Alfonso Barbarisi

FROM CELL-ECM INTERACTIONS TO TISSUE ENGINEERING

The extracellular matrix (ECM) consists of a complex mixture of structural and functional macromolecules and serves an important role in tissue and organ morphogenesis and in the maintenance of cell and tissue structure and function. The great diversity observed in the morphology and composition of the ECM contributes enormously to the properties and function of each organ and tissue. The ECM is also important during growth, development, and wound repair: its own dynamic composition acts as a reservoir for soluble signaling molecules and mediates signals from other sources to migrating, proliferating, and differentiating cells. Approaches to tissue engineering center on the need to provide signals to cell populations to promote cell proliferation and differentiation. These “external signals” are generated from growth factors, cell–ECM, and cell–cell interactions, as well as from physical‐chemical and mechanical stimuli. This review considers recent advances in knowledge about cell–ECM interactions. A description of the main ECM molecules and cellular receptors with particular care to integrins and their role in stimulation of specific types of signal transduction pathways is also explained. The general principles of biomaterial design for tissue engineering are considered, with same examples. J. Cell. Physiol. 199: 174–180, 2004© 2003 Wiley‐Liss, Inc.

Smart materials as scaffolds for tissue engineering

In this review, we focused our attention on the more important natural extracellular matrix (ECM) molecules (collagen and fibrin), employed as cellular scaffolds for tissue engineering and on a class of semi‐synthetic materials made from the fusion of specific oligopeptide sequences, showing biological activities, with synthetic materials. In particular, these new “intelligent” scaffolds may contain oligopeptide cleaving sequences specific for matrix metalloproteinases (MMPs), integrin binding domains, growth factors, anti‐thrombin sequences, plasmin degradation sites, and morphogenetic proteins. The aim was to confer to these new “intelligent” semi‐synthetic biomaterials, the advantages offered by both the synthetic materials (processability, mechanical strength) and by the natural materials (specific cell recognition, cellular invasion, and the ability to supply differentiation/proliferation signals). Due to their characteristics, these semi‐synthetic biomaterials represent a new and versatile class of biomimetic hybrid materials that hold clinical promise in serving as implants to promote wound healing and tissue regeneration.

A critical overview of ESEM applications in the biological field

Scanning Electron Microscope (SEM) is a powerful research tool, but since it requires high vacuum conditions, the wet materials and biological samples must undergo a complex preparation that limits the application of SEM on this kind of specimen and often causes the introduction of artifacts. The introduction of Environmental Scanning Electron Microscope (ESEM), working in gaseous atmosphere, represented a new perspective in biological research. Despite the fact that many biological applications have demonstrated the convenience of ESEM, the full potentialities of this technology are still under investigation. In this review, the exploration of the recent literature data confronted with the first results obtained in our experimental work suggest that ESEM represents an important extension of conventional scanning microscopy.

Cancer and anticancer therapy-induced modifications on metabolism mediated by carnitine system.

An efficient regulation of fuel metabolism in response to internal and environmental stimuli is a vital task that requires an intact carnitine system. The carnitine system, comprehensive of carnitine, its derivatives, and proteins involved in its transformation and transport, is indispensable for glucose and lipid metabolism in cells. Two major functions have been identified for the carnitine system: (1) to facilitate entry of long‐chain fatty acids into mitochondria for their utilization in energy‐generating processes; (2) to facilitate removal from mitochondria of short‐chain and medium‐chain fatty acids that accumulate as a result of normal and abnormal metabolism. In cancer patients, abnormalities of tumor tissue as well as nontumor tissue metabolism have been observed. Such abnormalities are supposed to contribute to deterioration of clinical status of patients, or might induce cancerogenesis by themselves. The carnitine system appears abnormally expressed both in tumor tissue, in such a way as to greatly reduce fatty acid beta‐oxidation, and in nontumor tissue. In this view, the study of the carnitine system represents a tool to understand the molecular basis underlying the metabolism in normal and cancer cells. Some important anticancer drugs contribute to dysfunction of the carnitine system in nontumor tissues, which is reversed by carnitine treatment, without affecting anticancer therapeutic efficacy. In conclusion, a more complex approach to mechanisms that underlie tumor growth, which takes into account the altered metabolic pathways in cancer disease, could represent a challenge for the future of cancer research. J. Cell. Physiol. 182:339–350, 2000.

