Different Sources of Mesenchymal Stem Cells for Tissue Regeneration: A Guide to Identifying the Most Favorable One in Orthopedics and Dentistry Applications.

The success of regenerative medicine in various clinical applications depends on the appropriate selection of the source of mesenchymal stem cells (MSCs). Indeed, the source conditions, the quality and quantity of MSCs, have an influence on the growth factors, cytokines, extracellular vesicles, and secrete bioactive factors of the regenerative milieu, thus influencing the clinical result. Thus, optimal source selection should harmonize this complex setting and ensure a well-personalized and effective treatment. Mesenchymal stem cells (MSCs) can be obtained from several sources, including bone marrow and adipose tissue, already used in orthopedic regenerative applications. In this sense, for bone, dental, and oral injuries, MSCs could provide an innovative and effective therapy. The present review aims to compare the properties (proliferation, migration, clonogenicity, angiogenic capacity, differentiation potential, and secretome) of MSCs derived from bone marrow, adipose tissue, and dental tissue to enable clinicians to select the best source of MSCs for their clinical application in bone and oral tissue regeneration to delineate new translational perspectives. A review of the literature was conducted using the search engines Web of Science, Pubmed, Scopus, and Google Scholar. An analysis of different publications showed that all sources compared (bone marrow mesenchymal stem cells (BM-MSCs), adipose tissue mesenchymal stem cells (AT-MSCs), and dental tissue mesenchymal stem cells (DT-MSCs)) are good options to promote proper migration and angiogenesis, and they turn out to be useful for gingival, dental pulp, bone, and periodontal regeneration. In particular, DT-MSCs have better proliferation rates and AT and G-MSC sources showed higher clonogenicity. MSCs from bone marrow, widely used in orthopedic regenerative medicine, are preferable for their differentiation ability. Considering all the properties among sources, BM-MSCs, AT-MSCs, and DT-MSCs present as potential candidates for oral and dental regeneration.

3D Bioprinting of Human Adipose-Derived Stem Cells and Their Tenogenic Differentiation in Clinical-Grade Medium.

Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 × 106 ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-ß3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE.

Adipose-derived stromal cell secretome reduces TNFα-induced hypertrophy and catabolic markers in primary human articular chondrocytes.

Recent clinical trials show the efficacy of Adipose-derived Stromal Cells (ASCs) in contrasting the osteoarthritis scenario. Since it is quite accepted that ASCs act predominantly through a paracrine mechanism, their secretome may represent a valid therapeutic substitute. The aim of this study was to investigate the effects of ASC conditioned medium (ASC-CM) on TNFα-stimulated human primary articular chondrocytes (CHs). CHs were treated with 10 ng/ml TNFα and/or ASC-CM (1:5 recipient:donor cell ratio). ASC-CM treatment blunted TNFα-induced hypertrophy, reducing the levels of Osteocalcin (-37%), Collagen X (-18%) and MMP-13 activity (-61%). In addition, it decreased MMP-3 activity by 59%. We showed that the reduction of MMP activity correlates to the abundance of TIMPs (Tissue Inhibitors of MMPs) in ASC secretome (with TIMP-1 exceeding 200 ng/ml and TIMP-2/3 in the ng/ml range) rather than to a direct down-modulation of the expression and/or release of these proteases. In addition, ASC secretome contains high levels of other cartilage protecting factors, i.e. OPG and DKK-1. ASC-CM comprises cartilage-protecting factors and exerts anti-hypertrophic and anti-catabolic effects on TNFα-stimulated CHs in vitro. Our results support a future use of this cell-derived but cell-free product as a therapeutic approach in the management of osteoarthritis.

Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs.

Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFß-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFß3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells.

Dose-Related and Time-Dependent Development of Collagenase-Induced Tendinopathy in Rats.

