D. Stanco

Isolation, characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models.

One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adipose-derived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous pre-clinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.

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.

Soft-Focused Extracorporeal Shock Waves Increase the Expression of Tendon-Specific Markers and the Release of Anti-inflammatory Cytokines in an Adherent Culture Model of Primary Human Tendon Cells

Focused extracorporeal shock waves have been found to upregulate the expression of collagen and to initiate cell proliferation in healthy tenocytes and to positively affect the metabolism of tendons, promoting the healing process. Recently, soft-focused extracorporeal shock waves have also been found to have a significant effect on tissue regeneration. However, very few in vitro reports have dealt with the application of this type of shock wave to cells, and in particular, no previous studies have investigated the response of tendon cells to this impulse. We devised an original model to investigate the in vitro effects of soft-focused shock waves on a heterogeneous population of human resident tendon cells in adherent monolayer culture. Our results indicate that soft-focused extracorporeal shock wave treatment (0.17 mJ/mm(2)) is able to induce positive modulation of cell viability, proliferation and tendon-specific marker expression, as well as release of anti-inflammatory cytokines. This could prefigure a new rationale for routine employment of soft-focused shock waves to treat the failed healing status that distinguishes tendinopathies.

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 two subpopulations of adipose‐derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of two subpopulations of ASC from the same isolation sites of non‐obese individuals. In particular, the behavior of ASC of obese versus 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. J. Cell. Physiol. 231: 668–679, 2016.

Multidifferentiation potential of human mesenchymal stem cells from adipose tissue and hamstring tendons for musculoskeletal cell-based therapy

AIM Adipose-derived stem cells (ASCs) have been deeply characterized for their usefulness in musculoskeletal tissue regeneration; recently, other mesenchymal stem cell (MSC) sources have also been proposed. This study compares for the first time human tendon stem/progenitor cells isolated from hamstring tendons with human ASCs. MATERIALS & METHODS Human TSPCs and ASCs were isolated from hamstring tendon portions and adipose tissue of healthy donors undergoing ACL reconstruction or liposuction, respectively (n = 7). Clonogenic ability, immunophenotype and multi-differentiation potential were assessed and compared. RESULTS Both populations showed similar proliferation and clonogenic ability and expressed embryonic stem cell genes and MSC surface markers. Tendon stem/progenitor cells showed lower adipogenic and osteogenic ability, but after the chondrogenic differentiation, they produced more abundant glycosaminoglycans and expressed higher levels of aggrecan with regards to ASCs. The tenogenic induction with BMP-12 upregulated SCX and DCN gene expression in both populations. CONCLUSION Our results demonstrate that waste hamstring tendon fragments could represent a convenient MSC source for musculoskeletal regenerative medicine.

Different culture conditions affect the growth of human tendon stem/progenitor cells (TSPCs) within a mixed tendon cells (TCs) population

BackgroundTendon resident cells (TCs) are a mixed population made of terminally differentiated tenocytes and tendon stem/progenitor cells (TSPCs). Since the enrichment of progenitors proportion could enhance the effectiveness of treatments based on these cell populations, the interest on the effect of culture conditions on the TSPCs is growing.In this study the clonal selection and the culture in presence or absence of basic fibroblast growth factor (bFGF) were used to assess their influences on the stemness properties and phenotype specific features of tendon cells.MethodsCells cultured with the different methods were analyzed in terms of clonogenic and differentiation abilities, stem and tendon specific genes expression and immunophenotype at passage 2 and passage 4.ResultsThe clonal selection allowed to isolate cells with a higher multi-differentiation potential, but at the same time a lower proliferation rate in comparison to the whole population. Moreover, the clones express a higher amounts of stemness marker OCT4 and tendon specific transcription factor Scleraxis (SCX) mRNA, but a lower level of decorin (DCN). On the other hand, the number of cells obtained by clonal selection was extremely low and most of the clones were unable to reach a high number of passages in cultures.The presence of bFGF influences TCs morphology, enhance their proliferation rate and reduce their clonogenic ability. Interestingly, the expression of CD54, a known mesenchymal stem cell marker, is reduced in presence of bFGF at early passages. Nevertheless, bFGF does not affect the chondrogenic and osteogenic potential of TCs and the expression of tendon specific markers, while it was able to downregulate the OCT4 expression.ConclusionThis study showed that clonal selection enhance progenitors content in TCs populations, but the extremely low number of cells produced with this method could represent an insurmountable obstacle to its application in clinical approaches. We observed that the addition of bFGF to the culture medium promotes the maintenance of a higher number of differentiated cells, reducing the proportion of progenitors within the whole population. Overall our findings demonstrated the importance of the use of specific culture protocols to obtain tendon cells for possible clinical applications.