L. De Girolamo

Human adipose-derived stem cells as future tools in tissue regeneration: osteogenic differentiation and cell-scaffold interaction.

Tissue engineering is now contributing to new developments in several clinical fields, and mesenchymal stem cells derived from adipose tissue (hASCs) may provide a novel opportunity to replace, repair and promote the regeneration of diseased or damaged musculoskeletal tissue. Our interest was to characterize and differentiate hASCs isolated from twenty-three donors. Proliferation, CFU-F, cytofluorimetric and histochemistry analyses were performed. HASCs differentiate into osteogenic, chondrogenic, and adipogenic lineages, as assessed by tissue-specific markers such as alkaline phosphatase, osteopontin expression and deposition of calcium matrix, lipid-vacuoles formation and Glycosaminoglycans production. We also compared osteo-differentiated hASCs cultured on monolayer and loaded on biomaterials routinely used in the clinic, such as hydroxyapatite, cancellous human bone fragments, deproteinized bovine bone granules, and titanium. Scaffolds loaded with pre-differentiated hASCs do not affect cell proliferation and no cellular toxicity was observed. HASCs tightly adhere to scaffolds and differentiated-hASCs on human bone fragments and bovine bone granules produced, respectively, 3.4- and 2.1-fold more calcified matrix than osteo-differentiated hASCs on monolayer. Moreover, both human and deproteinized bovine bone is able to induce osteogenic differentiation of CTRL-hASCs. Although our in vitro results need to be confirmed in in vivo bone regeneration models, our data suggest that hASCs may be considered suitable biological tools for the screening of innovative scaffolds that would be useful in tissue engineering.

Stemness and osteogenic and adipogenic potential are differently impaired in subcutaneous and visceral adipose derived stem cells (ASCs) isolated from obese donors.

Today adipose tissue is not just considered as the primary energy storage organ, but it is also recognized as an important endocrine tissue and an abundant source of mesenchymal stem cells (adipose-derived stem cells, ASCs). During the last decade, several studies have provided preclinical data on the safety and efficacy of ASCs, supporting their use in cell-based therapy for regenerative medicine purposes. Little is known about the effect of obesity on ASCs properties. Since ASCs differentiation and proliferation are determined by their niche, the differences in body fat distribution and the obesity-related co-morbidities may have several consequences. In this study we compared ASCs of subcutaneous adipose tissue from obese (obS-ASCs) and non-obese (nS-ASCs) donors in order to compare their immunophenotype and osteogenic and adipogenic potential. Moreover, in order to evaluate the possible difference between subcutaneous and visceral fat, obS-ASCs were also compared to ASCs derived from visceral adipose tissue of the same obese donors (obV-ASCs). Our results show that subcutaneous and visceral ASCs derived from obese donors have an impaired cell proliferation, clonogenic ability and immunophenotype. Nevertheless, obS-ASCs are able to differentiate toward osteogenic and adipogenic lineages, although to a small extent with respect to non-obese donors, whereas obV-ASCs lose most of their stem cell characteristics, including multi-differentiation potential. Taken together our findings confirm that not all ASCs present the same behavior, most likely due to their biological microenvironment in vivo. The specific stimuli which can play a key role in ASCs impairment, including the effects of the obesity-related inflammation, should be further investigated to have a complete picture of the phenomenon.

Role of autologous rabbit adipose‐derived stem cells in the early phases of the repairing process of critical bone defects

Adipose‐derived stem cells (ASCs) may represent a novel and efficient tool to promote bone regeneration. In this study, rabbit ASCs were expanded in culture and used for the regeneration of full‐thickness bone defects in the proximal epiphysis of tibia of 12 New Zealand rabbits. Defects were implanted with graft material as follows: untreated (control), empty hydroxyapatite (HA) disk, ASCs alone, and HA disk seeded with ASCs. Each isolated ASCs population was tested in vitro: they all showed a high proliferation rate, a marked clonogenic ability, and osteogenic differentiation potential. Eight weeks after implantation, macroscopic analyses of all the samples showed satisfactory filling of the lesions without any significant differences in term of stiffness between groups treated with or without cells (p > 0.05). In both the scaffold‐treated groups, a good osteointegration was radiographically observed. Even if HA was not completely reabsorbed, ASCs‐loaded HA displayed a higher scaffold resorption than the unloaded ones. Histological analyses showed that the osteogenic abilities of the scaffold‐treated defects was greater than those of scaffold‐free samples, and in particular new formed bone was more mature and more similar to native bone in presence of ASCs. These results demonstrated that autologous ASCs–HA constructs is a potential treatment for the regeneration of bone defects.

Two bone substitutes analyzed in vitro by porcine and human adipose-derived stromal cells.