Tendinopathy is a big burden in clinics and it represents 45% of musculoskeletal lesions. Despite the relevant social impact, both pathogenesis and development of the tendinopathy are still under-investigated, thus limiting the therapeutic advancement in this field. The purpose of this study was to evaluate the dose-dependent and time-related tissue-level changes occurring in a collagenase-induced tendinopathy in rat Achilles tendons, in order to establish a standardized model for future pre-clinical studies. With this purpose, 40 Sprague Dawley rats were randomly divided into two groups, treated by injecting collagenase type I within the Achilles tendon at 1 mg/mL (low dose) or 3 mg/mL (high dose). Tendon explants were histologically evaluated at 3, 7, 15, 30 and 45 days. Our results revealed that both the collagenase doses induced a disorganization of collagen fibers and increased the number of rounded resident cells. In particular, the high dose treatment determined a greater neovascularization and fatty degeneration with respect to the lower dose. These changes were found to be time-dependent and to resemble the features of human tendinopathy. Indeed, in our series, the acute phase occurred from day 3 to day 15, and then progressed towards the proliferative phase from day 30 to day 45 displaying a degenerative appearance associated with a very precocious and mild remodeling process. The model represents a good balance between similarity with histological features of human tendinopathy and feasibility, in terms of tendon size to create lesions and costs when compared to other animal models. Moreover, this model could contribute to improve the knowledge in this field, and it could be useful to properly design further pre-clinical studies to test innovative treatments for tendinopathy.

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.

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.

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.

In vitro characterization of stem/progenitor cells from semitendinosus and gracilis tendons as a possible new tool for cell-based therapy for tendon disorders.

PURPOSE: this study was conducted to characterize tendon stem/progenitor cells (TSPCs) isolated from human semitendinosus and gracilis tendons in terms of stemness properties and multi-differentiation potential. METHODS: TSPCs were isolated from waste portions of semitendinosus and gracilis tendons from three donors who underwent anterior cruciate ligament reconstruction. TSPCs were plated in culture until passage 4, when experiments to assess cell proliferation, viability and clonogenic ability were performed. The immunophenotype of TSPCs was evaluated by cytofluorimetric analysis. The in vitro osteogenic, chondrogenic, adipogenic and tenogenic potential was evaluated using biochemical, histological and gene expression analysis to detect specific differentiation markers. Statistical analysis was performed using Student's t-test. RESULTS: after a few passages in culture the cell populations showed a homogeneous fibroblast-like morphology typical of mesenchymal stem cells. The average doubling time of TSPCs increased from 52.4±4.8 at passage 2 to 100.8±23.4 hours at passage 4. The highest percentage of colonies was also found at passage 4 (4.7±2.3%). TSPCs showed the typical mesenchymal phenotype, with high expression of CD73, CD90 and CD105 and no expression of CD34 and CD45. Cells induced to differentiate toward osteogenic lineage showed significant upregulations of ALP activity (+189%, p<0.05) and calcified matrix deposition (+49%, p<0.05) compared with undifferentiated cells; culture in chondrogenic medium also provoked a significant increase in glycosaminoglycan levels (+108%, p<0.05). On the other hand, TSPCs were not able to respond to adipogenic stimuli. Scleraxis gene expression and decorin gene expression, considered tenogenic markers, were already very high in control cells, and culture in tenogenic medium further increased these values although not significantly. CONCLUSIONS: our data show that it is possible to isolate TSPCs from very small fragments of tissue and that they show the typical features of MSCs and multi-differentiation potential, above all toward osteogenic and chondrogenic lineages. CLINICAL RELEVANCE: this study can be considered one of the first attempts to clarify the biology of tendon cell populations, focusing in particular on the potential applicability of this cell source for future regenerative medicine purposes.

Adipose-derived stem cells and rabbit bone regeneration: histomorphometric, immunohistochemical and mechanical characterization.

BACKGROUND: In the last few years, several attempts have been made to treat large bone loss, including the use of tissue engineering with osteoinductive scaffolds and cells. This study highlights the role of mesenchymal stem cells from adipose tissue (ASCs; adipose-derived stem cells) in a rabbit bone regeneration model. METHODS: We compared the neoformed bone tissues achieved by treating critical tibial defects with either hydroxyapatite alone (HA, group I) or hydroxyapatite-autologous ASC constructs (ASCs-HA, group II), investigating their histomorphometric, immunohistochemical and biomechanical properties. RESULTS: After eight weeks of follow-up, we observed advanced maturation and a spatial distribution of new bone that was more homogeneous in the inner parts of the pores in group II, not just along the walls (as seen in group I). The new tissue expressed osteogenic markers, and biomechanical tests suggested that the newly formed bone in group II had a higher mineral content than that in group I. Although variability in differentiation was observed among the different cell populations in vitro, no differences in bone healing were observed in vivo; the variability seen in vitro was probably due to local microenvironment effects. CONCLUSIONS: Tibial defects treated with rabbit ASCs-HA showed an improved healing process when compared to the process that occurred when only the scaffold was used. We suggest that implanted ASCs ameliorate the bone reparative process either directly or by recruiting resident progenitor cells.

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.