Publications

INTRODUCTION: Mesenchymal stem cells ( MSCs) regulatecell processes by secreting growth factors, cytokines, chemokines,hormones and extracellular vesicles, known as secretome. This se-cretome has successfully exhibited therapeutic properties.1 Althoughsecretome can be directly administered in the target site, its fastclearance is still challenging. Therefore, new efforts have been madeto develop delivery platforms,1 that sustain secretome release andincrease its retention time.The aim of this work is to encapsulate secretome into nano-particles (NPs) for achieving a sustained release and to evaluate theirpotential in wound healing.EXPERIMENTAL METHODS: Secretome was obtained byculturing human umbilical cord MSCs under hypoxic conditions.Protein content was determined using a protein array. Secretome wasencapsulated in poly(lactic-co-glycolic) (PLGA) NPs by doubleemulsification. NPs size and zeta potential were measured using aZetasizer. The loading content and release was evaluated using amicroBCA. The secretome integrity was also assessed. NPs cellularcytocompatibility was studied using human dermal fibroblasts andkeratinocytes.RESULTS: The most expressed bioactive molecules detected inthe secretome were TIMP-2, TIMP-1, IL-6, IL-8, RANTES. NPsencapsulate between 7.2-13.5mg/mg NPs, having sizes of 300-400 nm and zeta potentials below -20 mV. NPs were biocompatibleand sustained the release for 7 days.DISCUSSION AND CONCLUSIONS: The secretome composi-tion evidenced its potential in wound healing. Secretome was suc-cessfully encapsulated in the NPs, showing a sustained release. Ourresults indicate that these nanoplatforms are promising woundhealing agents.

JTD Keywords: Msc, Nanoparticle, Secretome

Background Small cardiac tissue engineering constructs show promise for limiting post-infarct sequelae in animal models. This study sought to scale-up a 2-cm2 preclinical construct into a human-size advanced therapy medicinal product (ATMP; PeriCord), and to test it in a first-in-human implantation. Methods The PeriCord is a clinical-size (12–16 cm2) decellularised pericardial matrix colonised with human viable Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). WJ-MSCs expanded following good manufacturing practices (GMP) met safety and quality standards regarding the number of cumulative population doublings, genomic stability, and sterility. Human decellularised pericardial scaffolds were tested for DNA content, matrix stiffness, pore size, and absence of microbiological growth. Findings PeriCord implantation was surgically performed on a large non-revascularisable scar in the inferior wall of a 63-year-old male patient. Coronary artery bypass grafting was concomitantly performed in the non-infarcted area. At implantation, the 16-cm2 pericardial scaffold contained 12·5 × 106 viable WJ-MSCs (85·4% cell viability; <0·51 endotoxin units (EU)/mL). Intraoperative PeriCord delivery was expeditious, and secured with surgical glue. The post-operative course showed non-adverse reaction to the PeriCord, without requiring host immunosuppression. The three-month clinical follow-up was uneventful, and three-month cardiac magnetic resonance imaging showed ~9% reduction in scar mass in the treated area. Interpretation This preliminary report describes the development of a scalable clinical-size allogeneic PeriCord cardiac bioimplant, and its first-in-human implantation. Funding La Marató de TV3 Foundation, Government of Catalonia, Catalan Society of Cardiology, “La Caixa” Banking Foundation, Spanish Ministry of Science, Innovation and Universities, Institute of Health Carlos III, and the European Regional Development Fund.

JTD Keywords: Advanced therapy medicinal product (ATMP), Biofabrication, Cardiac tissue engineering, Myocardial infarction, Scaffold, Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs)