Nowadays, the repair of large bone defects is an important goal in orthopaedic and dental fields. Tissue engineering, applied to increase the bone regeneration process, combines suitable scaffolds with either terminally differentiated cells or Mesenchymal Stromal Cells. In vitro studies with Adipose-derived Stromal Cells (ASCs) may identify new bioactive supports, to be tested in preclinical model. In this study, we evaluated the biocompatibility and the osteoinductive properties of two bone substitutes, RegenOSS™ (RO-1) and a new generation scaffold (RO-2), on both porcine and human ASCs. Porcine ASCs need a prolonged initial phase to adapt to both substitutes; indeed, their growth was initially reduced respect to cells cultured in their absence. In contrast, human ASCs were not negatively affected. However, no toxicity of RO-1 and −2 was observed on both ASC populations which are able to stick to both biomaterials. RO-1 and −2 supported osteogenic differentiation of porcine and human ASCs in a different manner: the presence of RO-1 up-regulated both alkaline phosphatase (ALP) activity and collagen production of human ASCs, whereas in porcine ASCs, RO-2 seemed to up-regulate ALP activity, while the production of collagen is mainly stimulated by the presence of RO-1. We suggest to use not just human ASCs, but also animal ones to select suitable scaffolds to generate bio-constructs in vitro, which then need to be tested in animal model before reaching the market.

Autologous and heterologous adipose derived stem cells seeded on oligo(polyethylene glycol) fumarate hydrogel scaffold are effective in the regeneration of critical osteochondral defects in minipig model

Adequate cellular in-growth into biomaterials is one of the fundamental requirements in regenerative medicine. Type-I-collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support. However, adequate cell in-growth and cell seeding has been suboptimal. Different densities of collagen scaffolds (0.3% to 0.8% (w/v)) with/without polymer knitting (poly-caprolactone (PCL)) were prepared. The structure of collagen scaffolds was characterized using scanning electronic microscopy (SEM) and HE staining. The mechanical strength of hybrid scaffolds was determined using tensile strength analysis. Cellular penetration and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and urothelium seeding. SEM and HE analysis showed the honeycomb structure and the hybrid scaffolds were adequately connected. The hybrid scaffolds were much stronger than collagen alone. The distribution of the beads and cells were highly dependent on the collagen density: at lower densities the beads and cells were more evenly distributed and penetrated deeper into the scaffold. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a 0.4% hybrid scaffold strengthened with knitting achieved the best cellular distribution.Human adult heart harbors a population of resident progenitor cells that can be isolated by Sca-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes and vascular cells in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, the survival rate and retention is really poor, less than 1% of injected cells being detectable in the hosttissue within few weeks. The present study aimed at investigating the possibility to produce scaffoldless, thick cardiac progenitor cell-derived cardiac patches by thermo-responsive technology. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces obtained by poly-N-isopropylacrylamide (PNIPAAm) surface immobilization. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and produced abundant extracellular matrix, while preserving their phenotype and plasticity. Cell phenotype and viability within the 3D construct were followed for 1 week, showing that no significant differentiation or apoptotic events occurred within the construct. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such approach to deliver stem cells.Spinal cord injury and repair is one of the important focus areas in tissue regeneration. Mechanical trauma caused due to factors such as contusion, compression or involuntary stretching induce post-traumatic secondary tissue damage in many Spinal Cord Injury (SCI) patients. Therefore, there is a need for scaffolds that provide a conducive threedimensionsal (3D) environment for injured cells to attach and grow. In this study we propose to synthesize 3D polymeric scaffolds in order to study the mechanical and adhesive properties & the nature of the interactions between hyaluronan-based (HY) biomaterials and cells and tissues both in vitroandin vivo. Here we have synthesized 3D HY-based hydrogels with robust mechanical and adhesive properties and demonstrate the use of this material for neuronal-related applications such as the treatment of SCI. Cell culture and survivability studies were done with NSC-34 cells. Live/Dead assay performed on the cells revealed significant differences in the staining of live cells and showed increased viability and proliferation. The number of live cells in the HY-based hydrogels with 0.1% collagen showed higher cell numbers compared with the other hydrogels. In this study we show that Injectable HYbased hydrogels with high elasticity, comparable to the mechanical properties of nervous tissue have been used in this study to study their biocompatibility and neuroprotective properties and they show better affinity for neuronal cells.Calcium phosphates (CaP) obtained by biomineralisation in Simulated Boby Fluid have been used for decades to assess the mineralisation capability of biomaterials. Recently, they have been envisioned as potential agents to promote bone formation. In this study, we have fabricated and coated with calcium phosphate melt electrospun scaffolds whereby macropores permit adequate cell migration and nutrient transfer. We have systematically investigated the effect of coating and osteoinduction onto the response of ovine osteoblasts and we observed that the coating up-regulated alkaline phosphatase activity regardless of the in vitro culture conditions. Micro Computed Tomography revealed that only scaffolds cultured in an osteoinductive cocktail were capable of depositing mineralised matrix, and that CaP coated scaffolds were more efficient at promoting mineralisation. Theses scaffolds were subcutaneously implanted in athymic rats and this demonstrated that the osteoinduction was a pre-requisite for bone formation in this ectopic model. It showed that although the bone formation was not significantly different after 8 weeks, the CaP coated scaffolds were superior at inducing bone formation as evidenced by higher levels of mineralisation at earlier time points. This work demonstrated that CaP coating is not sufficient to induce bone formation; however the combination of osteoinduction and CaP coating resulted in earlier bone formation in an ectopic model.Introduction: Bladder regeneration using minced bladder mucosa is an alternative to costly and time-consuming conventional in vitro culturing of urothelial cells. In this method, the uroepithelium ...