Systematic review on spheroids from adipose-derived stem cells: Spontaneous or artefact state?

Three-dimensional (3D) cell cultures represent the spontaneous state of stem cells with specific gene and protein molecular expression that are more alike the in vivo condition. In vitro two-dimensional (2D) cell adhesion cultures are still commonly employed for various cellular studies such as movement, proliferation and differentiation phenomena; this procedure is standardized and amply used in laboratories, however their representing the original tissue has recently been subject to questioning. Cell cultures in 2D require a support/substrate (flasks, multiwells, etc.) and use of fetal bovine serum as an adjuvant that stimulates adhesion that most likely leads to cellular aging. A 3D environment stimulates cells to grow in suspended aggregates that are defined as "spheroids." In particular, adipose stem cells (ASCs) are traditionally observed in adhesion conditions, but a recent and vast literature offers many strategies that obtain 3D cell spheroids. These cells seem to possess a greater ability in maintaining their stemness and differentiate towards all mesenchymal lineages, as demonstrated in in vitro and in vivo studies compared to adhesion cultures. To date, standardized procedures that form ASC spheroids have not yet been established. This systematic review carries out an in-depth analysis of the 76 articles produced over the past 10 years and discusses the similarities and differences in materials, techniques, and purposes to standardize the methods aimed at obtaining ASC spheroids as already described for 2D cultures.

Human Spheroids from Adipose-Derived Stem Cells Induce Calvarial Bone Production in a Xenogeneic Rabbit Model.

ABSTRACT: Calvarial defects can result from several causes. Tissue engineering hold the potential to restore native form and protective function. We have recently shown that stemness and differentiation ability of spheroids from adipose-derived stem cells (S-ASCs) promotes osteoblasts growth within Integra in a small vertebral lesion. In our study, we aimed to test osteogenic potential of S-ASCs in aiding regeneration of a calvarial defect. Groups containing Integra showed increased bone regeneration at the calvarial defect-Integra interface compared with the control group. In particular, S-ASC-derived osteoblasts group showed a superior calvarial remodeling than undifferentiated S-ASCs group. Clusters of ossification were observed in these both groups with enhanced microvasculature density and fibrosis. In conclusion, seeding of S-ASCs in dermal regeneration templates enhanced bone healing in a rabbit calvarial defect model. These findings could prompt the elective use of S-ASCs with enhanced multilineage differentiation potential for tissue engineering purposes.

Immunomodulation in Vascularized Composite Allotransplantation: What Is the Role for Adipose-Derived Stem Cells?

Hand and face transplants are becoming increasingly common, recording progressively more penile, uterus, abdominal wall, and allotransplantation cases reported worldwide. Despite current protocols allow long-term survival of the allografts, the ultimate goal of donor-specific tolerance has not been achieved yet. In fact, the harmful adverse effects related to the lifelong administration of immunosuppressive agents are the main drawbacks for vascularized composite allotransplantations. Research is very active in investigating alternative methods to induce greater tolerance while minimizing toxicity. Adipose-derived stem cells (ASCs) represent promising cell therapies for immunomodulation in preclinical and clinical settings. Their clinical appeal is due to their easy harvest in large quantities through a noninvasive and well-accepted approach; they may well promote donor-specific tolerance and potentially reduce immunosuppression. Several experimental studies exist, but lacking review articles reporting current evidence. This work proposes a literature review on the immunomodulatory role of ASCs in vascularized composite allotransplantations. In vitro and in vivo evidence will be summarized. The role that cell passaging and upstream progenitors-the so-called spheroid ASCs-may play in modulating the immune response will also be discussed. Finally, this article will summarize current knowledge on biodistribution, migration, and homing of injected stem cells. This review may well provide useful information for preclinical and clinical studies, aiming at a breakthrough for donor-specific tolerance.

Spheroids from adipose-derived stem cells exhibit an miRNA profile of highly undifferentiated cells.

Two-dimensional (2D) cell cultures have been extensively used to investigate stem cell biology, but new insights show that the 2D model may not properly represent the potential of the tissue of origin. Conversely, three-dimensional cultures exhibit protein expression patterns and intercellular junctions that are more representative of their in vivo condition. Multiclonal cells that grow in suspension are defined as "spheroids," and we have previously demonstrated that spheroids from adipose-derived stem cells (S-ASCs) displayed enhanced regenerative capability. With the current study, we further characterized S-ASCs to further understand the molecular mechanisms underlying their stemness properties. Recent studies have shown that microRNAs (miRNAs) are involved in many cellular mechanisms, including stemness maintenance and proliferation, and adipose stem cell differentiation. Most studies have been conducted to identify a specific miRNA profile on adherent adipose stem cells, although little is still known about S-ASCs. In this study, we investigate for the first time the miRNA expression pattern in S-ASCs compared to that of ASCs, demonstrating that cell lines cultured in suspension show a typical miRNA expression profile that is closer to the one reported in induced pluripotent stem cells. Moreover, we have analyzed miRNAs that are specifically involved in two distinct moments of each differentiation, namely early and late stages of osteogenic, adipogenic, and chondrogenic lineages during long-term in vitro culture. The data reported in the current study suggest that S-ASCs have superior stemness features than the ASCs and they represent the true upstream stem cell fraction present in adipose tissue, relegating their adherent counterparts.

