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Bone allografts are commonly used by orthopedists to provide a mechanical support and template for cellular colonization and tissue repair. There is an increasing demand for bone graft substitutes that are safe and easy to store but which are equally effective in supporting new bone growth. In this study, we compared three different human bone allografts: (1) the cryopreserved allograft (frozen), (2) the gamma-irradiated and cryopreserved allograft (γ-irradiated), and (3) the solvent dehydrated and γ-irradiated-processed bone allograft (Tutoplast(®) Process Bone [TPB]). Human mesenchymal stromal cells (hMSCs) have the potential to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Our results showed that hMSC seeding efficiency was equivalent among the three bone allografts. However, differences were observed in terms of cell metabolism (viability), osteoblastic gene expression, and in vivo bone formation. Frozen allografts had the higher frequency of new bone formation in vivo (89%). Compared with frozen allografts, we demonstrated that TPB allografts allowed optimal hMSC viability, osteoblastic differentiation, and bone formation to occur in vivo (72%). Further, the frequency of successful bone formation was higher than that obtained with the γ-irradiated allograft (55%). Moreover, after hMSC osteoinduction, 100% of the TPB and frozen allografts formed bone in vivo whereas only 61% of the γ-irradiated allografts did. As healthcare teams around the world require bone-grafting scaffolds that are safe and easy to store, the TPB allograft appears to be a good compromise between efficient bone formation in vivo and convenient storage at room temperature.

Cholera toxin and isoproterenol (β-adrenergic receptor agonist) are largely used to enhance cell proliferation. The aim of the study was to assess the effects of cholera toxin and isoproterenol on growth and differentiation of cells cultured from human superficial limbal explants.Limbal epithelial cells were cultured from superficial limbal explantsin basal medium either supplemented with cholera toxin or isoproterenol for 3 weeks. Growth kinetics and morphometry were studied by light and confocal microscopy. Progenitor and differentiated epithelial cell markers were studied by immunocytochemistry, flow cytometry, Colony Formation Assay, and reverse transcription and polymerase chain reaction.Cell proliferation was significantly higher with 0.5 µg/ml (p = 0.049), 1 µg/ml (p = 0.005), and 2 µg/ml (p = 0.008) isoproterenol whereas, cholera toxin and 4 µg/ml isoproterenol did not significantly increase cell proliferation. Multilayered epithelial cell sheets were obtained in all culture conditions. Addition of isoproterenol resulted in smaller cell size (p0.05) 14 days after cells were cultured, whereas cholera toxin had no effects. Strong expression of cytokeratins 3 and 4/5/6/8/10/13/18 and lower expression of cytokeratin 19, vimentin, and Delta N p63α were observed after 3 weeks of culture with no significant differences in the percentage of positive cells according to culture medium. Colony-forming efficiencies were observed after 2 weeks in all culture condition but not after 3 weeks.Isoproterenol was more efficient than cholera toxin for enhancing cell proliferation and resulted in smaller cell size. It appears to be useful and safe for growing human limbal epithelial progenitors from limbal explants with no feeders before transplantation to patients with limbal deficiency.

Selon la monographie 2.6.27 de la pharmacopee europeenne (controle microbiologique des produits cellulaires), la comparaison interlaboratoires est une possibilite de validation de methodes et la participation a des etudes collaboratives permet a chaque laboratoire d’evaluer ses performances et de repondre a ces exigences de validation. En 2016, en relais de l’ANSM, le laboratoire de controle qualite de l’EFS Aquitaine-Limousin a organise deux campagnes de comparaison inter-laboratoires, l’un portant sur le controle microbiologique des milieux corneens, l’autre sur celui des produits de therapie cellulaire. Le materiel a contaminer etait dans le premier cas un milieu de transport de cornee, dans le deuxieme cas des residus de couches leuco-plaquettaires du sang total. Les germes contaminants ont ete choisis parmi les germes recommandes pour la validation de methode ou pour leur interet. Les laboratoires participants a ces etudes utilisent des techniques differentes (automatisee ou manuelle), des durees et des temperatures d’incubation differentes, toutes en accord avec les recommandations de la pharmacopee 2.6.27. Le controle des milieux de cornees a rassemble 7 participants; 86 % des laboratoires ont eu 100 % de reponses satisfaisantes: detection d’un echantillon sterile et identification correcte des deux echantillons contamines. Dix-sept laboratoires ont participe au controle des produits de therapie cellulaires. Quatre-vingt-deux pour cent des laboratoires ont eu 100 % de reponses satisfaisantes, les erreurs mises en evidence dans les autres laboratoires correspondent a une identification erronee des echantillons ou a une contamination croisee.

