FROM IN VITRO STUDIES TO A LARGE ANIMAL MODEL: A MULTISTEP DISSECTION ON THE FUTURE ROLE OF ADIPOSE-DERIVED STEM CELLS FOR MUSCULOSKELETAL TISSUE ENGINEERING.

Tissue engineering is an emerging interdisciplinary field, born with the purpose to provide an alternative solution for the regeneration of lesioned or lost tissues, combining cells, biocompatible scaffolds and bioactive factors. The cells for this approach should be non-immunoreactive and non-tumorigenic. Moreover, they should be available in large amount and possess, or be able to acquire, a specific protein expression pattern similar to that of the damaged tissue and/or act as a pool of trophic factors for resident cells. All these reasons, make mesenchymal stem cells (MSCs) good candidates for applications in regenerative medicine. Although bone marrow is still the most common source of MSCs, these cells could be harvested from all vascularised tissues, and, interestingly, from tissues that are normally discarded, such as fat, placenta or umbilical cord. One of the most convenient source of MSCs, is unequivocally, the adipose tissue due to the easily accessible anatomical location and the abundance of subcutaneous adipose tissue. Adipose-derived stem cells (ASCs) are similar to MSCs isolated from bone marrow in morphology, immunophenotype, and differentiation ability, and own interesting features such as immunoregolatory and anti-inflammatory properties. In the recent years, many strategies for the cure of musculoskeletal tissues critical lesions, mainly in orthopaedic, oral and maxillo-facial surgery, have been under investigations. In this contest, the regeneration of structures including different tissues, such as the periodontium and the osteochondral unit, are particularly challenging. Periodontal regeneration is especially demanding, as it requires regeneration of three quite diverse and unique tissues such as the alveolar bone, the periodontal ligament and the cementum, that have to interface with each other to restore their complex structure. Since the promising results obtained with ASCs in preclinical studies of periodontal diseases arouse the curiosity of maxillofacial and dental surgeons, we decided to identify a novel source of ASCs, i.e, the buccal fat pad, convenient for these specialists. For this purpose, we studied human adipose derived-stem cells from buccal fat pad (BFP-ASCs), comparing them with cells from the subcutaneous adipose tissue (SC-ASCs) of the same donor (n=2). In parallel, considering the need for preclinical studies in which the effect of allogenic cells should be tested, and swine as an accepted animal model in tissue engineering applications, we also characterized porcine cells (n=6). With preclinical and clinical application prospective, we also investigated ASC interactions with oral tissues, natural and synthetic scaffolds and Amelogenin, an oral bioactive molecule. First of all, we showed that it is feasible to isolate ASCs even starting from very limited amounts of tissue (0,5 ml) and that the cellular yield is influenced by species, but not by the site of harvesting (1.1x105±1.4x104 human BFP-ASCs/ml and 1.15x105±7.1x103 human SC-ASCs/ml; 3.0x104±9.3x103 porcine BFP-ASCs/ml and 5.5x104±3.3x104 porcine SC-ASCs/ml). Despite the lower yield, the pASCs great proliferation rate allows to obtain high number of cells (potentially, 108 - 109) after few (3, 4) passages in culture. After the isolation, a great amount of cells deriving from all the tissues, adhered to cell culture plates showing the MSC fibroblast like morphology, with only mild shape differences constituted by the higher elongation and dimension of human SC-ASCs. Moreover, all the cells are easily expandable and showed good clonogenic ability at early passages. Cells of the same species, from both the harvesting site, displayed the same surface markers profile, that, in particular for human ASCs, was the typical one of hMSC (CD90+, CD105+, CD73+, CD14-, CD31-, and CD34-). Human and porcine BFP-ASCs, as SC-ASCs, are multipotent; indeed, when induced towards osteogenic and adipogenic lineages, they up-regulated significantly ALP activity, collagen and calcified extracellular matrix deposition and lipid vacuoles productions, respectively, already after 14 days of differentiation in vitro. Next, since cell/scaffold interaction is fundamental for the outcome of a tissue engineering approach, in sight of a preclinical study, we combined porcine BFP and SC-ASCs to both clinical grade (titanium) and innovative [silicon carbide–plasma-enhanced chemical vapor deposition (SIC-PECVD)] biomaterials, and studied cell