An algorithm to improve outcomes of radial forearm flap donor site.

Abstarct Background: Due to the high rate of donor site complications the Radial Forearm Flap (RFF) has lost ground in favor of the Antero-lateral tight flap (ALT) and other flaps. We have designed a reconstruction algorithm for reconstruction of its donor site. The goal of this study was to retrospectively evaluate the impact of this algorithm on RFF donor site complication rates. METHODS: The authors analyzed retrospectively 31 patients who underwent free radial forearm flap reconstruction between November 2009 and May 2013. Donor site complications were compared with data from patients treated before introdutction of the algorithm. Within the group were compared patients in which the flap was harvested suprafascial with those in which the flap was harvested as subfascial. RESULTS: Before application of the algorithm, there was a 23.3% complication rate at the RFF donor site, in our experience. After introduction of the algorithm, complication rate has dropped to 3.2%, consisting in a partial skin graft necrosis treated by local wound-care and healed without further intervention. CONCLUSIONS: Application of the algorithm described has led to a significant reduction in RFF donor site complication rates. This demonstrates that if flap donor sites are analyzed and tailor treated in the same way as primary defects are, instead of being given secondary importance and just grafted, outcomes improve.

Seeding nerve sutures with minced nerve-graft (MINE-G): a simple method to improve nerve regeneration in rats.

BACKGROUND: The aim of this study was to assess the effect of seeding the distal nerve suture with nerve fragments in rats. METHODS: On 20 rats, a 15 mm sciatic nerve defect was reconstructed with a nerve autograft. In the Study Group (10 rats), a minced 1 mm nerve segment was seeded around the nerve suture. In the Control Group (10 rats), a nerve graft alone was used. At 4 and 12 weeks, a walking track analysis with open field test (WTA), hystomorphometry (number of myelinated fibers (n), fiber density (FD) and fiber area (FA) and soleus and gastrocnemius muscle weight ratios (MWR) were evaluated. The Student t-test was used for statistical analysis. RESULTS: At 4 and 12 weeks the Study Group had a significantly higher n and FD (p = .043 and .033). The SMWR was significantly higher in the Study Group at 12 weeks (p = .0207). CONCLUSIONS: Seeding the distal nerve suture with nerve fragments increases the number of myelinated fibers, the FD and the SMWR. The technique seems promising and deserves further investigation to clarify the mechanisms involved and its functional effects.

Characteristics, Management Techniques, and Outcomes of the Most Common Soft-Tissue Hand Tumors: A Literature Review and Our Experience.

INTRODUCTION: Diagnosis of the hand's soft-tissue tumors is often difficult because of the different anatomic structures present in this region and yet clinicians must be able to distinguish typical benign entities from life-threatening or limb-threatening malignant diseases. MATERIALS AND METHODS: At the Department of Plastic and Reconstructive Surgery at the University of Palermo, 629 patients with hand tumors were studied. Treatment was surgical for all of them; also radiotherapy and chemotherapy were necessary based on the histological diagnosis. CONCLUSIONS: Our retrospective study with a literature review aims to present the most commonly observed soft-tissue hand lesions, analyzing their causes, their objective and instrumental evaluation, and their treatments.

Variability and reliability of the vastus lateralis muscle anatomy.

PURPOSE: The aims of this study are to investigate the variability of the morphological and neurovascular anatomy of the vastus lateralis (VL) muscle and to describe the relationships among its intramuscular partitions and with the other muscles of the quadriceps femoris. Clinical implications in its reliability as a flap donor are also discussed. METHODS: In 2012, the extra- and intramuscular neurovascular anatomy of the VL was investigated in 10 cadaveric lower limbs. In three specimens, the segmental arterial pedicles were injected with latex of different colors to point out their anastomotic connections. The morphological anatomy was investigated with regard to the mutual relationship of the three muscular partitions and the relation of the VL with the other muscles of the quadriceps femoris. RESULTS: The VL has a segmental morphological anatomy. However, the fibers of its three partitions interconnect individually and with the other bellies of the quadriceps femoris, particularly, in several variable portions with the vastus intermedius and mainly in the posterior part of the VL. The lateral circumflex femoral artery and its branches have variable origin, but demonstrate constant segmental distribution. Intramuscular dissection and colored latex injections show a rich anastomotic vascular network among the three partitions. CONCLUSIONS: Moderate variability exists in both the myological and the neurovascular anatomy of the VL. Despite this variability, the anatomy of the VL always has a constant segmental pattern, which makes the VL a reliable flap donor. Detailed knowledge of the VL anatomy could have useful applications in a broad clinical field.

