Céline M. Desmet

Autologous Adipose Stromal Cells Seeded onto a Human Collagen Matrix for Dermal Regeneration in Chronic Wounds: Clinical Proof of Concept.

Background: Nonhealing wounds are unable to integrate skin autografts by avascular and fibrotic dermal tissue. Adipose-derived stromal cells can improve the local environment of the wound bed by angiogenesis and immunomodulation. This work aimed to develop a biological dressing made of adipose-derived stromal cells onto a human acellular collagen matrix. Methods: Adipose-derived stromal cells were isolated from human adipose tissue (n = 8). In vitro, the genetic stability during early and late passages (1, 4, 10, and 16) and vascular endothelial growth factor (VEGF) secretion were assessed. Adipose-derived stromal cell adhesion and spreading on collagen matrix were preliminarily studied. In vivo tumorigenicity, angiogenesis, and tissue oxygenation were assessed after implantation of the construct in nude rats (n = 10). The biological dressing was manufactured and implanted in three patients with chronic wounds. Results: In vitro, aneuploidies, but no clonal transformation, were detected up to late cellular passages. VEGF was secreted more during hypoxia (0.1% oxygen) than during normoxia (21% oxygen). Adipose-derived stromal cells can adhere and spread on the scaffold within 18 to 20 days. No tumor development occurred 3 months after implantation in immunocompromised rats. Vessel counts and tissue oxygenation were higher after adipose-derived stromal cell implantation. In patients, granulation tissue was found (276 percent of vessel density), followed by epithelialization or split-thickness skin engraftment up to 22 months after implantation. Conclusions: Implantation of adipose-derived stromal cells seeded onto human acellular collagen matrix (biological dressing) represents a promising therapy for nonhealing wounds, offering improvement in dermal angiogenesis and remodeling. This therapy using autologous stromal cells is safe, without significant genetic alterations after in vitro expansion.

Application of Electron Paramagnetic Resonance (EPR) Oximetry to Monitor Oxygen in Wounds in Diabetic Models

A lack of oxygen is classically described as a major cause of impaired wound healing in diabetic patients. Even if the role of oxygen in the wound healing process is well recognized, measurement of oxygen levels in a wound remains challenging. The purpose of the present study was to assess the value of electron paramagnetic resonance (EPR) oximetry to monitor pO2 in wounds during the healing process in diabetic mouse models. Kinetics of wound closure were carried out in streptozotocin (STZ)-treated and db/db mice. The pO2 was followed repeatedly during the healing process by 1 GHz EPR spectroscopy with lithium phthalocyanine (LiPc) crystals used as oxygen sensor in two different wound models: a full-thickness excisional skin wound and a pedicled skin flap. Wound closure kinetics were dramatically slower in 12-week-old db/db compared to control (db/+) mice, whereas kinetics were not statistically different in STZ-treated compared to control mice. At the center of excisional wounds, measurements were highly influenced by atmospheric oxygen early in the healing process. In pedicled flaps, hypoxia was observed early after wounding. While reoxygenation occurred over time in db/+ mice, hypoxia was prolonged in the diabetic db/db model. This observation was consistent with impaired healing and microangiopathies observed using intravital microscopy. In conclusion, EPR oximetry using LiPc crystals as the oxygen sensor is an appropriate technique to follow wound oxygenation in acute and chronic wounds, in normal and diabetic animals. Nevertheless, the technique is limited for measurements in pedicled skin flaps and cannot be applied to excisional wounds in which diffusion of atmospheric oxygen significantly affects the measurements.

Influence of Free Radicals Signal from Dental Resins on the Radio-Induced Signal in Teeth in EPR Retrospective Dosimetry