Carnitine: an osmolyte that plays a metabolic role.

Carnitine, gamma‐trimethyl‐beta‐hydroxybutyrobetaine, is a small molecule widely present in all cells from prokaryotic to eukaryotic ones. It is the sole source of carbon and nitrogen in some bacteria; it serves as osmoprotectant in others. It is a carrier of acyl moieties, and exclusively of long‐chain fatty acids for mitochondrial beta‐oxidation in mammals. The conspicuously similar composition of the intracellular milieu among widely different species in relation to organic osmolyte systems involves the methylamine family to which carnitine belongs. This prompted us to examine the osmolytic properties of carnitine in an attempt to clarify the metabolic functions carnitine has acquired during evolution. An understanding of the metabolic functions of this organic compatible solute impinge on research involving this compound. J. Cell. Biochem. 80:1–10, 2000.

17‐B estradiol elicits an autocrine leiomyoma cell proliferation: Evidence for a stimulation of protein kinase‐dependent pathway

The mechanism by which estradiol (E2) acts on cell proliferation is still unclear. In this paper, we report the results of a series of experiments in an attempt to elucidate the effector pathway(s) involved in coupling the E2 receptors binding to cellular growth response in leiomyoma cells (LSMC). Under conditions of E2‐dependent growth, E2 treatment of LSMC triggers rapid and transient activation of the MAP‐kinase pathway. Interestingly, we demonstrate that the early downstream signal transduction events determined by E2‐stimulation in quiescent LSMC, including the rapid protein tyrosine phosphorylation of a subset of intracellular proteins, such GAP, PI‐3‐K, and PLCγ, and the concomitant activation of ancillary protein kinases, are related to E2‐induced PDGF secretion. Moreover, we identify the PDGF, alone or in association with other growth factors, as the main growth factor involved in the proliferation response of LSMC to E2 stimulation. The addition of neutralizing antibodies anti‐PDGF was able to inhibit the mitogenic activity present in LSMC conditioned media samples. On the other hand, E2 did not affect the constitutive expression as well as the ligand affinity of PDGF receptors on LSMC plasmamembrane. Cell treatment with the antiestrogen ICI 182780 correlate both with a perturbation of E2‐induced transductional circuit and with the disappearance of the mitogenic factor, PDGF, in LSMC conditioned media; the latter therefore, represents the main autocrine mediator of cell growth modulation, upregulated by E2 and down‐regulated by antiestrogenic compound. Our experiments suggest that growth factor secretion is an initial and integral part of the signaling events mediated by the estradiol receptors, not related, at least in part, to E2 transcriptional modulation.

Therapy with autologous adipose-derived regenerative cells for the care of chronic ulcer of lower limbs in patients with peripheral arterial disease.

BACKGROUND An ulcer is a trophic lesion with loss of tissue that often has a multifactorial genesis. It typically diverges from the physiologic processes of regeneration because it rarely tends to heal spontaneously. In this study, we used purified adipose-derived stem and regenerative cells (ADRCs) extracted from autologous fat, for the care of chronic ulcers of the lower limbs of arteriopathic patients. The primary objective of this study was complete re-epithelization of chronic ulcers; the secondary objective was a decrease in diameter and depth. METHODS From January 2010 to January 2012, 20 patients with peripheral arterial disease, with an ankle-brachial index between 0.30-0.40, in the age range 60-70 y (14 men and six women), with chronic ulcers of the lower limb, were involved in the study. Only 10 arteriopathic patients (seven men and three women) with chronic ulcers of the lower limb were surgically treated. Using the Celution system, we isolated a solution of ADRCs in about 150 min. The isolated cells were injected through a 10-mL syringe into the edges of the ulcer, taking care to spread it in all directions. Using a small amount of Celution extract, we performed cell characterization by flow cytometry analysis and cell viability assay. RESULTS We monitored patients treated with ADRC or untreated at 4, 10, 20, 60, and 90 d. In all cases treated with ADRC, we found a reduction in both diameter and depth of the ulcer, which led to a decrease in pain associated with the ulcer process. In six of 10 cases there was complete healing of the ulcer. Characterization of the cells by FACS clearly showed that the ADRC cells contained adipose-derived stem cells. Viability assays demonstrated that partial or total closure of the ulcer was attributable exclusively to ADRC cells present in the Celution extract, and not to growth factors extracted during the process of purification of the Celution and injected together with the cells. CONCLUSIONS For the first time, the Celution method has been applied for the care of chronic ulcers in the lower extremity of patients with peripheral arterial disease. Our results demonstrate that the technique is feasible for autologous cell application and is not associated with adverse events. Moreover, the transplantation of autologous stem cells extracted with Celution may represent a valuable method for the treatment of chronic ulcers in lower limbs of arteriopathic patients.

