 in 170 patients with myeloid disorders and we identified a minimal region of loss in 7q31.1 spanning between the D7S2554 and D7S2460 markers. The closest gene to our most deleted microsatellite, D7S2554, is the human I-mfa domain containing (HIC) gene, alias MyoD family inhibitor domain containing (MDFIC). We investigated the involvement of HIC in myeloid neoplasms by screening for mutations the coding regions and the intron–exon boundaries of this gene in 15 patients who presented chromosome 7 deletions in the region of HIC. No mutations were found in the coding region of this gene.

We studied deletion and monosomy of chromosome 7 in 150 patients with myeloproliferative diseases. We found 8/150 patients with monosomy 7 by cytogenetics and 4/150 with deletions of the long arm of chromosome 7 by restriction fragment length polymorphism (RFLP) analysis performed with Southern and polymerase chain reaction. To overcome limitation of RFLP analysis, we restricted loss of heterozygosity study with microsatellites to 45 patients, observing deletion 7q31.1 in 7/45 patients. In all patients with molecular alterations the deletion was observed only in myeloid cells, while the monosomy was detected in both myeloid precursor and lymphocytes. This finding suggests a CD34-totipotent stem cell origin for the monosomy and a colony forming unit – granulocyte, erytrocyte, monocyte, megakaryocytes (CFU–GEMM) stem cell origin for the deletions.