adhesion and their differentiation ability. All the cells nicely grew on both scaffolds and, when osteoinduced, significantly increased the amount of calcified ECM compared to control cells; interestingly, titanium is osteoinductive even per se on pASCs (+284% and +91 for BFP- and SC-ASCs). Considering the importance of cell interaction with tissue of the lesion site, and with materials commonly used during surgical practices, we studied human BFP- and SC-ASC adherence to several supports. SEM analysis confirmed that both cell type nicely stick on alveolar bone, periodontal ligament, collagen membrane and polyglycolic acid filaments. Finally, we found that amelogenin, the most abundant enamel matrix protein seems to be an early osteoinductive factor for BFP-hASCs, whereas this effect is not manifested for SC-hASCs. For future cellular therapy, and since the use of FBS pose the risk of xenogenic contaminations leading to immunological complications during transplant, we tested cells growth in the presence of autologous supplements. Interestingly, both hASCs adapted rapidly to human serum, increasing their proliferation rates compared to standard culture condition, while porcine autologous or heterologous sera, did not improve pASC growth. In conclusion, we identified a cell population derived from a tissue easily available to dentists and maxillofacial surgeons, whose multipotent features and interaction with clinical grade scaffolds make proper candidate for future uses in tissue engineering approaches of periodontal diseases. In parallel, part of my PhD project was focused on the study of a critical osteochondral defect regeneration performed in a large animal preclinical model. The main obstacles for clinicians in treating this defect arises from the disparity concerning anatomy, composition and, most importantly, rate of healing of the articular cartilage (AC) and the subchondral bone. The key points of our study are the use of an innovative hydrogel of oligo(polyethylene glycol)fumarate (OPF) to fill the osteochondral defect, and of either porcine, or human ASCs, to create bioconstructs to be implanted in non-immuno-compromised minipigs. In particular, four critical osteochondral defects (diameter 9mm, depth 8mm) were created in the peripheral part of the trochlea of seven animals (defect n=28), and then treated with the different pre-made constructs. Untreated defects and defects filled by just scaffold were included as controls. No side-effects have been observed during the six-moths follow-up. At the end of this period, animals were sacrificed and knees explanted. Gross appearance analyses showed quite satisfactory filling of all the lesions, with the exception of one animal, whose joint appeared infected and not healed. MRI analyses revealed that in all the scaffold treated groups an overall improvement of the tissue quality at the osteochondral lesion site, was induced. More accurate evaluations (histological and immunohistochemistry analyses) revealed that some important tissue features were significantly improved by the association of OPF and ASCs. Indeed, regarding the subchondral bone, in all the OPF+ASCs groups, a mature bone appeared, with higher deposition of collagen type I compared to untreated or unseeded OPF groups. Moreover, the use of ASCs associated to scaffolds induced an improvement in newly formed cartilage features such as collagen type II deposition, and histological scores associated to these samples indicated a significant increase in matrix staining, tissue morphology and formation of tidemark, together with a reduction in vascularisation (a positive aspect in cartilage) compared to unseeded scaffolds. However, the histology indicated that in all the samples cartilage regeneration was still immature, most likely due to the limited time of follow up and/or the insufficient stimuli for cartilage complete regeneration. Despite this, biomechanical tests revealed that the neo-cartilage found in the cell-loaded scaffold groups possessed poroelastic behaviour, as well as indentation modulus and creep curves comparable to native cartilage. This important result suggest that the ASC presence at the lesion site, is able to enhance newly formed cartilage functionality. In conclusion, this in vivo study provide the evidence that both porcine and human adipose-derived stem cells associated to OPF hydrogel improve osteochondral defect regeneration, even though, at the moment, we are not able to define if the implanted ASCs are responsible per se of the new tissue formation or if they help spontaneous regeneration process by paracrine actions.