Laser treatment of giant xanthelasma palpebrarum.

Xanthelasma palpebrarum is the most common cutaneous xanthoma. It typically presents in middle-aged and older adults, most often around the eyelids. The diagnosis is made clinically. Giant xanthelasmas palpebrarum are xanthelasmas that extensively affect the superior and inferior bilateral eyelids. Many techniques have been put forward for treating these lesions (surgical, laser, and chemical techniques), but we describe our experience in the treatment of giant xanthelasmas by ultrapulsed CO2 laser. Between 2009 and 2012, in the Division of Plastic and Reconstructive Surgery at the University of Palermo, 12 patients with giant xanthelasmas were treated using a CO2 laser. The laser parameters are as follows: frequency 20 Hz, energy 75 mJ, and power 1.5 W. Each laser session lasts 15 min; the treatment consists of three or four sessions that are carried out at intervals of 15 days. Patients were followed up after 2, 6, and 12 months. This technique is rapid and it is accepted very well by patients. The only disadvantage is a long healing time (10-15 days). The ultrapulsed CO2 laser, in experienced hands, is an excellent device that enables the complete removal of giant xanthelasmas with a minimally invasive but very effective technique.

Prophylactic chimera anterolateral thigh/vastus lateralis flap: preventing complications in high-risk head and neck reconstruction.

PURPOSE: In high-risk head and neck cases treated with tumor resection and associated radical neck dissection, orocutaneous fistulas and wound breakdowns in the neck are relatively frequent and can have serious consequences, such as carotid blowout syndrome (CBS), the need for salvage reoperations, and prolonged recovery time. The authors present the application of a prophylactic chimeric anterolateral thigh (ALT) and vastus lateralis (VL) flap to prevent complications. MATERIALS AND METHODS: A retrospective review was performed of a historical group (96 patients) of patients with head and neck cancer treated with tumor resection, radical neck dissection, and microsurgical reconstruction of the tumor site only and a prospective cohort (21 patients) in which a chimeric ALT-VL flap was used to simultaneously reconstruct the tumor site and sternocleidomastoid muscle to fill dead space and protect the carotid artery. RESULTS: The rate of complications was higher in the historical group: CBS occurred in 4.1% and orocutaneous fistulas in 11.5% of patients; 5.2% of patients required major salvage surgery for a wound complication. In the cohort group, no CBS or orocutaneous fistula occurred and no major salvage surgical procedure was needed. CONCLUSIONS: Prophylactic ALT-VL flaps in high-risk head and neck cancers provide adequate and long-lasting soft tissue coverage for the carotid artery, with minimal additional morbidity, and could be beneficial in preventing serious and life-threatening wound complications and the need for reoperation.

Reconstruction of nasal alar defects with freestyle facial artery perforator flaps.

In 2009, we have described the use of freestyle facial artery perforator flaps for one-stage nose reconstruction. Since then, several articles have reported the use of facial artery perforator flaps for nose reconstruction. The purpose of this article is to provide an update of the published technique after 10 years of experience. Since 2004, 21 patients have been treated with a freestyle facial artery perforator flap for one-stage reconstruction of the nasal ala. The flaps were 16 propellers, 4 V-Y, and 1 island transposition. A single venous congestion leading to a minor flap tip necrosis and a wound dehiscence was observed. All other flaps healed uneventfully. The V-Y design and multiple subunit reconstruction gave suboptimal results. It was concluded that indications for freestyle facial artery perforator flaps are total nasal alar subunit reconstruction or reconstruction of lateral alar defects when perforator anatomy allows. In these cases, freestyle facial artery perforator flaps are the first choice technique at our institution because they allow excellent results in one-stage operation. One-stage nasal ala reconstruction with freestyle facial artery perforator flaps.

A propeller flap for single-stage nose reconstruction in selected patients: supratrochlear artery axial propeller flap.