New polyelectrolyte hydrogels for biomedical applications

Abstract Copolymerisation of charged and neutral monomers is a well-tested strategy to introduce charged moieties in a polymeric chain and obtain polyelectrolyte polymers. We have synthesized new cationic and anionic polyelectrolytes by bulk radical copolymerisation of 2-hydroxyethyl methacrylate with [2-methacryloyloxyethyltrimethyl ammonium chloride (METAC)] or [2-acrylamido-2-methylpropane-sulphonic acid (AMPS)] monomers. The chemical structure of the synthesized copolymers was confirmed by FT-IR spectroscopy. Swelling studies on synthesized copolymers showed a high water content in the swollen state and a “smart behaviour” upon changes in external stimuli (pH and ionic strength). Cytotoxicity and cytocompatibility studies demonstrated that synthesized materials were not toxic. Moreover, the cationic copolymer showed good cell adhesion, whereas the anionic copolymer showed poor cell adhesion on its surface. X-ray photoelectron spectroscopy (XPS) showed that the disparate behaviour was due to the chemical nature of charged groups on the copolymer surfaces.

Preventing seroma formation after axillary dissection for breast cancer: a randomized clinical trial.

BACKGROUND Seroma formation after axillary dissection remains the most common early sequel to breast cancer surgery. Different surgical approaches have been performed to reduce seroma collection. Therefore, we aimed to assess the outcome of patients operated on using an ultrasound scalpel according to a standardized operative technique before accepting it as a routine procedure. METHODS A randomized controlled trial was designed to compare the outcome of patients undergoing breast surgery and axillary dissection using either standard scalpel blades, scissors, ligations, and electrocautery or the ultrasound scalpel only. Each arm of the trial consisted of 30 patients. RESULTS A statistically significant benefit in terms of axillary and chest wall drainage volume, the number of axilla seromas, intraoperative bleeding, and hospitalization stay was recorded in the harmonic scalpel group. No significant differences were found between the 2 groups in terms of operative time. Finally, no postoperative hematoma, wound infections, and chest wall seroma were observed. CONCLUSIONS The use of the harmonic scalpel was shown to reduce the magnitude of seromas in axilla and hospitalization stay. The harmonic scalpel can be used alone in axillary dissection with a safe and effective hemostasis. Our results must be confirmed by larger series.

Growth and endothelial differentiation of adipose stem cells on polycaprolactone

Adipose tissue is a readily available source of multipotent adult stem cells for use in tissue engineering/regenerative medicine. Various growth factors have been used to stimulate acquisition of endothelial characteristics by adipose-derived stem cells (ADSC). Herein, we study the growth and endothelial differentiation potential of ADSC seeded onto a porous polycaprolactone (PCL) scaffold. The objective of this study is to demonstrate that PCL is a good material to be used as a scaffold to support reconstruction of new endothelial tissue using adipose stem cells. We found that undifferentiated ADSC adhere and grow on PCL. We show that, after culture in endothelial differentiation medium, ADSC were positive to LDL uptake and expressed molecular markers characteristic of endothelial cells (CD31; eNOS and vWF). In addition, our study defines the time required for the differentiation of ADSC directly onto PCL. This study suggests that PCL can be used as a scaffold to generate endothelial tissue in vitro. PLC has excellent mechanical properties and a slow degradation rate. Moreover, based on our results, we propose that PCL could be used to graft scaffolds coated with endothelial cells derived from ADSC stem cells. Endothelial cells-coated PCL could find several applications to replace damaged area of the body; for example, a possible use could be the generation of vascular grafts.