Edoardo Raposio

A novel and effective strategy for the isolation of adipose-derived stem cells: minimally manipulated adipose-derived stem cells for more rapid and safe stem cell therapy.

Summary: Adipose-derived stem cells are an ideal mesenchymal stem cell population for regenerative medical application. The isolation procedure is performed by mechanical isolation under a laminar air flow bench without using serum or animal-derived reagents; cells were characterized by flow cytometric analysis. Cell availability is improved compared with enzymatic digestion procedures. The adipose-derived stem cell mechanical isolating procedure presented here is easier, safer, cheaper, and faster than traditional currently performed enzymatic procedures.

A Comparative Study of Proliferation and Hepatic Differentiation of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells possess a lot of stem cell characteristics, so they may be considered a source of stem cell population. On the basis of that, we have investigated the hepatic potential of adipose-derived stem cells, obtained from liposuction, following two differentiation protocols. In the first procedure, medium was supplemented with epidermal growth factor (EGF), basic fibroblast growth factor, hepatocyte growth factor (HGF) and nicotinamide; the second involved the addition of factors such as dexametasone, EGF, insulin-transferrin-sodium selenite, HGF, dimethyl sulfoxide and oncostatin. In parallel, we carried out our study in the Hep G2 cell line, as human hepatic differentiated in vitro model. Immunocytochemical analysis and RT-PCR were performed using hepatic markers to evaluate cell differentiation. DNA content, MTT test and carboxyl fluorescein succinimidyl ester staining were carried out to evaluate cell proliferation. We reported the evidence of basal hepatic marker in undifferentiated adipose-derived stem cells, which confirmed their multipotency. A strong expression of albumin and α-fetoprotein was observed in hepatic-induced adipose-derived stem cells following both differentiation procedures. Morphological aspects of the two types of hepatic adipose-derived stem cells were alike. Proliferation index suggested that the first differentiation procedure promoted better growth than the second. These preliminary findings suggest adipose-derived stem cells may be induced into hepatic lineage, and the most significant difference between the two standard differentiation procedures concerns proliferation rate. This aspect is to be considered when adipose-derived stem cells are employed in research and clinical studies.

In vitro polydeoxyribonucleotide effects on human pre-adipocytes

Abstract.  Objectives: Adipose tissue is the most abundant and accessible source of adult stem cells. Human processed lipoaspirate contains pre‐adipocytes that possess one of the a characteristic pathways of multipotent adult stem cells and are able to differentiate in vitro into mesenchymal and also neurogenic lineages. Because stem cells have great potential for use in tissue repair and regeneration, it would be significant to be able to obtain large amounts of these cells in vitro. As demonstrated previously, purine nucleosides and nucleotides mixtures can act as mitogens for several cell types. The aim of this study was to evaluate the effects of polydeoxyribonucleotides (PDRN), at appropriate concentrations, on human pre‐adipocytes grown in a controlled medium, also using different passages, so as to investigate the relationship between the effect of this compound and cellular senescence, which is the phenomenon when normal diploid cells lose the ability to divide further. Materials and methods: Human pre‐adipocytes were obtained by liposuction. Cells from different culture passages (P6 and P16) were treated with PDRN at different experimental times. Cell number was evaluated for each sample by direct counting after trypan blue treatment. DNA assay and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide test were also carried out in all cases. Results and Conclusions: PDRN seemed to promote proliferation of human pre‐adipocytes at both passages, but cell population growth increased in pre‐adipocyte at P16, after 9 days as compared to control. Our data suggest that PDRN could act as a pre‐adipocyte growth stimulator.

Effects of galeotomies on scalp flaps.

The aim of the present study was to evaluate how much a relaxing incision of the galea aponeurotica affects the biomechanical properties of a scalp flap to quantify the surgery-related advantages provided by this procedure. Twenty scalp flaps, created by a reverse-Y incision down to and through the galea aponeurotica together with undermining (in the layer between the galea and the pericranium) to within 1 cm of the external auditory canal were studied. Data were collected by stepwise loading the scalp flaps before and after performing three full-thickness galeotomies lengthwise and parallel to the sagittal scalp incision. The tension/extension ratio characteristics were computed, and loading curves as well as mean stiffness values were measured. A statistically significant difference (−16.6 g per millimeter) was found between the slope (computed as Youngs modulus) of the curves obtained before and after performing the galeotomies. This value corresponded to a mean 40% reduction of the closing tension attained with each galeotomy. In the closing-tension interval 500 g to 1,500 g, the mean gain of length of the flap per galeotomy was 1.67 mm. These data confirm the usefulness of galeotomies for lengthening the scalp flaps and for diminishing the tension on wound margins when closing scalp defects.

Pre-adipocytes commitment to neurogenesis 1: Preliminary localisation of cholinergic molecules

A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimers disease and motor neuron disease). A stem cell population (i.e. pre‐adipocytes) is present in the adipose stromal compartment. Pre‐adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre‐adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre‐adipocytes, in parallel with neural morphology. The passage of the choline‐acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic‐like phenotype from pre‐adipocytes.