In case of radiological accident, retrospective dosimetry is needed to reconstruct the absorbed dose of overexposed individuals not wearing personal dosimeters at the onset of the incident. In such a situation, emergency mass triage will be required. In this context, it has been shown that Electron Paramagnetic Resonance (EPR) spectroscopy would be a rapid and sensitive method, on the field deployable system, allowing dose evaluation of a great number of people in a short time period. This methodology uses tooth enamel as a natural dosimeter. Ionising radiations create stable free radicals in the enamel, in a dose dependent manner, which can be detected by EPR directly in the mouth with an appropriate resonator. Teeth are often subject to restorations, currently made of synthetic dimethacrylate-based photopolymerizable composites. It is known that some dental composites give an EPR signal which is likely to interfere with the dosimetric signal from the enamel. So far, no information was available about the occurrence of this signal in the various composites available on the market, the magnitude of the signal compared to the dosimetric signal, nor its evolution with time. In this study, we conducted a systematic characterization of the signal (intensity, kinetics, interference with dosimetric signal) on 19 most widely used composites for tooth restoration, and on 14 experimental resins made with the most characteristic monomers found in commercial composites. Although a strong EPR signal was observed in every material, a rapid decay of the signal was noted. Six months after the polymerization, the signal was negligible in most composites compared to a 3 Gy dosimetric signal in a tooth. In some cases, a stable atypical signal was observed, which was still interfering with the dosimetric signal.

Adipose tissue-derived stem cells in a fibrin implant enhance neovascularization in a peritoneal grafting site: a potential way to improve ovarian tissue transplantation.

STUDY QUESTION Do two different concentrations of human adipose tissue-derived stem cells (ASCs) embedded inside a fibrin scaffold have the potential to differentiate into vessels and aid vascularization in a peritoneal grafting site intended for ovarian tissue transplantation? SUMMARY ANSWER Human ASCs in low and high concentrations differentiated into vessels when transplanted to mouse peritoneum inside a fibrin matrix, but only high ASC concentrations significantly increased human vessel area 14 days after transplantation. WHAT IS KNOWN ALREADY ASCs have multilineage differentiation potential, including proangiogenic properties and have been used in tissue engineering to enhance vascularization in transplanted tissues. Fibrin has been studied and used as an ASC-compatible biomaterial. STUDY DESIGN, SIZE, DURATION In vivo experimental model using 22 severe combined immunodeficient mice. In total, 16 mice (eight per group) were intraperitoneally grafted with a fibrin scaffold loaded with two different human ASC concentrations (either 150 000 [L-ASC] or 1 500 000 [H-ASC] cells) and lithium phthalocyanine (LiPc) crystals as oxygen-sensitive probes. Six mice were grafted with an empty fibrin (EF) implant containing only LiPc and served as controls. Levels of partial pressure of oxygen (pO2) in implants were monitored in vivo by electron paramagnetic resonance oximetry (EPR). ASC identification, proliferation, and host and human vascularization were analyzed by immunohistochemistry (IHC). All analyses were performed on post-grafting Days 3, 7 and 14. PARTICIPANTS/MATERIALS, SETTING, METHODS Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), as well as histological (hematoxylin-eosin staining) and IHC (anti-human vimentin, anti-human Ki67, anti-mouse and human double CD34) analyses. MAIN RESULTS AND THE ROLE OF CHANCE A significant increase in pO2 in implants was observed in all groups between Days 3 and 7 (P < 0.001). ASC-loaded implants displayed a tendency towards increased pO2 levels from Days 7 to 14, not observed in EF implants. ASC-loaded implants showed differentiation into human CD34-positive vessels. Total CD34-positive endothelial area was correlated to pO2 values obtained by EPR oximetry (r = 0.6506, P = 0.0019). In the H-ASC group, a greater human CD34-positive vascular surface area was found compared to the L-ASC group 14 days after transplantation (P < 0.0049). LARGE SCALE DATA N/A. LIMITATIONS REASONS FOR CAUTION As demonstrated by our results, ASCs transplanted inside a fibrin matrix can differentiate into CD34-positive human vessels. However, other possible mechanisms involved in ASC angiogenic behavior remain to be investigated. WIDER IMPLICATIONS OF THE FINDINGS High concentrations of ASCs loaded inside a fibrin scaffold could serve as a substrate to prepare a peritoneal grafting site over 14 days, in order to enhance vascularization once human ovarian tissue is grafted. Our proposed preparation of the grafting site would not only benefit ovarian tissue transplantation, but also other experimental avascular grafting procedures. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, Télévie Grant no. 7.6515.16F awarded to DDM and Grant 5/4/150/5 awarded to M.M.D. [CAA is FRS-FNRS research associate]), Fonds Spéciaux de Recherche, and Fondation St Luc, Foundation Against Cancer, and donations from the Ferrero family. None of the authors have any competing interests to declare.