The paramedian forehead flap is the gold standard technique for nose reconstruction. It requires two different surgical operations which prolonged the postoperative dressing and care. We present our 5-year experience with a propeller flap based on the supratrochlear artery, which allows one-stage transfer of the forehead skin to the nose without the need for pedicle division. This technique is indicated in a selected group of patients who are not suitable for multiple-stage reconstructions because they have concurrent medical conditions, reduced mobility, or live far away from specialized medical centers. We have renamed this procedure as supratrochlear artery axial propeller flap, from the acronym STAAP flap, to stress the axial, well known and constant, vascularization of the flap. In the past 5 years, we have been performing 25 STAAP flaps; full-thickness nasal reconstruction was performed in 11 cases. The patients were 16 males and 9 females, with a mean age of 79.5 years. All patients had multiple comorbidities. Complete flap survival was observed in 23 cases and healing was complete in 7 days. In two cases, there was a partial distal necrosis of the flap treated conservatively. Cosmetic results were good and the patient's satisfaction was significant. These results indicate that the STAAP flap is a reliable and useful technique in selected cases, as old or noncompliant patients who benefit from a one-stage technique of nose reconstruction.

Advancement perforator cheek flap for aesthetic one-stage reconstruction of postoncological extended split-thickness defects of the nasal sidewall.

Aesthetic reconstruction of soft tissue nasal sidewall loss has an important influence on the appearance of the nose. The unique character of this subunit and the complex relationships with a number of different facial or nasal subunits make the excision of large tumors difficult to manage. Numerous techniques are described in the literature, but a primary reconstruction with a final good result is not often possible. The authors develop an advancement cheek flap for an aesthetic one-stage reconstruction of postoncological extended nasal sidewall defects. Between January 2009 and July 2012, 16 patients (mean age, 63.3 yr) underwent excision of skin tumors of nasal sidewall and immediate reconstruction with an advancement cheek flap nourished by perforators from the transverse facial branch of the superficial temporal artery. The tumors were excised with 0.4-0.6 cm lateral margins and defects size ranged from 2.6 × 2.6 cm to 3.5 × 5 cm. Oncological radicality was obtained in all cases. The aesthetic results were excellent in all patients. No scar revision was needed. The authors' advancement cheek flap can be considered the first choice for reconstruction of split-thickness defect of nasal sidewall larger than 2.5 cm because it reestablishes in one stage the nasal contour detail.

Spheroids of adipose derived stem cells show their potential in differentiating towards the angiogenic lineage.

INTRODUCTION: Adipose derived stem cells (ASCs) are a mesenchymal stem cell population of great scientific interest due to their abundance and easiness in obtaining them from adipose tissue. Recently, several techniques for three dimensional (3D) ASCs cultivation have been developed to obtain spheroids of adipose stem cells (SASCs). It was already proved that ASCs are able to differentiate towards the endothelial lineage thus, for the first time, we investigated the ability of our 3D SASCs to differentiate endothelially and the effects of not differentiated SASC secreted factors on specific cultured cells. MATERIALS AND METHODS: SASCs were differentiated with a specific medium towards endothelial lineage. Cell viability, gene and protein expression of typical endothelial markers were analysed. Moreover, tube formation, wound healing and migration assays were performed to investigate the ability in migration and angiogenic networks formation of endothelially differentiated cells. SASCs secretome were also tested. RESULTS: We showed the ability of SASCs to differentiate towards the endothelial lineage with an increase in cell viability of 15-fold and 8-fold at 14 and 21 days of differentiation respectively. Moreover, we showed the upregulation of VEGF-A and CD31 mRNAs of 9-fold and 1300-fold in SASCs endothelially differentiated cells, whilst protein expression was different. VEGF-A protein expression was upregulated whilst CD31 protein wasn't translated. In addition, ICAM1, VCAM1, ANGPT1, CD62E protein levels remain unchanged. SASCs were also able to organize themselves into angiogenic networks after 7 days of culturing themon ECMatrix. Secreted factors from undifferentiated 3D SASCs acted in a paracrine way on HUVECs and endothelially differentiated ASCs seeded on ECMatrix to promote angiogenic events. CONCLUSIONS: SASCs, thanks to their multilineage differentiation potential, also possess the ability to differentiate towards endothelial lineage and to organize themselves into angiogenic networks. Moreover, they are able to promote angiogenesis through their secreted factors.

An analysis of the immunomodulatory properties of human spheroids from adipose-derived stem cells.