Background Pneumonectomy is well known for a high risk of postoperative death. The alternative, sleeve lobectomy, is sometimes technically inaccessible, and is associated with locoregional recurrence. In certain situations, the use of a bronchial substitute would allow longer bronchial resections with better security margins. Previous experiments demonstrated that aortic grafts are valuable tracheal and carinal substitutes. The present study evaluated bronchial replacement with arterial allografts. Methods Fifteen female sheep underwent a left bilobectomy with replacement of the bronchus intermedius with arterial allografts: 5 received a fresh graft (group 1) and 10 received cryopreserved (group 2). A bronchial silicone stent was used to confer rigidity. Evaluation was conducted on clinical and histologic criteria at regular intervals up to 18 months. Results There were no perioperative deaths. Atelectasis, the only early postoperative complication (n = 2), was successfully treated by fiberscopic aspiration. The late postoperative period was uneventful in 12 sheep. Complications included 1 bronchopneumonia, 1 pulmonary abscess, and 1 distortion of the bronchial stent. Fiberscopic examination revealed 3 sheep with granuloma formation. The bronchial stent was removed in 3 sheep, 1 at 9 months and 2 at 12 months, without clinical complications or stenosis of the graft. Histologic analysis showed regeneration of new bronchial tissue, comprising epithelium and cartilage. Conclusions This study confirmed that an arterial allograft could be a valuable bronchial substitute. The use of a bronchial substitute offers new perspectives in surgical resection of lung cancer because it would avoid pneumonectomy in some patients.

Importance Airway transplantation could be an option for patients with proximal lung tumor or with end-stage tracheobronchial disease. New methods for airway transplantation remain highly controversial. Objective To establish the feasibility of airway bioengineering using a technique based on the implantation of stented aortic matrices. Design, Setting, and Participants Uncontrolled single-center cohort study including 20 patients with end-stage tracheal lesions or with proximal lung tumors requiring a pneumonectomy. The study was conducted in Paris, France, from October 2009 through February 2017; final follow-up for all patients occurred on November 2, 2017. Exposures Radical resection of the lesions was performed using standard surgical techniques. After resection, airway reconstruction was performed using a human cryopreserved (−80°C) aortic allograft, which was not matched by the ABO and leukocyte antigen systems. To prevent airway collapse, a custom-made stent was inserted into the allograft. In patients with proximal lung tumors, the lung-sparing intervention of bronchial transplantation was used. Main Outcomes and Measures The primary outcome was 90-day mortality. The secondary outcome was 90-day morbidity. Results Twenty patients were included in the study (mean age, 54.9 years; age range, 24-79 years; 13 men [65%]). Thirteen patients underwent tracheal (n = 5), bronchial (n = 7), or carinal (n = 1) transplantation. Airway transplantation was not performed in 7 patients for the following reasons: medical contraindication (n = 1), unavoidable pneumonectomy (n = 1), exploratory thoracotomy only (n = 2), and a lobectomy or bilobectomy was possible (n = 3). Among the 20 patients initially included, the overall 90-day mortality rate was 5% (1 patient underwent a carinal transplantation and died). No mortality at 90 days was observed among patients who underwent tracheal or bronchial reconstruction. Among the 13 patients who underwent airway transplantation, major 90-day morbidity events occurred in 4 (30.8%) and included laryngeal edema, acute lung edema, acute respiratory distress syndrome, and atrial fibrillation. There was no adverse event directly related to the surgical technique. Stent removal was performed at a postoperative mean of 18.2 months. At a median follow-up of 3 years 11 months, 10 of the 13 patients (76.9%) were alive. Of these 10 patients, 8 (80%) breathed normally through newly formed airways after stent removal. Regeneration of epithelium and de novo generation of cartilage were observed within aortic matrices from recipient cells. Conclusions and Relevance In this uncontrolled study, airway bioengineering using stented aortic matrices demonstrated feasibility for complex tracheal and bronchial reconstruction. Further research is needed to assess efficacy and safety. Trial Registration clinicaltrials.gov Identifier:NCT01331863