Tooth Retrospective Dosimetry Using Electron Paramagnetic Resonance: Influence of Irradiated Dental Composites

In the aftermath of a major radiological accident, the medical management of overexposed individuals will rely on the determination of the dose of ionizing radiations absorbed by the victims. Because people in the general population do not possess conventional dosimeters, after the fact dose reconstruction methods are needed. Free radicals are induced by radiations in the tooth enamel of victims, in direct proportion to dose, and can be quantified using Electron Paramagnetic Resonance (EPR) spectrometry, a technique that was demonstrated to be very appropriate for mass triage. The presence of dimethacrylate based restorations on teeth can interfere with the dosimetric signal from the enamel, as free radicals could also be induced in the various composites used. The aim of the present study was to screen irradiated composites for a possible radiation-induced EPR signal, to characterize it, and evaluate a possible interference with the dosimetric signal of the enamel. We investigated the most common commercial composites, and experimental compositions, for a possible class effect. The effect of the dose was studied between 10 Gy and 100 Gy using high sensitivity X-band spectrometer. The influence of this radiation-induced signal from the composite on the dosimetric signal of the enamel was also investigated using a clinical L-Band EPR spectrometer, specifically developed in the EPR center at Dartmouth College. In X-band, a radiation-induced signal was observed for high doses (25-100 Gy); it was rapidly decaying, and not detected after only 24h post irradiation. At 10 Gy, the signal was in most cases not measurable in the commercial composites tested, with the exception of 3 composites showing a significant intensity. In L-band study, only one irradiated commercial composite influenced significantly the dosimetric signal of the tooth, with an overestimation about 30%. In conclusion, the presence of the radiation-induced signal from dental composites should not significantly influence the dosimetry for early dose assessment.

EPR monitoring of wound oxygenation as a biomarker of response to gene therapy encoding hCAP‐18/LL37 peptide

To investigate the value of electron paramagnetic resonance oximetry to follow oxygenation in wounds treated by a plasmid‐encoding host defense peptide hCAP‐18/LL37.

Nanomedicines and gene therapy for the delivery of growth factors to improve perfusion and oxygenation in wound healing.

ABSTRACT Oxygen plays a key role in wound healing, and hypoxia is a major cause of wound healing impairment; therefore, treatments to improve hemodynamics and increase wound oxygenation are of particular interest for the treatment of chronic wounds. This article describes the roles of oxygen and angiogenesis in wound healing as well as the tools used to evaluate tissue oxygenation and perfusion and then presents a review of nanomedicines and gene therapies designed to improve perfusion and oxygenation and accelerate wound healing.

Two-step transplantation with adipose tissue-derived stem cells increases follicle survival by enhancing vascularization in xenografted frozen–thawed human ovarian tissue