AIMS: Current methods to induce tolerance following allotransplantation or in autoimmunity carry significant morbidity, and research is very active in investigating alternative methods which could minimize toxicity. Spheroids from adipose stem cells (SASCs) are increasingly gaining interest, they hold a great proliferative and differentiating potential. An immunomodulatory effect has not been investigated on SASCs yet. In this study, we analysed the immunomodulatory properties of SASCs and compared them to ADSCs. MAIN METHODS: Adipose stem cells (SASCs and ADSCs) and peripheral blood mononuclear cells (PBMCs) were collected from healthy individuals. We analysed the cytokine production and proliferation of T cells co-cultured with adipose samples or conditioned medium. KEY FINDINGS: SASCs modulated cytokines production and proliferation of heterologous and autologous T cells. In the heterologous assays, we observed a reduction of IFNγ and IL-17 production and an increase of IL-9 in γδ T cells. The soluble factors present in SASCs sovranatants were also able to induce a slight reduction of IFNγ and an increase of IL-9, IL-10 and IL-17 while they could not modulate the proliferative ability of γδ T cells. In the autologous assays, we observed a reduction of the proliferative ability of T cells in co-culture at different ratios with SASCs. Analysis of the SASCs secretome showed an increased IL-5, IL-10, IL-4 and IL-13 production compared to the ADSCs one, demonstrating greater anti-inflammatory properties. SIGNIFICANCE: Our preliminary results support the idea that SASCs exert more pronounced biological immune modulation compared to the classical adherent ADSCs, especially in heterologous experimental settings.

κ-Carrageenan and PVA blends as bioinks to 3D print scaffolds for cartilage reconstruction.

3D printing of polymeric scaffolds and autologous stem cells is a promising tool for damaged facial cartilage reconstruction surgeries. To this end, suitable bioinks are needed to generate scaffolds with the required morphological and functional features. We formulated hydrogel bioinks using k-Carrageen (kC) and poly(vinyl alcohol) (PVA) in three different weight ratios. The kC gives the systems the ability to undergo rapid sol-to-gel transitions upon cooling from 60 °C and above to body temperature, while the PVA is used as rheology modifier and porogen. The latter is crosslinked after molding or printing by freeze-thaw cycling for 1 day (FT1) or 5 days (FT5). To select the most suitable formulation for 3D printing, the sol-to-gel transition and the physico-chemical, mechanical and morphological properties of obtained hydrogels were studied. Moreover, the absence of cytotoxic effects of the material on SASCs was assessed in both stemness-preserving or chondro-inductive media. Printing trials were performed to identify optimal process parameters and co-printing and post-printing seeding approaches of SASCs were evaluated. Cells were found to be viable after co-printing and also after the FT1 treatment. Viable adherent cells were also found in the FT5 system, where cells were plated after freezing and thawing treatment.

Injectable xyloglucan hydrogels incorporating spheroids of adipose stem cells for bone and cartilage regeneration.

Cartilage or bone regeneration approaches based on the direct injection of mesenchymal stem cells (MSCs) at the lesion site encounter several challenges, related to uncontrolled cell spreading and differentiation, reduced cell viability and poor engrafting. This work presents a simple and versatile strategy based on the synergic combination of in-situ forming hydrogels and spheroids of adipose stem cells (SASCs) with great potential for minimally invasive regenerative interventions aimed to threat bone and cartilage defects. Aqueous dispersions of partially degalactosylated xyloglucan (dXG) are mixed with SASCs derived from liposuction and either a chondroinductive or an osteoinductive medium. The dispersions rapidly set into hydrogels when temperature is brought to 37 °C. The physico-chemical and mechanical properties of the hydrogels are controlled by polymer concentration. The hydrogels, during 21 day incubation at 37 °C, undergo significant structural rearrangements that support cell proliferation and spreading. In formulations containing 1%w dXG cell viability increases up to 300% for SASCs-derived osteoblasts and up to 1000% for SASCs-derived chondrocytes if compared with control 2D cultures. The successful differentiation into the target cells is supported by the expression of lineage-specific genes. Cell-cell and cell-matrix interactions are also investigated. All formulations resulted injectable, and the incorporated cells are fully viable after injection.

Cell quality evaluation with gene expression analysis of spheroids (3D) and adherent (2D) adipose stem cells.

Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (SASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by "cell-quality"; for this reason, the involvement of stress signals and cellular aging need to be further investigated. Here, we performed a comparative analysis of genes connected with stemness, aging, telomeric length and oxidative stress, in 3D and 2D primary cultures. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P < 0.001) in SASCs-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P < 0.001). The 3D and 2D cultures also had a different gene expression profile for genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P < 0.01 and P < 0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P < 0.05, P < 0.01 and P < 0.001) and presented a striking large variation in their cellular redox state. Based on our findings, we propose a "cell quality" model of SASCs, highlighting a precise molecular expression of several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance.