In JoVE (1)

Other Publications (29)

Articles by Delfo D'Alessandro in JoVE

 JoVE Bioengineering

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

1Dept. of Clinical and Experimental Medicine, University of Pisa, 2OtoLab, Azienda Ospedaliero-Universitaria Pisana (AOUP), 3Dept. of Civil and Industrial Engineering, University of Pisa, 4Immunohematology Operative Unit, Azienda Ospedaliero-Universitaria Pisana (AOUP), 5Dept. Of Surgical, Medical, Molecular Pathology and Emergency Medicine, University of Pisa, 6II Orthopedic and Traumatologic Clinic, Azienda Ospedaliero-Universitaria Pisana (AOUP)


JoVE 54845

Other articles by Delfo D'Alessandro on PubMed

Cellular and Subcellular Localization of the Small G Protein RhoA in the Human and Rat Embryonic and Adult Kidney

Acta Histochemica. 2003  |  Pubmed ID: 12666992

Rho proteins, a subgroup of the Ras GTPase superfamily, control many cellular processes and morphogenetic events by acting as signaling molecules in the transduction pathways of various receptors. Among the "Rho-dependent" receptors are the extracellular matrix- and growth factor-binding sites; these are particularly involved in the modulation of renal development since they control the epithelial-mesenchymal interactions that drive kidney organogenesis. The present study has addressed the immunohistochemical localization of RhoA in developing and adult kidneys of rats and humans because: a) Rho proteins are known to have a morphogenetic role, b) data in the literature on expression of Rho GTPases during mammalian histogenesis and organogenesis are scarce, and c) their involvement in the transduction pathways of receptors is implicated in kidney development. In particular, RhoA peptide was found to be localized in the mesonephric duct and vesicles in both rats and humans; metanephric anlagen were mainly stained in ampullar-derived cells. Periglomerular tubules of fetal and adult kidneys as well as collecting ducts of adult kidneys showed intense staining. Therefore, the present study provides new information on the distribution patterns of RhoA during early stages of mammalian kidney development suggesting that this signaling molecule may take part in epithelial-mesenchymal induction processes that control kidney organogenesis. RhoA expression in adult structures may be linked with renewal of renal epithelial cells and the maintenance of their morphology and polarity.

Carboxy-terminal Fragment of Osteogenic Growth Peptide Regulates Myeloid Differentiation Through RhoA

Journal of Cellular Biochemistry. Dec, 2004  |  Pubmed ID: 15486974

The carboxy-terminal fragment of osteogenic growth peptide, OGP(10-14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10-14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10-14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP-binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM-CSF had a comparable OGP(10-14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10-14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out.

Kidney Expression of RhoA, TGF-beta1, and Fibronectin in Human IgA Nephropathy

Nephron. Experimental Nephrology. 2005  |  Pubmed ID: 15925904

The Rho/transforming growth factor-beta (TGF-beta) system plays a crucial role in the progression of renal damage due to stimulation of extracellular matrix molecule deposition. In fact, the in vitro TGF-beta-mediated production of fibronectin, one of the major TGF-beta-regulated extracellular components, has recently been correlated with Rho protein signalling molecules. Although a close relationship between increased renal tissue levels of TGF-beta1 and fibronectin has been reported in IgA nephropathy, no data are available on renal tissue expression of Rho proteins.

Immediate Structural Changes of Porcine Renal Arteries After Angioplasty: a Histological and Morphometric Study

Micron (Oxford, England : 1993). 2006  |  Pubmed ID: 16361101

The aim of this research was to characterize the immediate alterations induced by angioplasty and to compare the results of the application of two types of balloons. Ten porcine renal arteries were dilated with a compliant balloon, and ten with a non-compliant balloon. After angioplastic treatment arterial specimens were wax embedded for light microscopy. Sections were stained with the orcein-Van Gieson method, orcein, haematoxylin-eosin, and PAS. Image analysis was performed taking into consideration the following parameters: thickness of the entire wall, of the tunica media and of the inner elastic lamina. The major axes of the smooth muscle cells nuclei were also measured. The effects of the two types of balloon resulted in changes consisting in thinning of the entire arterial wall, reduction of the tunica media, distension of reticular fibers, presence of wide spaces between smooth muscle cells, stretching of smooth muscle cells, inner elastic lamina thickening. Both angioplasty devices used can modify the vascular wall. The identification of the tunica media structural damages might be useful in order to estimate the behavior of the vascular wall in the follow-up after angioplasty, because the entity of modifications could be predictive of restenosis that often takes place weeks or months after angioplasty.

