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\ub11.4x104 human BFP-ASCs/ml and 1.15x105\ub17.1x103 human SC-ASCs/ml; 3.0x104\ub19.3x103 porcine BFP-ASCs/ml and 5.5x104\ub13.3x104 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\u2013plasma-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

Differential Proteomic Analysis Predicts Appropriate Applications for the Secretome of Adipose-Derived Mesenchymal Stem/Stromal Cells and Dermal Fibroblasts

The adult stem cell secretome is currently under investigation as an alternative to cell-based therapy in regenerative medicine, thanks to the remarkable translational opportunity and the advantages in terms of handling and safety. In this perspective, we recently demonstrated the efficient performance of the adipose-derived mesenchymal stem/stromal cell (ASC) secretome in contrasting neuroinflammation in a murine model of diabetic neuropathy, where the administration of factors released by dermal fibroblasts (DFs) did not exert any effect. Up to now, the complex mixture of the constituents of the conditioned medium from ASCs has not been fully deepened, although its appropriate characterization is required in the perspective of a clinical use. Herein, we propose the differential proteomic approach for the identification of the players accounting for the functional effects of the cell secretome with the aim to unravel its appropriate applications. Out of 967 quantified proteins, 34 and 62 factors were found preponderantly or exclusively secreted by ASCs and DFs, respectively. This approach led to the recognition of distinct functions related to the conditioned medium of ASCs and DFs, with the former being involved in the regulation of neuronal death and apoptosis and the latter in bone metabolism and ossification. The proosteogenic effect of DF secretome was validated in vitro on human primary osteoblasts, providing a proof of concept of its osteoinductive potential. Besides discovering new applications of the cell type-specific secretome, the proposed strategy could allow the recognition of the cocktail of bioactive factors which might be responsible for the effects of conditioned media, thus providing a solid rationale to the implementation of a cell-free approach in several clinical scenarios involving tissue regeneration

Hypoxia promotes the inflammatory response and stemness features in visceral fat stem cells from obese subjects

Low-grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of 2 subpopulations of adipose-derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of 2 subpopulations of ASC from the same isolation sites of non-obese individuals. In particular, the behavior of ASC of obese vs non-obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV-ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation-related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue-specific NF-kB activation and an NF-kB-mediated increase in inflammatory and fibrogenic responses. Moreover, obV-ASC, which showed a less stem-like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV-ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF-kB inhibitors. These novel findings suggest that the deregulated hyper-responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low-grade chronic inflammation, which has been implicated in obesity-related morbidity

Does freeze-thawing influence the effects of platelet concentrates? An in vitro study on human adipose-derived stem cells

Human adipose-derived stem cells (hASCs) have been proposed as a possible therapy for tissue regeneration in aesthetic, plastic, and reconstructive surgery. Today, platelet concentrates are used in a wide range of disciplines, but their storage has become a controversial aspect. The purpose of this in vitro study was to evaluate the effect of plasma rich in growth factors (PRGF), after a freeze-thawing cycle, on the proliferation and biological activity of progenitor cells involved in soft tissue healing. Different formulations of activated PRGF were added to hASCs cultured in serum-free medium. Cell proliferation was assessed by MTT test and cell count up to 7 and 12-day incubation. Osteo-differentiation ability of hASCs was also tested after 7 and 14-day incubation by alkaline phosphatase assay. The effects of 4 PRGF preparations (fresh/frozen and with/without platelets) were compared with corresponding formulations of plasma poor in growth factors and with standard medium. hASCs cultured in the presence of platelet concentrates increased proliferation rate with respect to cells grown in standard medium without significant differences among all the tested plasma formulations on cell viability up to 12 days of culture. PRGF activity is preserved after cryopreservation and platelet-rich preparations promoted osteo-differentiation of hASCs at day 7. In conclusion, PRGF supports the proliferation and the differentiation of progenitor cells in vitro also when applied after cryopreservation. Platelet concentrates, either alone or in combination with mesenchymal stem cells, might be a valuable tool in the field of tissue regeneration

Nitrogen Containing Bisphosphonates Impair the Release of Bone Homeostasis Mediators and Matrix Production by Human Primary Pre-Osteoblasts

Bisphosphonates (BPs) represent the first-line treatment for a wide array of bone disorders. Despite their well-known action on osteoclasts, the effects they induce on osteoblasts are still unclear. In order to shed light on this aspect we evaluated the impact of two nitrogen containing bisphosphonates, Alendronate (ALN) and Zoledronate (ZOL), on human primary pre-osteoblasts. At first, we showed an inhibitory effect on cell viability and alkaline phosphatase activity starting from \u3bcM concentrations of both drugs. In addition, an inhibitory trend on mineralized nodules deposition was observed. Then low doses of both ALN and ZOL rapidly increased the release of the pro-inflammatory mediators TNF\u3b1 and IL-1\u3b2, while increased DKK-1 and Sclerostin, both inhibitors of osteoblastogenesis. Finally, ALN and 10-7M ZOL decreased the expression of type I Collagen and Osteopontin, while both drugs slightly stimulated SPARC production. With these results, we would like to suggest a direct inhibitory action on bone-forming cells by nitrogen containing bisphosphonates

Porcine adipose-derived stem cells from buccal fat pad and subcutaneous adipose tissue for future preclinical studies in oral surgery

Introduction: Adipose-derived stem cells (ASCs) are progenitor cells used in bone tissue engineering and regenerative medicine. Despite subcutaneous adipose tissue being more abundant, the buccal fat pad (BFP) is easily accessible for dentists and maxillofacial surgeons. For this reason, considering the need for preclinical study and the swine as an optimal animal model in tissue engineering applications, we compared the features of porcine ASCs (pASCs) from both tissue-harvesting sites. Methods: ASCs were isolated from interscapular subcutaneous adipose tissue (ScI) and buccal fat pads of six swine. Cells were characterized for their stemness and multipotent features. Moreover, their osteogenic ability when cultured on titanium disks and silicon carbide-plasma-enhanced chemical vapor-deposition fragments, and their growth in the presence of autologous and heterologous serum were also assessed. Results: Independent of the harvesting site, no differences in proliferation, viability, and clonogenicity were observed among all the pASC populations. Furthermore, when induced toward osteogenic differentiation, both ScI- and BFP-pASCs showed an increase of collagen and calcified extracellular matrix (ECM) production, alkaline phosphatase activity, and osteonectin expression, indicating their ability to differentiate toward osteoblast-like cells. In addition, they differentiated toward adipocyte-like cells, and chondrogenic induced pASCs were able to increase glycosaminoglycans (GAGs) production over time. When cells were osteoinduced on synthetic biomaterials, they significantly increased the amount of calcified ECM compared with control cells; moreover, titanium showed the osteoinductive effect on pASCs, also without chemical stimuli. Finally, these cells grew nicely in 10% FBS, and no benefits were produced by substitution with swine serum. Conclusions: Swine buccal fat pad contains progenitor cells with mesenchymal features, and they also osteo-differentiate nicely in association with synthetic supports. We suggest that porcine BFP-ASCs may be applied in preclinical studies of periodontal and bone-defect regeneration

Mesenchymal stem cells from Bichat's Fat pad: in vitro comparison with adipose-derived stem cells from subcutaneous tissue

Adipose-derived stem/stromal cells (ASCs) are progenitor cells used in bone tissue engineering and regenerative medicine. Since Bichat's fat pad is easily accessible for dentists and maxillo-facial surgeons, we compared the features of ASCs from Bichat's fat pad (BFP-ASCs) with human ASCs from subcutaneous adipose tissue (SC-ASCs). BFP-ASCs isolated from a small amount of tissue were characterized for their stemness and multidifferentiative ability. They showed an important clonogenic ability and the typical mesenchymal stem cell immunophenotype. Moreover, when properly induced, osteogenic and adipogenic differentiation markers, such as alkaline phosphatase activity, collagen deposition and lipid vacuoles formation, were promptly observed. Growth of both BFP-ASCs and SC-ASCs in the presence of human serum and their adhesion to natural and synthetic scaffolds were also assessed. Both types of ASCs adapted rapidly to human autologous or heterologous sera, increasing their proliferation rate compared to standard culture condition, and all the cells adhered finely to bone, periodontal ligament, collagen membrane, and polyglycol acid filaments that are present in the oral cavity or are commonly used in oral surgery. At last, we showed that amelogenin seems to be an early osteoinductive factor for BFP-ASCs, but not SC-ASCs, in vitro. We conclude that Bichat's fat pad contains BFP-ASCs with stemness features that are able to differentiate and adhere to biological supports and synthetic materials. They are also able to proliferate in the presence of human serum. For all these reasons we propose BFP-ASCs for future therapies of periodontal defects and bone regeneration