A new protocol for cryopreservation of arteries frozen at −80°C was compared to the reference protocol for cryopreservation at −150°C and to freshly harvested arteries. The aim of the study is to evaluate both protocols as global procedures to freeze and thaw arteries commonly used in tissue banks. Changes in mechanical properties of rabbit common carotid arteries were studied. Vascular segments were tested in vitro under dynamics loading conditions. Pressure and diameter were recorded simultaneously by a high fidelity transducer and an echotracking device, respectively. The pressure–diameter relationship was fitted by the arctangent Langewouters' model and the arterial thickness was derived from histological measurements. Histological sections showed that the fresh and −80°C groups were less damaged by hemodynamic load and histological preparation than the −150°C group ( p

Background Seven years of experimental research provided a valuable tracheal substitute, the aortic allograft, which can promote the regeneration of epithelium and cartilage. In human application, both fresh and preserved aortic allografts could be used. The optimal method of aortic allograft preservation remains to be evaluated. This study assessed the use of cryopreserved, decellularized, or glutaraldehyde-treated aortic allografts as tracheal substitutes. Methods Twenty-two sheep underwent tracheal replacement using cryopreserved (n = 10), decellularized (n = 7) or glutaraldehyde-treated (n = 5) allografts, supported by a temporary stent to prevent airway collapse. Aortic segments were retrieved at regular intervals up to 12 months after implantation to analyze the regenerative process. Results All animals survived the operation. Major complications such as infection, stent migration, or obstruction were predominantly encountered in the decellularized group. The lack of major inflammatory response within the aortic graft observed in the glutaraldehyde group was associated with the absence of tracheal regeneration. Histologic examinations showed a progressive transformation of the aorta into a tracheal tissue comprising respiratory epithelium and cartilage only in the cryopreserved group. Conclusions This study demonstrated that regeneration of a functional tissue could be obtained after tracheal replacement with a cryopreserved aortic allograft. The regenerative process followed the same pattern as previously described for fresh allografts. Cryopreserved aortic allografts present major advantages: availability in tissue banks, permanent storage, and no need for immunosuppression. This offers a new field of perspectives for clinical application in patients with extensive tracheal cancer.

BACKGROUND: Weak D Types 1, 2, and 3 recipients cannot be immunized when exposed to D antigen. Molecular biology is very efficient to type weak D variants but rarely implemented in daily practice. The serologic typing practice of weak D in a Caucasian patient population was analyzed and a transfusion strategy is proposed. STUDY DESIGN AND METHODS: Samples typed either ddCcee or ddccEe in routine laboratories were tested with the indirect antiglobulin test (Du test). Du-positive samples were screened for weak D alleles Types 1, 2, and 3 and further tested with immunoglobulin M (IgM) anti-D reagents, used in a fully automated device. RESULTS: A total of 468 of 55,162 samples were found to be ddCcee or ddccEe. Ninety-three expressed weak D after the Du test leading to D+ assignment for transfusion. Seventy-three percent of Du-positive samples were weak D alleles Type 1, 2, or 3. Almost all weak D Types 1, 2, and 3 were positive with IgM reagents in gel matrix with an automated device. Other variants that could be potentially associated with anti-D alloimmunization, however, were also positive. CONCLUSION: Serology is very sensitive to detect weak D Types 1, 2, and 3, but there is no cutoff to distinguish variants of clinical significance. When molecular analysis is not available, it is proposed that a D+ status for blood recipients found to be weak D with a sensitive method be assigned, except for women of childbearing age or younger, because of the remaining possibility to be partial D or other rare weak D who can be immunized.