STUDY QUESTION Do adipose tissue-derived stem cells (ASCs) enhance vascularization and follicle survival in xenografted ovarian tissue using a two-step transplantation approach? SUMMARY ANSWER Higher rates of oxygenation and vascularization of ovarian tissue, as well as increased follicle survival rates, were detected in the early post-grafting period. WHAT IS KNOWN ALREADY ASCs have multilineage differentiation potential, proangiogenic properties and enhance vascularization in a peritoneal grafting site. Some studies suggest that using ASCs may improve ovarian tissue quality by enhancing graft angiogenesis. STUDY DESIGN, SIZE, DURATION A total of 15 severe combined immunodeficient (SCID) mice were intraperitoneally grafted with frozen-thawed human ovarian tissue (OT) from five different patients. A peritoneal transplantation site had been previously prepared in a first step using either empty fibrin (Fi+OT group [n = 5]) or ASC-loaded fibrin (Fi/ASCs+OT group [n = 5]) for 14 days prior to grafting. Five mice underwent the standard one-step transplantation procedure and served as controls (OT group). Lithium phthalocyanine (LiPc) crystals were inserted into all grafted human ovarian tissue before transplantation. Levels of partial pressure of oxygen (pO2) in grafts were monitored in vivo by electron paramagnetic resonance (EPR) oximetry on Days 3 and 7. Samples for histology and immunohistochemistry (IHC) were collected after euthanizing the mice on Day 7 following EPR. One piece of ovarian tissue per patient was fixed for analysis to serve as non-grafted controls. PARTICIPANTS/MATERIALS, SETTING, METHODS Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), histological (haematoxylin and eosin staining), immunohistochemistry (anti-mouse and human double CD34 and anti-human Ki-67) and TUNEL analyses. MAIN RESULTS AND THE ROLE OF CHANCE A significant increase in pO2 was observed in all groups between Days 3 and 7 (P < 0.001). A significantly higher pO2 level was observed in the Fi/ASCs+OT group compared to the OT group on Day 7 (P = 0.028). Total CD34-positive vessel area on Day 7 was greater in the Fi/ASCs+OT group than in any other group (vs non-grafted group: P = 0.0014; vs OT group: P = 0.013; vs Fi+OT group: P = 0.018). Primordial follicle survival rates after grafting were higher in the Fi/ASCs+OT group than in the OT (P = 0.0059) or Fi+OT groups (P = 0.0307). TUNEL-positive follicle percentages after grafting were significantly lower in the Fi/ASCs+OT group than in any other grafted tissue (vs OT group: P = 0.045; vs Fi+OT group: P = 0.0268). Percentages of Ki-67-positive primordial follicles were significantly higher in all grafted groups compared to non-grafted tissue controls (P < 0.01). LIMITATIONS REASONS FOR CAUTION As demonstrated by our results, the proposed two-step ovarian tissue transplantation procedure using ASCs enhances vascularization in the early post-grafting period, leading to increased follicle survival rates and decreased apoptosis. However, mechanisms involved in the proangiogenic behavior of ASCs remain to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest that the proposed transplantation procedure with ASCs is a promising step towards potentially solving the problem of massive follicle loss after ovarian tissue grafting. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, grant Télévie No. 7.6515.16 F to DDM and grant 5/4/150/5 awarded to MMD and CAA is research associate, FRS-FNRS), Fonds Spéciaux de Recherche, Fondation St Luc, and Foundation Against Cancer, and donations from the Ferrero family.

Biomarkers of tumour redox status in response to modulations of glutathione and thioredoxin antioxidant pathways

Abstract The ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with aggressiveness and drug resistance. Since the glutathione (GSH) and thioredoxin (TRX) systems cooperate to a tight regulation of ROS in cell physiology, and to a stimulation of tumour initiation and progression, modulation of the GSH and TRX pathways are emerging as new potential targets in cancer. In vivo methods to assess changes in tumour redox status are critically needed to assess the relevance of redox-targeted agents. The current study assesses in vitro and in vivo biomarkers of tumour redox status in response to treatments targeting the GSH and TRX pathways, by comparing cytosolic and mitochondrial redox nitroxide electron paramagnetic resonance (EPR) probes, and cross-validation with redox dynamic fluorescent measurement. For that purpose, the effect of the GSH modulator buthionine sulfoximine (BSO) and of the TRX reductase inhibitor auranofin were measured in vitro using both cytosolic and mitochondrial EPR and roGFP probes in breast and cervical cancer cells. In vivo, mice bearing breast or cervical cancer xenografts were treated with the GSH or TRX modulators and monitored using the mito-TEMPO spin probe. Our data highlight the importance of using mitochondria-targeted spin probes to assess changes in tumour redox status induced by redox modulators. Further in vivo validation of the mito-tempo spin probe with alternative in vivo methods should be considered, yet the spin probe used in vivo in xenografts demonstrated sensitivity to the redox status modulators.

Factors Affecting the Quality of Tooth Enamel for In Vivo EPR-Based Retrospective Biodosimetry.

In vivo electron paramagnetic resonance biodosimetry on tooth enamel is likely to be an important technology for triage of overexposed individuals after a major radiological incident. The accuracy and robustness of the technique relies on various properties of the enamel such as the geometry of the tooth, the presence of restorations, whitening treatments or exposition to sunlight. Those factors are reviewed, and their influence on dosimetry specifically for triage purposes is discussed.