The use of human mesenchymal stromal cells (hMSC) for treating diseased tissues with poor vascularization has received significant attention, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have also been suggested as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. In this study, calcium-releasing particles and hMSC were combined within a hydrogel to examine their vasculogenic potential in vitro and in vivo. The particles provided sustained calcium release and showed proangiogenic stimulation in a chorioallantoic membrane (CAM) assay. The number of hMSC encapsulated in a degradable RGD-functionalized PEG hydrogel containing particles remained constant over time and IGF-1 release was increased. When implanted in the epidydimal fat pad of immunocompromised mice, this composite material improved cell survival and stimulated vessel formation and maturation. Thus, the combination of hMSC and calcium-releasing glass-ceramics represents a new strategy to achieve vessel stabilization, a key factor in the revascularization of ischemic tissues. Statement of Significance: Increasing blood vessel formation in diseased tissues with poor vascularization is a current clinical challenge. Cell therapy using human mesenchymal stem cells has received considerable interest, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have been explored as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. By incorporating both human mesenchymal stem cells and glass-ceramic particles in an implantable hydrogel, this study provides insights into the vasculogenic potential in soft tissues of the combined strategies. Enhancement of vessel formation and maturation supports further investigation of this strategy.

JTD Keywords: Calcium, Glass-ceramic particles, Vascularization, hMSC, Hydrogel

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

JTD Keywords: Bioengineering, Dendrimer, Nanopattern, Arginine-Glycine-Aspartic Acid (RGD), Atomic Force Microscopy (AFM), Cell Adhesion, Mesenchymal Stem Cells (Mscs), Chondrogenesis

Background: Current therapies for bone cancers - either primary or metastatic – are difficult to implement and unfortunately not completely effective. An alternative therapy could be found in cold plasmas generated at atmospheric pressure which have already demonstrated selective anti-tumor action in a number of carcinomas and in more relatively rare brain tumors. However, its effects on bone cancer are still unknown. Methods: Herein, we employed an atmospheric pressure plasma jet (APPJ) to validate its selectivity towards osteosarcoma cell line vs. osteoblasts & human mesenchymal stem cells. Results: Cytotoxicity following direct interaction of APPJ with cells is comparable to indirect interaction when only liquid medium is treated and subsequently added to the cells, especially on the long-term (72 h of cell culture). Moreover, following contact of the APPJ treated medium with cells, delayed effects are observed which lead to 100% bone cancer cell death through apoptosis (decreased cell viability with incubation time in contact with APPJ treated medium from 24 h to 72 h), while healthy cells remain fully viable and unaffected by the treatment. Conclusions: The high efficiency of the indirect treatment indicates that an important role is played by the reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the gaseous plasma stage and then transmitted to the liquid phase, which overall lead to lethal and selective action towards osteosarcoma cells. These findings open new pathways for treatment of metastatic bone disease with a minimally invasive approach.

JTD Keywords: Atmospheric pressure plasma jet, Bone cancer, hMSC, HOb, Liquids, Osteoblasts, Osteosarcoma, SaOS-2

The current study reports on the manufacturing by rapid prototyping technique of three-dimensional (3D) scaffolds based on silicon substituted hydroxyapatite with Elastin-like Recombinamers (ELRs) functionalized surfaces. Silicon doped hydroxyapatite (Si-HA), with Ca10(PO4)5.7(SiO4)0.3(OH)1.7h0.3 nominal formula, was surface functionalized with two different types of polymers designed by genetic engineering: ELR-RGD that contain cell attachment specific sequences and ELR-SNA15/RGD with both hydroxyapatite and cells domains that interact with the inorganic phase and with the cells, respectively. These hybrid materials were subjected to in vitro assays in order to clarify if the ELRs coating improved the well-known biocompatible and bone regeneration properties of calcium phosphates materials. The in vitro tests showed that there was a total and homogeneous colonization of the 3D scaffolds by Bone marrow Mesenchymal Stromal Cells (BMSCs). In addition, the BMSCs were viable and able to proliferate and differentiate into osteoblasts. Statement of Significance Bone tissue engineering is an area of increasing interest because its main applications are directly related to the rising life expectancy of the population, which promotes higher rates of several bone pathologies, so innovative strategies are needed for bone tissue regeneration therapies. Here we use the rapid prototyping technology to allow moulding ceramic 3D scaffolds and we use different bio-polymers for the functionalization of their surfaces in order to enhance the biological response. Combining the ceramic material (silicon doped hydroxyapatite, Si-HA) and the Elastin like Recombinamers (ELRs) polymers with the presence of the integrin-mediate adhesion domain alone or in combination with SNA15 peptide that possess high affinity for hydroxyapatite, provided an improved Bone marrow Mesenchymal Stromal Cells (BMSCs) differentiation into osteoblastic linkage.