Contexte Les greffes allogeniques se font souvent dans un contexte d’incompatibilite ABO majeure, necessitant le calcul du volume de globules rouges (GR) a partir de l’hematocrite or l’hyperleucocytose des greffons peut etre responsable d’une surestimation de l’Ht. Une etude multicentrique realisee sur des prelevements de CSH compare les resultats d’Ht obtenus sur des automates d’hematologie (AH) ainsi que les Ht calcules a partir des GR ou de l’hemoglobine(Hb). L’effet «matrice» sur la mesure de l’Ht a ete egalement evaluee. Materiel et methodes Les parametres sanguins d’echantillons de CSH peripheriques ont ete mesures sur differents AH par des unites de therapie cellulaire. L’impact de la matrice hyperleucocytaire a ete evaluee sur une suspension de GB prepares a partir de couches leucoplaquettaires additionnees de 5 % de GR. La mesure de l’Ht a ete realisee sur 3 AHC et par methode manuelle. Resultats Les hematocrites different suivant la technologie des AH. Le biais dose–dependante augmente avec la concentration croissante des GB dans les echantillons. Les comparaisons entre les Ht mesures sur l’AH de reference et les Ht calcules a partir du nombre des GR (Ht = GR × 9) ou de l’Hb (Ht = Hb × 3) montrent une diminution du biais. Par ailleurs, on constate une augmentation progressive de l’Ht sur la «matrice» diluee + 5 % de GR. Conclusion On observe une surestimation variable de l’Ht suivant les AH plus marquee sur les produits tres hyperleucocytaires. Les Ht mesures sur les AH combinant impedance et focalisation hydrodynamique sont les plus faibles. L’etude permet de faire un etat des lieux des techniques et de leur performance au regard de la determination de l’Ht dabs les PTC.

Stem cells may represent an excellent strategy to improve the healing of skin ulcers. Today the administration mode of stem cells to skin defects remains unsatisfactory. Delivering stem cells with topical treatments represents a new strategy and answering the patients' need. Mesenchymal stromal cells (MSC) have been shown to improve wound healing of cutaneous lesions and amniotic membrane (AM) is known to represent a natural scaffold for cells. The aim of this study is to develop a tissue-engineered product combining MSC and AM for clinical use. In this work we investigated whether the stromal matrix of intact human AM could constitute a scaffold for human MSC derived from either bone marrow (BM) or adipose tissue (AT). For this purpose, clinical-grade AM, MSC, and culture medium were used. We performed experiments of short-term adherence and proliferation for 15 days after the seeding of the cells. Morphological aspects and secretion profiles of MSC onto AM were studied, respectively, by scanning electron microscopy and Luminex analysis. Results demonstrated that the stromal matrix allow the adherence in much greater amount of MSC from BM or AT compared to 2D material. Experiments of proliferation showed that both kinds of MSC could proliferate on the stromal matrix and remain viable 15 days after the seeding of the cells. The 3D analysis of MSC culture demonstrated that both types of MSC invaded the stromal matrix and grew in multiple layers while retaining their fibroblastic morphology. By studying the secretion profile of MSC onto the stromal matrix, we found that both kinds of MSC secrete important cytokines and growth factors for wound healing of cutaneous lesions, such as vascular endothelial growth factor, hepatocyte growth factor, and basic fibroblast growth factor. In conclusion, these results suggest that the stromal matrix of AM seeded with MSC represents a bioactive scaffold that should be evaluated in patients with a nonhealing cutaneous wound.