Expression of neural markers by undifferentiated mesenchymal-like stem cells from different sources

The spontaneous expression of neural markers, already demonstrated in bone marrow (BM) mesenchymal stem cells (MSCs), has been considered as evidence of the MSCs' predisposition to differentiate toward neural lineages, supporting their use in stem cell-based therapy for neural repair. In this study we have evaluated, by immunocytochemistry, immunoblotting, and flow cytometry experiments, the expression of neural markers in undifferentiated MSCs from different sources: human adipose stem cells (hASCs), human skin-derived mesenchymal stem cells (hS-MSCs), human periodontal ligament stem cells (hPDLSCs,) and human dental pulp stem cells (hDPSCs). Our results demonstrate that the neuronal markers \u3b2III-tubulin and NeuN, unlike other evaluated markers, are spontaneously expressed by a very high percentage of undifferentiated hASCs, hS-MSCs, hPDLSCs, and hDPSCs. Conversely, the neural progenitor marker nestin is expressed only by a high percentage of undifferentiated hPDLSCs and hDPSCs. Our results suggest that the expression of \u3b2III-tubulin and NeuN could be a common feature of stem cells and not exclusive to neuronal cells. This could result in a reassessment of the use of \u3b2III-tubulin and NeuN as the only evidence proving neuronal differentiation. Further studies will be necessary to elucidate the relevance of the spontaneous expression of these markers in stem cells

Adult stem cell as new advanced therapy for experimental neuropathic pain treatmen

Neuropathic pain (NP) is a highly invalidating disease resulting as consequence of a lesion or disease affecting the somatosensory system. All the pharmacological treatments today in use give a long lasting pain relief only in a limited percentage of patients before pain reappears making NP an incurable disease. New approaches are therefore needed and research is testing stem cell usage. Several papers have been written on experimental neuropathic pain treatment using stem cells of different origin and species to treat experimental NP. The original idea was based on the capacity of stem cell to offer a totipotent cellular source for replacing injured neural cells and for delivering trophic factors to lesion site; soon the researchers agreed that the capacity of stem cells to contrast NP was not dependent upon their regenerative effect but was mostly linked to a bidirectional interaction between the stem cell and damaged microenvironment resident cells. In this paper we review the preclinical studies produced in the last years assessing the effects induced by several stem cells in different models of neuropathic pain. The overall positive results obtained on pain remission by using stem cells that are safe, of easy isolation, and which may allow an autologous transplant in patients may be encouraging for moving from bench to bedside, although there are several issues that still need to be solved

Systemic administration of human adipose-derived stem cells reverts nociceptive hypersensitivity in an experimental model of neuropathy

Over the last decade it has been proved that Mesenchymal Stem Cells (MSCs) elicit anti-inflammatory effects. Mesenchymal Stem Cells from adipose tissue (hASCs) differentiate into cells of mesodermal lineage and trans-differentiate into ectodermal origin cells. Though there are various etiologies to chronic pain, one common feature is that painful states are associated with increased inflammation. We believe in hASCs as an therapeutic tool also in pathologies involving neuro-inflammation and neuronal-tissue damage. We have investigated the effect of hASCs injected in a model of neuropathic pain (mouse sciatic nerve Chronic Constriction Injury-CCI). hASCs from 5 donors were characterized, and no major differences were depicted. hASCs were cryopreserved and grown on demand. 1x106, 3x106 and 6x106 hASCs were intravenously injected into normal immunocompetent mice. No mouse died and no macroscopic toxicity or behavioral changes were observed, confirming the safety of hASCs. hASCs, i.v. injected into C57BL/6 mice, when the neuropathic pain was already established, induced a significant reduction in mechanical allodynia and a complete reversion of thermal hyperalgesia in a dose response fashion, already 1 day after administration. Moreover, the hASCs effect can be boosted by repeated administrations, allowing a prolonged therapeutic effect. Treatment decreased the level of the CCI-induced pro-inflammatory cytokine IL-1\u3b2 and activated the anti-inflammatory cytokine IL-10 in the lesioned nerve. hASC treatment also restored normal inducible Nitric Oxide Synthase (iNOS) expression in the CCI animals spinal cord. Our data suggest that hASCs are worthy further studies as anti-inflammatory therapy in the treatment of neuropathic pain or chronic inflammatory diseases