Pilot in vivo toxicological investigation of boron nitride nanotubes

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

Tympanic Membrane Collagen Expression by Dynamically Cultured Human Mesenchymal Stromal Cell/Star-Branched Poly(ε-Caprolactone) Nonwoven Constructs

The tympanic membrane (TM) primes the sound transmission mechanism due to special fibrous layers mainly of collagens II, III, and IV as a product of TM fibroblasts, while type I is less represented. In this study, human mesenchymal stromal cells (hMSCs) were cultured on star-branched poly("-caprolactone) (*PCL)-based nonwovens using a TM bioreactor and proper dierentiating factors to induce the expression of the TM collagen types. The cell cultures were carried out for one week under static and dynamic conditions. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were used to assess collagen expression. A Finite Element Model was applied to calculate the stress distribution on the scaolds under dynamic culture. Nanohydroxyapatite (HA) was used as a filler to change density and tensile strength of *PCL scaolds. In dynamically cultured *PCL constructs, fibroblast surface marker was overexpressed, and collagen type II was revealed via IHC. Collagen types I, III and IV were also detected. Von Mises stress maps showed that during the bioreactor motion, the maximum stress in *PCL was double that in HA/*PCL scaolds. By using a *PCL nonwoven scaold, with suitable physico-mechanical properties, an oscillatory culture, and proper dierentiative factors, hMSCs were committed into fibroblast lineage-producing TM-like collagens

Piezoelectric Signals in Vascularized Bone Regeneration

The demand for bone substitutes is increasing in Western countries. Bone graft substitutes aim to provide reconstructive surgeons with off-the-shelf alternatives to the natural bone taken from humans or animal species. Under the tissue engineering paradigm, biomaterial scaffolds can be designed by incorporating bone stem cells to decrease the disadvantages of traditional tissue grafts. However, the effective clinical application of tissue-engineered bone is limited by insufficient neovascularization. As bone is a highly vascularized tissue, new strategies to promote both osteogenesis and vasculogenesis within the scaffolds need to be considered for a successful regeneration. It has been demonstrated that bone and blood vases are piezoelectric, namely, electric signals are locally produced upon mechanical stimulation of these tissues. The specific effects of electric charge generation on different cells are not fully understood, but a substantial amount of evidence has suggested their functional and physiological roles. This review summarizes the special contribution of piezoelectricity as a stimulatory signal for bone and vascular tissue regeneration, including osteogenesis, angiogenesis, vascular repair, and tissue engineering, by considering different stem cell sources entailed with osteogenic and angiogenic potential, aimed at collecting the key findings that may enable the development of successful vascularized bone replacements useful in orthopedic and otologic surgery

Development of tissue-engineered constructs for ossicular chain replacement

Ossicular chain (OC) is the bony part of middle ear dedicated to sound transmission. Chronic inflammations, infections and traumas occurring in the lifespan result in a set of severe diseases known as “conductive hearing loss”. To recover an acceptable hearing threshold, the damaged OC have to be surgically replaced with artificial prostheses. However, despite many efforts aimed at fabricating optimal replacements, all the synthetic prostheses are subject to extrusion, i.e., a type of rejection due to lack of biointegration. For these reasons, it is necessary to envision novel strategies for the OC substitution. In recent years, we have proposed an approach for OC reconstruction based on tissue engineering (TE), in which mesenchymal stromal cells (MSCs) are cultured under osteogenic differentiation regimen on bioresorbable 3D scaffolds up to obtain new bone substitutes with appropriate shape and dimensions (Danti et al., 2009; Danti et al., 2010). In this study, human MSCs were osteo-differentiated on different types of OC scaffolds fabricated in our laboratories. TE constructs were analyzed via biochemical assays, molecular biology and histo-morphological methods. An extensive analysis on native ossicles was performed to compare the results obtained in the constructs with the mature tissues. The results showed that the cells were viable, colonized the scaffolds and produced extracellular matrix molecules at intra- and extra-cellular level. MSC differentiation towards the osteogenic lineage was demonstrated by the production of mineralized matrix and specific osteogenic markers. Moreover, we assessed that all the investigated molecules were also expressed in the native tissues, even if at different expression levels, indicating that it was obtained a preliminary step for the creation of TE constructs to be employed, in perspective, as OC substitutes in the otologic surgery

In vitro study on the generation of tympanic membrane substitutes via tissue engineering

The tympanic membrane (TM) is an anatomical structure with unique histological and physiological features playing a fundamental role in sound transmission. In particular, the middle layer of the pars tensa, which represents the widest and thickest surface portion of the TM, consists of connective tissue mainly composed of collagen types II and III fibers, while collagen type I is present at a lesser extent [1]. Several pathologies affect the TM, including otitis media, tympanosclerosis, cholesteatoma and perforation that require reconstructive surgery depending on the lesion extent [2]. To this purpose, the temporalis fascia is currently considered as the gold standard material. However, due to limited graft availability, fully synthetic substitutes are also applied, with poorly satisfactory outcomes. For these reasons new strategies for TM replacement are still needed. In this study, we employed a tissue engineering (TE) approach for the regeneration of TM substitutes selecting some biocompatible and bioresorbable polymeric matrices to be cultured with human bone marrow-derived mesenchymal stem cells (MSCs). We set up a cell differentiation protocol using an appropriate mix of growing factors to obtain the in vitro differentiation of MSCs into TM fibroblasts. Furthermore, because of the role played by mechanical forces in TM motion, these engineered substitutes underwent mechanical stress during the culture. The obtained biohybrid constructs were characterized about cellular viability assays, gene expression quantification as well as histochemical and immunohistochemical analyses. Moreover, native TMs from cadavers were investigated for assessment and optimization of the engineered constructs. Our results showed that MSCs were able to grow and differentiate properly on the selected biomaterials and to synthesize appropriate extracellular matrix molecules. Moreover, the applied mechanical forces seem to promote TM-fibroblastic differentiation, increasing the production of collagen type II, that is a peculiarity of TM structure

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

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

Mesenchymal Stromal Cell culture in autologous conditions for orthopedic applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products. Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions — namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor’s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation. © 2016 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License