JTD Keywords: Bone marrow Mesenchymal Stromal Cells (BMSCs), Bone repair, Elastin-like Recombinamers (ELRs), Rapid prototyped 3D scaffolds, Silicon doped hydroxyapatite (Si-HA), Tissue engineering

Surface modification stands out as a versatile technique to create instructive biomaterials that are able to actively direct stem cell fate. Chemical functionalization of titanium has been used in this work to stimulate the differentiation of human mesenchymal stem cells (hMSCs) into the osteoblastic lineage, by covalently anchoring a synthetic double-branched molecule (PTF) to the metal that allows a finely controlled presentation of peptidic motifs. In detail, the effect of the RGD adhesive peptide and its synergy motif PHSRN is studied, comparing a random distribution of the two peptides with the chemically-tailored disposition within the custom made synthetic platform, which mimics the interspacing between the motifs observed in fibronectin. Contact angle measurement and XPS analysis are used to prove the efficiency of functionalization. We demonstrate that, by rationally designing ligands, stem cell response can be efficiently guided towards the osteogenic phenotype: In vitro, PTF-functionalized surfaces support hMSCs adhesion, with higher cell area and formation of focal contacts, expression of the integrin receptor α5β1 and the osteogenic marker Runx2, and deposition a highly mineralized matrix, reaching values of mineralization comparable to fibronectin. Our strategy is also demonstrated to be efficient in promoting new bone growth in vivo in a rat calvarial defect. These results highlight the efficacy of chemical control over the presentation of bioactive peptides; such systems may be used to engineer bioactive surfaces with improved osseointegrative properties, or can be easily tuned to generate multi-functional coatings requiring a tailored disposition of the peptidic motifs. Statement of significance Organic coatings have been proposed as a solution to foster osseointegration of orthopedic implants. Among them, extracellular matrix-derived peptide motifs are an interesting biomimetic strategy to harness cell-surface interactions. Nonetheless, the combination of multiple peptide motifs in a controlled manner is essential to achieve receptor specificity and fully exploit the potentiality of synthetic peptides. Herein, we covalently graft to titanium a double branched molecule to guide stem cell fate in vitro and generate an osseoinductive titanium surface in vivo. Such synthetic ligand allows for the simultaneous presentation of two bioactive motifs, thus is ideal to test the effect of synergic sequences, such as RGD and PHSRN, and is a clear example of the versatility and feasibility of rationally designed biomolecules.

JTD Keywords: hMSCs, Integrin-binding peptides, Osseointegration, RGD-PHSRN, Titanium

Bone is the main store of calcium and progenitor cells in the body. During the resorption process, the local calcium concentration reaches 8-40 mM, and the surrounding cells are exposed to these fluctuations in calcium. This stimulus is a signal that is detected through the calcium sensing receptor (CaSR), which modulates chemotactic and proliferative G protein-dependent signaling pathways. The objective of the present work is to evaluate the roles of extracellular calcium ([Ca2+]o) and the CaSR in osteoinduction. Rat bone marrow mesenchymal stromal cells (rBMSCs) were stimulated with 10 mM of Ca2+. Several experiments were conducted to demonstrate the effect of [Ca2+]o on chemotaxis, proliferation and differentiation on the osteoblastic lineage. It was found that [Ca2+]o induces rBMSCs to migrate and proliferate in a concentration-dependent manner. Real-time polymerase chain reaction and immunofluorescence also revealed that 10 mM Ca2+ stimulates overexpression of osteogenic markers in rBMSCs, including alkaline phosphatase (ALP), bone sialoprotein, collagen Ia1 and osteocalcin. Functional assays determining ALP activity and mineralization tests both corroborate the increased expression of these markers in rBMSCs stimulated with Ca2+. Moreover, CaSR blockage inhibited the cellular response to stimulation with high concentrations of [Ca2+]o, revealing that the CaSR is a key modulator of these cellular responses.

JTD Keywords: Calcium sensing receptor (CaSR), Extracellular calcium, Mesenchymal stromal cells (MSCs), Osteoinduction, Regenerative medicine