After more than 50 years of research, airway transplantation remains a major challenge in the fields of thoracic surgery and regenerative medicine. Five principal types of tracheobronchial substitutes, including synthetic prostheses, bioprostheses, allografts, autografts and bioengineered conduits have been evaluated experimentally in numerous studies. However, none of these works have provided a standardized technique for the replacement of the airways. More recently, few clinical attempts have offered encouraging results with ex vivo or stem cell–based engineered airways and tracheal allografts implanted after heterotopic revascularization. In 1997, we proposed a novel approach: the use of aortic grafts as a biological matrix for extensive airway reconstruction. In vivo regeneration of epithelium and cartilage were demonstrated in animal models. This led to the first human applications using cryopreserved aortic allografts that present key advantages because they are available in tissue banks and do not require immunosuppressive therapy. Favorable results obtained in pioneering cases have to be confirmed in larger series of patients with extensive tracheobronchial diseases.

Background Pneumonectomies for lung cancer are associated with a high postoperative mortality, especially when right-sided, after neoadjuvant radiochemotherapy, and in patients over 70 years of age. Preliminary studies in our laboratory have shown that aortic grafts could be valuable airway substitutes. We report the first human bronchial transplantation of a cryopreserved aortic allograft used as a biologic airway substitute to prevent a pneumonectomy for lung cancer. Methods The procedure was performed in a high-risk 78-year old patient with an extensive right bronchopulmonary malignant tumor pretreated with chemotherapy. After a complete resection of the lung cancer using an upper bilobectomy with lymph node removal, mobilization procedures did not allow for a primary end-to-end bronchial anastomosis. A stent-supported cryopreserved aortic allograft from a certified tissue bank was interposed to restore the bronchial continuity with sparing of the lower lobe. Results The postoperative course was eventful for a supraventricular arrhythmia leading to mild pulmonary edema that resolved using standard medical therapy, and a right lower lobe atelectasis with bacterial colonization that required fiberoptic bronchoscopies in addition to antibiotic treatment. A 1-year postoperative evaluation found a well-functioning reimplanted lower lobe with no complications related to the cryopreserved aortic allograft or the stent. The patient recovered to his baseline activity with a satisfying health-related quality of life. Conclusions We demonstrate the feasibility of this surgical innovation to prevent the high-risk procedure of pneumonectomy in a single case. If confirmed in larger series of selected patients, it could bring new perspectives in conservative lung cancer surgery.

A new protocol for cryopreservation of arteries frozen at -80 degrees C was compared to the reference protocol for cryopreservation at -150 degrees C and to freshly harvested arteries. The aim of the study is to evaluate both protocols as global procedures to freeze and thaw arteries commonly used in tissue banks. Changes in mechanical properties of rabbit common carotid arteries were studied. Vascular segments were tested in vitro under dynamics loading conditions. Pressure and diameter were recorded simultaneously by a high fidelity transducer and an echotracking device, respectively. The pressure-diameter relationship was fitted by the arctangent Langewouters' model and the arterial thickness was derived from histological measurements. Histological sections showed that the fresh and -80 degrees C groups were less damaged by hemodynamic load and histological preparation than the -150 degrees C group (p0.05). No differences between fresh and cryopreserved arteries regarding the structural (diameter, intimal-media thickness) and mechanical parameters (distensibility, circumferential stress, elastic modulus) were found. The isobaric circumferential stress was reduced in frozen arteries. These results demonstrate that the cryopreservation at -80 degrees C preserves the histological structure and mechanical properties better than the cryopreservation at -150 degrees C, suggesting that the new cryopreservation protocol at -80 degrees C is a method of choice for treating vessel replacement in vascular surgery.

Weak D Types 1, 2, and 3 recipients cannot be immunized when exposed to D antigen. Molecular biology is very efficient to type weak D variants but rarely implemented in daily practice. The serologic typing practice of weak D in a Caucasian patient population was analyzed and a transfusion strategy is proposed.Samples typed either ddCcee or ddccEe in routine laboratories were tested with the indirect antiglobulin test (D(u) test). D(u)-positive samples were screened for weak D alleles Types 1, 2, and 3 and further tested with immunoglobulin M (IgM) anti-D reagents, used in a fully automated device.A total of 468 of 55,162 samples were found to be ddCcee or ddccEe. Ninety-three expressed weak D after the D(u) test leading to D+ assignment for transfusion. Seventy-three percent of D(u)-positive samples were weak D alleles Type 1, 2, or 3. Almost all weak D Types 1, 2, and 3 were positive with IgM reagents in gel matrix with an automated device. Other variants that could be potentially associated with anti-D alloimmunization, however, were also positive.Serology is very sensitive to detect weak D Types 1, 2, and 3, but there is no cutoff to distinguish variants of clinical significance. When molecular analysis is not available, it is proposed that a D+ status for blood recipients found to be weak D with a sensitive method be assigned, except for women of childbearing age or younger, because of the remaining possibility to be partial D or other rare weak D who can be immunized.

A rare erythrocyte phenotype is characterized by the absence of expression of a high-frequency antigen on red blood cells or the absence of several antigens within the same blood group. In France, a blood group is considered rare when the frequency in the general population is less than 1/4000. Allo-immunization against lacking antigens through transfusion or pregnancy requires providing adapted transfusion-support in order to secure further transfusions. A national file of individuals including both patients and donors has been implemented for more than 20 years, along with a national rare blood bank (BNSPR) designed to provide rare blood 24 hours a day. The organization is based on cooperation between the National Blood Service (EFS) and the National Reference Center for Blood Groups (CNRGS). Rare blood donation is anonymous, homologous and non paid. Special dispensations are applied compared to the standard blood donation rules: age of donor, absence of leucodepletion, specific agreement for markers except HIV. The BNSPR is in charge of the rare blood unit management (reception, freezing, storage, thawing, distribution, delivering, and shipping) and of the sample storage. RBCs are freezed under −80°C with glycerol according to the Valeri’s method. The post wash storage period is limited to 24h in the open system of cryopreservation because the deglycerolization step is performed in an open system. In the automated closed system, RBCs can be both glycerolized and deglycerolized, and then the post wash storage in SAGM at +4°C is extended to 7 days. In both systems, the mean value of freeze-thaw wash process recovery is 79.5%. The mean percentage of haemolysis and residual extracellular glycerol, measured on the day of deglycerolization, are 0.2% and 0.15g respectively. From 2002 to 2007, 4064 units were frozen, 1076 were thawed for 211 patients and 2426 were destroyed for storage regulation. The aim of regulation is to reach for almost all units the standard blood donation rules, but also to improve the phenotypic and genotypic characteristics of the units. As an example, in the FY blood group, only the RH:−20, KEL: −6 units will be maintained in the frozen stock in order to avoid allo-immunization against low frequency antigens, difficult to detect in a routine laboratory. At the end of 2007, 9600 individuals are listed in the national file, 5470 blood units corresponding to 1603 donors are frozen. 97% of the donors are typed at least in the RH, KEL, FY, JK and MNS. More than 125 rare specificities are represented with the following repartition of the units: FY: −1, −2 (25%), KEL: −2 (18%), YT: −1 (15%), rare RH (8%), Vel-negative (6.5%), MNS: −3, −4, −5 (6%) LU: −2 (5%). Units from Afro-Caribbean donors represent 35% of the stock. 83% of the units are stored for less than 10 years and 84% are tested for HIV and HCV NAT. 2% of the units present biological abnormality markers, 2% have medical counter-indications in the donors and 0.9 % are not leuco-depleted. The mean number of transfused units per patient is 5.8 (min=1, max=36 for a KEL: −2 individual). The mean number of transfusion episodes per patient is 2.5 (min=1, max=16 for a FY: −1, −2 individual). Since 2002, the BNSPR has been helping other countries and 16 units have been shipped to different regions e.g. Canada, The Netherlands, Belgium, Germany, Switzerland. France should now define its policy regarding the shipping of rare blood units to foreign countries in order to extend this service to longer populations. This may be done in the cooperation frame of the European Council.