Development of the inframammary fold and ptosis in breast reconstruction with textured tissue expanders

Abstract. Tissue expansion has become the most important method for postmastectomy breast reconstruction. However, well-defined inframammary fold and ptosis are difficult to achieve with this technique. This study was performed to evaluate the inframammary fold and ptosis achieved in breast reconstruction using a textured tissue expander, later replaced by a textured implant. In ten postmastectomy patients, a textured tissue expander was inserted into a submuscular pocket. Every two to three weeks the volume of the expander was increased by about 30%. About three months after the last filling, the expander was removed and replaced with a permanent textured, gel-filled implant. The profile of the reconstructed breast was recorded before and after the tissue expansion, as well as before and after the change of the implant. The results showed that the inframammary fold did not move significantly upwards or downwards during the expansion period when a textured tissue expander was used. Waiting three months after the last inflation of the expander before replacing it with the permanent implant resulted in a more ptotic breast mound. Usually, however, no real ptosis was achieved, meaning that the angle between the lower part of the breast and the lower chest wall was more than 90 degrees. These findings indicate that a textured expander could help create a pronounced inframammary fold, but without ptosis. A three-month waiting period before inserting the permanent implant may improve the development of an inframammary fold.

The biological and clinical basis for the use of adipose-derived stem cells in the field of wound healing

Worldwide, hard-to-heal lower limb wounds are estimated to affect 1.5–3% of the adult population with a treatment-related annual cost of

Undermining of the Scalp: Quantitative Effects

10 billion. Thus, chronic skin ulcers of the lower limb are a matter of economic and public concern. Over the years, multiple medical and surgical approaches have been proposed but they are still inadequate, and no effective therapy yet exists. Regenerative medicine and stem cell-based therapies hold great promise for wound healing. Recently, many plastic surgeons have studied the potential clinical application of adipose-derived stem cells (ASCs), which are a readily available adult stem cell population that can undergo multilineage differentiation and secrete growth factors that can enhance wound-healing processes by promoting angiogenesis, and hence increase local blood supply. ASCs have been widely studied in vitro and in vivo in animal models. However, there are few randomized clinical trials on humans, and these are still ongoing or recruiting patients. Moreover, there is no consensus on a common isolation protocol that is clinically feasible and which would ensure reproducible results. The authors aim to provide readers with an overview of the biological properties of ASCs as well as their clinical application, to help better understanding of present and future strategies for the treatment of hard-to-heal wounds by means of stem cell-based therapies.

Procedure, applications, and outcomes of autologous fat grafting

&NA; The aim of the present study was to evaluate to what extent undermining affects the closing‐tension of scalp defects to quantify the surgery‐related benefits provided by this procedure. Data were collected by stepwise loading in 10 patients, 20 scalp flaps (obtained by a reversed Y scalp incision), and three different degrees of subgaleal undermining (1,5, and 15 cm). The obtained data confirmed the value of undermining to diminish the tension on wound margins when closing a scalp defect. There was a progressive decrease in tension required to advance the wound edge when the amount of undermining was sequentially increased. Most of this reduction occurred with the 5‐cm undermining, although statistically the 15‐cm undermining also resulted in a significant decrease in the tension required to close the defect. Mean 83.3‐ and 92.2‐percent reductions of the closing tension were obtained with 5 cm and 15 cm of undermining, respectively, compared with that achieved by the 1‐cm undermining with the same width of defect. (Plast. Reconstr. Surg. 101: 1218, 1998.)

Autologous fat transplantation for breast reconstruction: A literature review

Objective To systematically review the procedure, applications, and outcomes of autologous fat grafting, a promising technique with various clinical applications. Patients and methods Literature review of publications concerning autologous fat grafting. Results Since its introduction, lipofilling has become increasingly popular; however, its results are variable and unpredictable. Several modifications have been made to the procedures of fat harvesting, processing, and injecting. Surgical excision and low negative-pressure aspiration with large-bore cannulas minimize adipocyte damage during fat harvesting. The “wet” method of fat harvesting involves fluid injection at the donor site and facilitates lipoaspiration while minimizing pain and ecchymosis. For fat processing, centrifugation at a low speed is preferable to high-speed centrifugation, gravity separation or filtration. Fat injection at the recipient site should be performed using small-gauge cannulas in a fanning out pattern over multiple sessions, rather than a single session. Fat grafts exhibit not only dermal filler properties but also regenerative potential owing to the presence of stem cells in fat tissue. Thus, the clinical applications of autologous fat grafting include correction of secondary contour defects after breast reconstruction, release of painful scar contractures, and treatment of burn scars and radiodermatitis. Lipofilling is also used in aesthetic surgery, such as facial and hand rejuvenation, augmentation rhinoplasty, and breast and gluteal augmentation. The complications of lipofilling are minimal and include bruising, swelling, pain, infection, necrosis, and calcification. Conclusions Lipofilling is a low-risk procedure that can be used to correct soft-tissue defects in the face, trunk, and extremities, with minimal discomfort for patients.