Interaction of Human Gingival Fibroblasts with PVA/gelatine Sponges

Micron (Oxford, England : 1993). Jul, 2008  |  Pubmed ID: 17702585

Tissue engineering scaffolds should be able to reproduce optimal microenvironments in order to support cell attachment, three-dimensional growth, migration and, regarding fibroblasts, must also promote extracellular matrix production. Various bioactive molecules are employed in the preparation of spongy scaffolds to obtain biomimetic matrices by either surface-coating or introducing them into the bulk composition of the biomaterial. The biomimetic properties of a spongy matrix composed of PVA combined with the natural component gelatine were evaluated by culturing human gingival fibroblasts on the scaffold. Cell adhesion, morphology and distribution within the scaffold were assessed by histology and electron microscopy; viability and metabolic activity as well as extracellular matrix production were analyzed by MTT assay, cytochemistry and immunocytochemistry. Fibroblasts interacted positively with PVA/gelatine. They adhered to the PVA/gelatine matrix in which they had good spreading activity and active metabolism; fibroblasts were also able to produce extracellular matrix molecules (type I collagen, fibronectin and laminin) compared to bi-dimensionally grown cells. The in situ creation of a biological matrix by human fibroblasts together with the ability to produce growth factor TGF-beta1 and the intracellular signal transduction molecule RhoA, suggests that this kind of PVA/gelatine sponge may represent a suitable support for in vitro extracellular matrix production and connective tissue regeneration.

Human Autologous Plasma-derived Clot As a Biological Scaffold for Mesenchymal Stem Cells in Treatment of Orthopedic Healing

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Feb, 2008  |  Pubmed ID: 17868116

Recent advances in the isolation, expansion, and characterization of human mesenchymal stem cells (hMSCs) have raised the possibility of using them in cell therapies and tissue engineering for bone reconstruction. hMSCs, isolated from the bone marrow of eight normal adult patients, were minimally expanded ex vivo and pulsed twice toward osteogenic lineage. The cells were then included into autologous plasma-derived clots. Cytofluorimetric analysis, immunocytochemistry (osteopontin), histochemistry (alkaline phosphatase, Alcian blue, Von Kossa, and alizarin red staining), and viable/proliferation tests were performed to study both stem and differentiating cells. Although two short inductions increased osteogenic markers in hMSCs, inside the clot the cells were able to terminally differentiate into osteoblasts. Moreover, we show that the clot is able to sustain cell proliferation under appropriate cell culture conditions. Our results suggested that clot could be useful for hMSC delivery into the site of the lesion to promote bone formation. Moreover, the plasticity of this material allowed good in vitro hMSC spreading and proliferation. The advantages of using this autologous biological material are its biocompatibility and reabsorption; furthermore, using a gel as scaffold, it is possible to mold it to the shape of a bone cavity.

Gelatin/PLLA Sponge-like Scaffolds Allow Proliferation and Osteogenic Differentiation of Human Mesenchymal Stromal Cells

Macromolecular Bioscience. Sep, 2008  |  Pubmed ID: 18504804

Tissue engineering has the potential to supply constructs capable of restoring the normal function of native tissue following injury. Poly(L-lactic acid) (PLLA) scaffolds are amongst the most commonly used biodegradable polymers in tissue engineering and previous studies performed on ovine fibroblasts have showed that addition of gelatin creates a favorable hydrophilic microenvironment for the growth of these cells. The attractiveness of using mesenchymal stromal cells (MSCs) in tissue regeneration is that they are able to differentiate into several lines including osteoblasts. In this study, we investigated the ability of gelatin/PLLA sponges to support the adhesion, proliferation, and osteogenic differentiation of human MSCs isolated from the bone marrow of four donors. [Figure: see text].

Good Manufacturing Practice-grade Fibrin Gel is Useful As a Scaffold for Human Mesenchymal Stromal Cells and Supports in Vitro Osteogenic Differentiation

Transfusion. Oct, 2008  |  Pubmed ID: 18657082

Recently, there has been an increased interest in using mesenchymal stromal cells (MSCs) in bone tissue engineering coupled with a suitable scaffold of both biological and synthetic origin. The cells and these constructs can be combined in vitro or directly in vivo to enhance tissue repair. MSCs are spindle-shaped cells capable of self-renewal and can be induced to differentiate mainly into osteo-, chondro-, and adipogenic-progeny types. Several biomaterials are currently available and, among them, fibrin-based constructs seem to be suitable for guiding the cells during tissue repair or regeneration due to their biocompatibility and biodegradability.

Morpho-functional Characterization of Human Mesenchymal Stem Cells from Umbilical Cord Blood for Potential Uses in Regenerative Medicine

Stem Cells and Development. Mar, 2009  |  Pubmed ID: 18444788

Mesenchymal stem cells (MSCs) represent a promising source of progenitor cells having the potential to repair and to regenerate diseased or damaged skeletal tissues. Bone marrow (BM) has been the first source reported to contain MSCs. However, BM-derived cells are not always acceptable, due to the highly invasive drawing and the decline in MSC number and differentiative capability with increasing age. Human umbilical cord blood (UCB), obtainable by donation with a noninvasive method, has been introduced as an alternative source of MSCs. Here human UCB-derived MSCs isolation and morpho-functional characterization are reported. Human UCB-derived mononuclear cells, obtained by negative immunoselection, exhibited either an osteoclast-like or a mesenchymal-like phenotype. However, we were able to obtain homogeneous populations of MSCs that displayed a fibroblast-like morphology, expressed mesenchym-related antigens and showed differentiative capacities along osteoblastic and early chondroblastic lineages. Furthermore, this study is one among a few papers investigating human UCB-derived MSC growth and differentiation on three-dimensional scaffolds focusing on their potential applications in regenerative medicine and tissue engineering. UCB-derived MSCs were proved to grow on biodegradable microfiber meshes; additionally, they were able to differentiate toward mature osteoblasts when cultured inside human plasma clots, suggesting their potential application in orthopedic surgery.

Novel Biological/biohybrid Prostheses for the Ossicular Chain: Fabrication Feasibility and Preliminary Functional Characterization

Biomedical Microdevices. Aug, 2009  |  Pubmed ID: 19294514

Alternatives for ossicular replacements were fabricated in order to overcome persisting rejections in middle ear prosthetization. Unlike the synthetic prostheses in fashion, we propose biological and biohybrid replacements containing extra cellular matrix (ECM) molecules to improve biointegration. In this study, ECM-containing devices shaped as Partial Ossicular Replacement Prostheses (PORPs) were fabricated reproducing the current synthetic models. Biological PORPs were obtained from human decellularized cortical bone allografts by computer numerically controlled ultraprecision micromilling. Moreover, porous PORP-like scaffolds were produced and cultured with osteoinduced human mesenchymal stromal cells to generate in vitro bone ECM within the scaffold porosity (biohybrid PORPs). The acoustic responses of such devices were investigated and compared to those of commercial prostheses. Results showed that biological PORPs transmit mechanical signals with appropriate frequencies, amplitudes, and with early extinction time. Although signal transmission in biohybrid PORPs showed insufficient amplitude, we believe that tissue engineered constructs represent the new challenge in ossiculoplasty.

Development of Tissue-engineered Substitutes of the Ear Ossicles: PORP-shaped Poly(propylene Fumarate)-based Scaffolds Cultured with Human Mesenchymal Stromal Cells

Journal of Biomedical Materials Research. Part A. Mar, 2010  |  Pubmed ID: 19353559

This is a novel study aimed at exploring possible tissue engineering (TE) options for fabricating middle ear ossicle replacements. Alternatives to prosthetic replacements currently used in ossiculoplasty are desirable, considering that current devices are known to suffer from a persistent rejection phenomenon, known as extrusion. In this study a biocompatible and biodegradable polymer, poly(propylene fumarate)/poly(propylene fumarate)-diacrylate (PPF/PPF-DA), was chosen to assess the fabrication feasibility of highly porous devices shaped as partial ossicular replacement prostheses (PORPs). PORP-like scaffolds were produced, and their poral features (porosity and pore interconnectivity) were evaluated via micro-CT. In addition, their capability to support human mesenchymal stromal cell (hMSC) colonization and osteoblastic differentiation in vitro was investigated with both quantitative and qualitative analyses. This report summarizes and discusses all the fundamental issues associated with ossicle prosthetization as well as the challenging opportunities potentially offered to middle ear reconstruction by TE; moreover it demonstrates that PPF/PPF-DA PORP-like scaffolds can be appropriately fabricated to allow both the colonization of hMSCs and their osteoblastic maturation in vitro. Specifically, the expression patterns of the main osteogenic markers (alkaline phosphatase, calcium) and of various matrix biomolecules (glycoproteins, glycosaminoglycans, collagen I) were studied. These preliminarily obtained outcomes may launch a new trend in otology dedicated to TE ossicle development to improve on the performance of current prosthetic replacements.

Assessing Cytotoxicity of Boron Nitride Nanotubes: Interference with the MTT Assay

Biochemical and Biophysical Research Communications. Apr, 2010  |  Pubmed ID: 20226164

Thanks to a non-covalent wrapping with glycol-chitosan, highly biocompatible and highly concentrated dispersions of boron nitride nanotubes were obtained and tested on human neuroblastoma cells. A systematic investigation of the cytotoxicity of these nanovectors with several complementary qualitative and quantitative assays allowed a strong interference with the MTT metabolic assay to be highlighted, similar to a phenomenon already observed for carbon nanotubes, that would wrongly suggest toxicity of boron nitride nanotubes. These results confirm the high complexity of these new nanomaterials, and the needing of extensive investigations on their exciting potential applications in the biomedical field.

Barium Titanate Nanoparticles: Highly Cytocompatible Dispersions in Glycol-chitosan and Doxorubicin Complexes for Cancer Therapy

Nanoscale Research Letters. 2010  |  Pubmed ID: 20596329

In the latest years, innovative nanomaterials have attracted a dramatic and exponentially increasing interest, in particular for their potential applications in the biomedical field. In this paper, we reported our findings on the cytocompatibility of barium titanate nanoparticles (BTNPs), an extremely interesting ceramic material. A rational and systematic study of BTNP cytocompatibility was performed, using a dispersion method based on a non-covalent binding to glycol-chitosan, which demonstrated the optimal cytocompatibility of this nanomaterial even at high concentration (100 μg/ml). Moreover, we showed that the efficiency of doxorubicin, a widely used chemotherapy drug, is highly enhanced following the complexation with BTNPs. Our results suggest that innovative ceramic nanomaterials such as BTNPs can be realistically exploited as alternative cellular nanovectors.

Enhancement of Neurite Outgrowth in Neuronal-like Cells Following Boron Nitride Nanotube-mediated Stimulation

ACS Nano. Oct, 2010  |  Pubmed ID: 20925390

In this paper, we propose an absolutely innovative technique for the electrical stimulation of cells, based on piezoelectric nanoparticles. Ultrasounds are used to impart mechanical stress to boron nitride nanotubes incubated with neuronal-like PC12 cells. By virtue of their piezoelectric properties, these nanotubes can polarize and convey electrical stimuli to the cells. PC12 stimulated with the present method exhibit neurite sprout 30% greater than the control cultures after 9 days of treatment.

Human Dental Pulp Stem Cells Protect Mouse Dopaminergic Neurons Against MPP+ or Rotenone

Brain Research. Jan, 2011  |  Pubmed ID: 20854799

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive death of substantia nigra dopaminergic neurons that results in a regional loss of striatal dopamine (DA) levels. Dental pulp contains ex vivo-expandable cells called dental pulp stem cells (DPSCs), with the capacity to differentiate into multiple cell lineages. More interestingly, due to their embryonic origin, DPSCs express neurotrophic factors such as brain-derived neurotrophic factor, nerve growth factor and glial cell-derived neurotrophic factor. The aim of the present study was to investigate the neuroprotective effects of DPSCs against MPP+ (2.5, 5, and 10 μM) and rotenone (0.25, 0.5 and 1 μM) in an in vitro model of PD, using an indirect co-culture system with mesencephalic cell cultures. When mesencephalic cultures were challenged with MPP+ or rotenone, in the presence of DPSCs a statistically significant protective effect was observed at all the tested doses in terms of DA uptake. DPSCs protective effect on DA neurons was also confirmed by immunocytochemistry: an increased number of spared tyrosine hydroxylase (TH)+ cells was observed in co-culture conditions compared to controls, and neurons showed longer processes in comparison with mesencephalic cells grown without DPSCs. In conclusion, the co-culture with DPSCs significantly attenuated MPP+ or rotenone-induced toxicity in primary cultures of mesencephalic neurons. Considering that the direct contact between the two cell types was prevented, it can be speculated that neuroprotection could be due to soluble factors such as BDNF and NGF, released by DPSCs. Blocking BDNF and NGF with neutralizing antibodies, the neuroprotecting effect of DPSCs was completely abolished. Therefore DPSCs can be viewed as possible candidates for studies on cell-based therapy in neurodegenerative disorders.

Good Manufacturing Practices--grade Preformed Ossicular Prostheses from Banked Bone Via Computer Numerically Controlled Micromilling

The Annals of Otology, Rhinology, and Laryngology. Jan, 2011  |  Pubmed ID: 21370675

The aim of this study was the fabrication of ossicular replacement prostheses (ORPs) from decellularized banked cortical bone via computer numerically controlled (CNC) ultraprecision micromilling, in order to obtain preformed clinical-grade tissue products, reproducing shape, size, and details perfectly comparable to those of synthetic devices.

Pilot in Vivo Toxicological Investigation of Boron Nitride Nanotubes

International Journal of Nanomedicine. 2012  |  Pubmed ID: 22275819

Boron nitride nanotubes (BNNTs) have attracted huge attention in many different research fields thanks to their outstanding chemical and physical properties. During recent years, our group has pioneered the use of BNNTs for biomedical applications, first of all assessing their in vitro cytocompatibility on many different cell lines. At this point, in vivo investigations are necessary before proceeding toward realistic developments of the proposed applications. In this communication, we report a pilot toxicological study of BNNTs in rabbits. Animals were injected with a 1 mg/kg BNNT solution and blood tests were performed up to 72 hours after injection. The analyses aimed at evaluating any acute alteration of hematic parameters that could represent evidence of functional impairment in blood, liver, and kidneys. Even if preliminary, the data are highly promising, as they showed no adverse effects on all the evaluated parameters, and therefore suggest the possibility of the realistic application of BNNTs in the biomedical field.

Transferrin-conjugated Boron Nitride Nanotubes: Protein Grafting, Characterization, and Interaction with Human Endothelial Cells

International Journal of Pharmaceutics. Oct, 2012  |  Pubmed ID: 22732669

In this paper we report on a covalent grafting of boron nitride nanotubes with human transferrin. After silanization of the nanotube wall, transferrin was linked to the nanotubes through carbamide binding. The obtained transferrin-conjugated boron nitride nanotubes (tf-BNNTs) resulted stable in aqueous environments and were characterized in terms of scanning electron microscopy, transmission electron microscopy, size distribution analysis and Z-potential measurement. Effective covalent grafting of transferrin was demonstrated by Fourier transform infrared spectroscopy and UV-Vis spectrophotometry. The obtained tf-BNNTs were thereafter tested on human umbilical vein endothelial cells (HUVECs); in particular cellular up-take was investigated by confocal, scanning and transmission electron microscopy, demonstrating the key role of transferrin during the internalization process. Here reported for the first time in the literature, the covalent BNNT functionalization with a targeting ligand represents a fundamental step towards BNNT exploitation as smart and selective nanocarriers in a number of nanomedicine applications.

Histologic Characterization of Human Ear Ossicles for the Development of Tissue-engineered Replacements

Otology & Neurotology : Official Publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology. Oct, 2012  |  Pubmed ID: 22975908

Precise knowledge of the expression and distribution of extracellular matrix (ECM) molecules and osteochondrogenic markers helps target the proper in vitro regeneration of novel ossicular chain (OC) replacements via tissue engineering (TE).

Effects of Barium Titanate Nanoparticles on Proliferation and Differentiation of Rat Mesenchymal Stem Cells

Colloids and Surfaces. B, Biointerfaces. Feb, 2013  |  Pubmed ID: 23006571

Nanomaterials hold great promise in the manipulation and treatments of mesenchymal stem cells, since they allow the modulation of their properties and differentiation. However, systematic studies have to be carried out in order to assess their potential toxicological effects. The present study reports on biocompatibility evaluation of glycol-chitosan coated barium titanate nanoparticles (BTNPs) on rat mesenchymal stem cells (MSCs). BTNPs are a class of ceramic systems which possess interesting features for biological applications thanks to their peculiar dielectric and piezoelectric properties. Viability was evaluated up to 5 days of incubation (concentrations in the range 0-100 μg/ml) both quantitatively and qualitatively with specific assays. Interactions cells/nanoparticles were further investigated with analysis of the cytoskeleton conformation, with SEM and TEM imaging, and with AFM analysis. Finally, differentiation in adipocytes and osteocytes was achieved in the presence of high doses of BTNPs, thus highlighting the safety of these nanostructures towards mesenchymal stem cells.

Growing Bone Tissue-engineered Niches with Graded Osteogenicity: an in Vitro Method for Biomimetic Construct Assembly

Tissue Engineering. Part C, Methods. Dec, 2013  |  Pubmed ID: 23537352

The traditional bone tissue-engineering approach exploits mesenchymal stem cells (MSCs) to be seeded once only on three-dimensional (3D) scaffolds, hence, differentiated for a certain period of time and resulting in a homogeneous osteoblast population at the endpoint. However, after achieving terminal osteodifferentiation, cell viability is usually markedly compromised. On the other hand, naturally occurring osteogenesis results from the coexistence of MSC progenies at distinct differentiative stages in the same microenvironment. This diversification also enables long-term viability of the mature tissue. We report an easy and tunable in vitro method to engineer simple osteogenic cell niches in a biomimetic fashion. The niches were grown via periodic reseeding of undifferentiated MSCs on MSC/scaffold constructs, the latter undergoing osteogenic commitment. Time-fractioning of the seeded cell number during differentiation time of the constructs allowed graded osteogenic cell populations to be grown together on the same scaffolds (i.e., not only terminally differentiated osteoblasts). In such cell-dynamic systems, the overall differentiative stage of the constructs could also be tuned by varying the cell density seeded at each inoculation. In this way, we generated two different biomimetic niche models able to host good reservoirs of preosteoblasts and other osteoprogenitors after 21 culture days. At that time, the niche type resulting in 40.8% of immature osteogenic progenies and only 59.2% of mature osteoblasts showed a calcium content comparable to the constructs obtained with the traditional culture method (i.e., 100.03 ± 29.30 vs. 78.51 ± 28.50 pg/cell, respectively; p=not significant), the latter colonized only by fully differentiated osteoblasts showing exhausted viability. This assembly method for tissue-engineered constructs enabled a set of important parameters, such as viability, colonization, and osteogenic yield of the MSCs to be balanced on 3D scaffolds, thus achieving biomimetic in vitro models with graded osteogenicity, which are more complex and reliable than those currently used by tissue engineers.

Use of Autologous Human Mesenchymal Stromal Cell/fibrin Clot Constructs in Upper Limb Non-unions: Long-term Assessment

PloS One. 2013  |  Pubmed ID: 24023694

Tissue engineering appears to be an attractive alternative to the traditional approach in the treatment of fracture non-unions. Mesenchymal stromal cells (MSCs) are considered an appealing cell source for clinical intervention. However, ex vivo cell expansion and differentiation towards the osteogenic lineage, together with the design of a suitable scaffold have yet to be optimized. Major concerns exist about the safety of MSC-based therapies, including possible abnormal overgrowth and potential cancer evolution.

Boron Nitride Nanotubes and Primary Human Osteoblasts: in Vitro Compatibility and Biological Interactions Under Low Frequency Ultrasound Stimulation

Nanotechnology. Nov, 2013  |  Pubmed ID: 24150892

In this paper we investigated a novel and non-invasive approach for an endogenous osteoblast stimulation mediated by boron nitride nanotubes (BNNTs). Specifically, following the cellular uptake of the piezoelectric nanotubes, cultures of primary human osteoblasts (hOBs) were irradiated with low frequency ultrasound (US), as a simple method to apply a mechanical input to the cells loaded with BNNTs. This in vitro study was aimed at investigating the main interactions between hOBs and BNNTs and to study the effects of the 'BNNTs + US' stimulatory method on the osteoblastic function and maturation.A non-cytotoxic BNNT concentration to be used in vitro with hOB cultures was established. Moreover, investigation with transmission electron microscopy/electron energy loss spectroscopy (TEM/EELS) confirmed that BNNTs were internalized in membranal vesicles. The panel of investigated osteoblastic markers disclosed that BNNTs were capable of fostering the expression of late-stage bone proteins in vitro, without using any mineralizing culture supplements. In our samples, the maximal osteopontin expression, with the highest osteocalcin and Ca(2+) production, in the presence of mineral matrix with nodular morphology, was observed in the samples treated with BNNTs + US. In this group was also shown a significantly enhanced synthesis of TGF-β1, a molecule sensitive to electric stimulation in bone. Finally, gene deregulations of the analyzed osteoblastic genes leading to depletive cellular effects were not detected. Due to their piezoelectricity, BNNT-based therapies might disclose advancements in the treatment of bone diseases.

Processing Large-diameter Poly(L-lactic Acid) Microfiber Mesh/mesenchymal Stromal Cell Constructs Via Resin Embedding: an Efficient Histologic Method

Biomedical Materials (Bristol, England). Aug, 2014  |  Pubmed ID: 25029413

In this study, we performed a complete histologic analysis of constructs based on large diameter ( >100 μm) poly-L-lactic acid (PLLA) microfibers obtained via dry-wet spinning and rat Mesenchymal Stromal Cells (rMSCs) differentiated towards the osteogenic lineage, using acrylic resin embedding. In many synthetic polymer-based microfiber meshes, ex post processability of fiber/cell constructs for histologic analysis may face deterring difficulties, leading to an incomplete investigation of the potential of these scaffolds. Indeed, while polymeric nanofiber (fiber diameter = tens of nanometers)/cell constructs can usually be embedded in common histologic media and easily sectioned, preserving the material structure and the antigenic reactivity, histologic analysis of large polymeric microfiber/cell constructs in the literature is really scant. This affects microfiber scaffolds based on FDA-approved and widely used polymers such as PLLA and its copolymers. Indeed, for such constructs, especially those with fiber diameter and fiber interspace much larger than cell size, standard histologic processing is usually inefficient due to inhomogeneous hardness and lack of cohesion between the synthetic and the biological phases under sectioning. In this study, the microfiber/MSC constructs were embedded in acrylic resin and the staining/reaction procedures were calibrated to demonstrate the possibility of successfully employing histologic methods in tissue engineering studies even in such difficult cases. We histologically investigated the main osteogenic markers and extracellular matrix molecules, such as alkaline phosphatase, osteopontin, osteocalcin, TGF-β1, Runx2, Collagen type I and the presence of amorphous, fibrillar and mineralized matrix. Biochemical tests were employed to confirm our findings. This protocol permitted efficient sectioning of the treated constructs and good penetration of the histologic reagents, thus allowing distribution and expression of almost all the tested molecules to be revealed. Our results demonstrated that it is possible to perform histologic analyses of large-diameter PLLA-based microfiber scaffold/MSC constructs that face the failure of standard histologic procedures.

Plasticity of Human Dental Pulp Stromal Cells with Bioengineering Platforms: a Versatile Tool for Regenerative Medicine

Micron (Oxford, England : 1993). Dec, 2014  |  Pubmed ID: 25180486

In recent years, human dental pulp stromal cells (DPSCs) have received growing attention due to their characteristics in common with other mesenchymal stem cells, in addition to the ease with which they can be harvested. In this study, we demonstrated that the isolation of DPSCs from third molar teeth of healthy individuals allowed the recovery of dental mesenchymal stem cells that showed self-renewal and multipotent differentiation capability. DPSCs resulted positive for CD73, CD90, CD105, STRO-1, negative for CD34, CD45, CD14 and were able to differentiate into osteogenic and chondrogenic cells. We also assayed the angiogenic potential of DPSCs, their capillary tube-like formation was assessed using an in vitro angiogenesis assay and the uptake of acetylated low-density lipoprotein was measured as a marker of endothelial function. Based on these results, DPSCs were capable of differentiating into cells with phenotypic and functional features of endothelial cells. Furthermore, this study investigated the growth and differentiation of human DPSCs under a variety of bioengineering platforms, such as low frequency ultrasounds, tissue engineering and nanomaterials. DPSCs showed an enhanced chondrogenic differentiation under ultrasound application. Moreover, DPSCs were tested on different scaffolds, poly(vinyl alcohol)/gelatin (PVA/G) sponges and human plasma clots. We showed that both PVA/G and human plasma clot are suitable scaffolds for adhesion, growth and differentiation of DPSCs toward osteoblastic lineages. Finally, we evaluated the interactions of DPSCs with a novel class of nanomaterials, namely boron nitride nanotubes (BNNTs). From our investigation, DPSCs have appeared as a highly versatile cellular tool to be employed in regenerative medicine.

Interfacing Polymeric Scaffolds with Primary Pancreatic Ductal Adenocarcinoma Cells to Develop 3D Cancer Models

Biomatter. 2014  |  Pubmed ID: 25482337

We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.

Boron Nitride Nanotube-functionalised Myoblast/microfibre Constructs: a Nanotech-assisted Tissue-engineered Platform for Muscle Stimulation

Journal of Tissue Engineering and Regenerative Medicine. Jul, 2015  |  Pubmed ID: 24596180

In this communication, we introduce boron nitride nanotube (BNNT)-functionalised muscle cell/microfibre mesh constructs, obtained via tissue engineering, as a three-dimensional (3D) platform to study a wireless stimulation system for electrically responsive cells and tissues. Our stimulation strategy exploits the piezoelectric behaviour of some classes of ceramic nanoparticles, such as BNNTs, able to polarize under mechanical stress, e.g. using low-frequency ultrasound (US). In the microfibre scaffolds, C2C12 myoblasts were able to differentiate into viable myotubes and to internalize BNNTs, also upon US irradiation, so as to obtain a nanotech-assisted 3D in vitro model. We then tested our stimulatory system on 2D and 3D cellular models by investigating the expression of connexin 43 (Cx43), as a molecule involved in cell crosstalk and mechanotransduction, and myosin, as a myogenic differentiation marker. Cx43 gene expression revealed a marked model dependency. In control samples (without US and/or BNNTs), Cx43 was upregulated under 2D culture conditions (10.78 ± 1.05-fold difference). Interactions with BNNTs increased Cx43 expression in 3D samples. Cx43 mRNA dropped in 2D under the 'BNNTs + US' regimen, while it was best enhanced in 3D samples (3.58 ± 1.05 vs 13.74 ± 1.42-fold difference, p = 0.0001). At the protein level, the maximal expressions of Cx43 and myosin were detected in the 3D model. In contrast with the 3D model, in 2D cultures, BNNTs and US exerted a synergistic depletive effect upon myosin synthesis. These findings indicate that model dimensionality and stimulatory regimens can strongly affect the responses of signalling and differentiation molecules, proving the importance of developing proper in vitro platforms for biological modelling.

Multiscale Fabrication of Biomimetic Scaffolds for Tympanic Membrane Tissue Engineering

Biofabrication. May, 2015  |  Pubmed ID: 25947357

The tympanic membrane (TM) is a thin tissue able to efficiently collect and transmit sound vibrations across the middle ear thanks to the particular orientation of its collagen fibers, radiate on one side and circular on the opposite side. Through the combination of advanced scaffolds and autologous cells, tissue engineering (TE) could offer valuable alternatives to autografting in major TM lesions. In this study, a multiscale approach based on electrospinning (ES) and additive manufacturing (AM) was investigated to fabricate scaffolds, based on FDA approved copolymers, resembling the anatomic features and collagen fiber arrangement of the human TM. A single scale TM scaffold was manufactured using a custom-made collector designed to confer a radial macro-arrangement to poly(lactic-co-glycolic acid) electrospun fibers during their deposition. Dual and triple scale scaffolds were fabricated combining conventional ES with AM to produce poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer scaffolds with anatomic-like architecture. The processing parameters were optimized for each manufacturing method and copolymer. TM scaffolds were cultured in vitro with human mesenchymal stromal cells, which were viable, metabolically active and organized following the anisotropic character of the scaffolds. The highest viability, cell density and protein content were detected in dual and triple scale scaffolds. Our findings showed that these biomimetic micro-patterned substrates enabled cell disposal along architectural directions, thus appearing as promising substrates for developing functional TM replacements via TE.

Bovine Bone Matrix/poly(l-lactic-co-ε-caprolactone)/gelatin Hybrid Scaffold (SmartBone(®)) for Maxillary Sinus Augmentation: A Histologic Study on Bone Regeneration

International Journal of Pharmaceutics. Oct, 2016  |  Pubmed ID: 27769886

The ideal scaffold for bone regeneration is required to be highly porous, non-immunogenic, biostable until the new tissue formation, bioresorbable and osteoconductive. This study aimed at investigating the process of new bone formation in patients treated with granular SmartBone(®) for sinus augmentation, providing an extensive histologic analysis. Five biopsies were collected at 4-9 months post SmartBone(®) implantation and processed for histochemistry and immunohistochemistry. Histomorphometric analysis was performed. Bone-particle conductivity index (BPCi) was used to assess SmartBone(®) osteoconductivity. At 4 months, SmartBone(®) (12%) and new bone (43.9%) were both present and surrounded by vascularized connective tissue (37.2%). New bone was grown on SmartBone(®) (BPCi=0.22). At 6 months, SmartBone(®) was almost completely resorbed (0.5%) and new bone was massively present (80.8%). At 7 and 9 months, new bone accounted for a large volume fraction (79.3% and 67.4%, respectively) and SmartBone(®) was resorbed (0.5% and 0%, respectively). Well-oriented lamellae and bone scars, typical of mature bone, were observed. In all the biopsies, bone matrix biomolecules and active osteoblasts were visible. The absence of inflammatory cells confirmed SmartBone(®) biocompatibility and non-immunogenicity. These data indicate that SmartBone(®) is osteoconductive, promotes fast bone regeneration, leading to mature bone formation in about 7 months.

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