Application of a three-dimensional graft of autologous osteodifferentiated adipose stem cells in patients undergoing minimally invasive transforaminal lumbar interbody fusion: clinical proof of concept.

BACKGROUND: The authors applied a scaffold-free osteogenic three-dimensional (3D) graft made of adipose-derived mesenchymal stem cells (AMSCs) in patients undergoing minimally invasive transforaminal lumbar interbody fusion (MI-TLIF). METHODS: Three patients (two patients and one patient with 1 and 2 levels, respectively) with degenerative spondylolisthesis underwent MI-TLIF with 3D graft made of AMSCs. To obtain the AMSCs, fatty tissue was collected from the abdomen by lipoaspiration and differentiated afterwards in our Cell/Tissue bank. Clinical outcomes, including the Oswestry Disability Index (ODI) and visual analog scale (VAS) as well as fusion status were assessed preoperatively and up to 12 months postoperatively. RESULTS: At 12 months, all four operated AMSC levels could be assessed (n = 4). Grade 3 fusion could be confirmed at two levels out of four. Mean VAS score improved from 8.3 to 2 and ODI also improved from 47 to 31%. No donor site complication was observed. The final AMSC osteogenic product was stable, did not rupture with forceps manipulation, and was easily implanted directly into the cage with no marked modification of operating time. CONCLUSIONS: A scaffold-free 3D graft made of AMSCs can be manufactured and used as a promising alternative for spinal fusion procedures. Nevertheless, further studies of a larger series of patients are needed to confirm its effectiveness.

A genomic-based approach identifies FXYD domain containing ion transport regulator 2 (FXYD2)gammaa as a pancreatic beta cell-specific biomarker.

AIMS/HYPOTHESIS: Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers. METHODS: We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays. RESULTS: After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na(+)-K(+)-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2gammaa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2gammaa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2gammaa and loss of insulin-positive cells. CONCLUSIONS/INTERPRETATION: We propose human FXYD2gammaa as a novel beta cell-specific biomarker.

Human tissue allograft processing: impact on in vitro and in vivo biocompatibility.

This work investigates the impact of chemical and physical treatments on biocompatibility for human bone/tendon tissues. Nontreated and treated tissues were compared. In vitro testing assessed indirect and direct cytotoxicity. Tissues were subcutaneously implanted in rats to assess the immunological, recolonization, and revascularization processes at 2-4 weeks postimplantation. No significant cytotoxicity was found for freeze-dried treated bones and tendons in comparison to control. The cellular adhesion was significantly reduced for cells seeded on these treated tissues after 24 h of direct contact. A significant cytotoxicity was found for frozen treated bones in comparison to freeze-dried treated bones. Tissue remodeling with graft stability, no harmful inflammation, and neo-vascularization was observed for freeze-dried chemically treated bones and tendons. Frozen-treated bones were characterized by a lack of matrix recolonization at 4 weeks postimplantation. In conclusion, chemical processing with freeze-drying of human tissues maintains in vitro biocompatibility and in vivo tissue remodeling for clinical application.

Nutrient control of insulin secretion in perifused adult pig islets.

OBJECTIVES: Xenotransplantation of pig islets is a potential solution to the shortage of human islets, but our knowledge of how these islets secrete insulin in response to nutrients is still fragmentary. This was the question addressed in the present study. METHODS: After 24 h culture adult pig islets were perifused to characterize the dynamics of insulin secretion. Some responses were compared to those in human islets. RESULTS: Increasing glucose from 1 to 15 mM weakly (approximately 2x) stimulated insulin secretion, which was potentiated (approximately 12x) by the cAMP-producing agent, forskolin. The effect of glucose was concentration-dependent (threshold at 3-5 mM and maximum at approximately 10 mM). The pattern of secretion was biphasic with a small first phase and an ascending second phase, and a paradoxical increase when the glucose concentration was abruptly lowered. Diazoxide abolished glucose-induced insulin secretion and tolbutamide reversed the inhibition. Glucose also increased secretion when islets were depolarized with tolbutamide or KCl. Insulin secretion was increased by leucine+glutamine, arginine, alanine or a mixture of amino acids, but their effect was significant only in the presence of forskolin. Upon stimulation by glucose alone, human islets secreted approximately 10x more insulin than pig islets, and the kinetics was characterized by a large first phase, a flat second phase, and rapid reversibility. CONCLUSIONS: Compared with human islets, in vitro insulin secretion by adult pig islets is characterized by a different kinetics and a major quantitative deficiency that can be corrected by cAMP.

Non-heart-beating donor, 10-year experience in a Belgian transplant center.

All over the world, transplant teams are looking for ways to increase and improve the donor pool. Non-heart-beating donation may increase the number of donors, even if some technical, logistical, and emotional problems are still encountered. The results obtained by our team should stimulate other centers to implement this kind of donation in their hospitals. Herein we have described our experience with non-heart-beating donation.

[Correction of a diabetes mellitus type 1 on primate with encapsulated islet of pig pancreatic transplant]. / Correction d'un diabète de type I chez le primate par greffe d'îlots pancréatiques porcins encapsulés.

The allogeneic transplantation of pancreatic islet cells into diabetic patients is severely restricted by the recurrent lack of organs. To the lack of donors is added the fact that several pancreas donors are often needed in order to treat a single diabetic recipient. Another source of cells that can produce insulin would therefore be of major interest. The pig constitutes a possible option to be taken seriously. Nonetheless, grafting pig islets might prove difficult because of the species barrier. The principal obstacle to the xenotransplantation of cells remains the necessity for powerful immunosuppression, probably inapplicable in man. Therefore, we have centred our work around the encapsulation of islets in order to evaluate the possibility of using pig cells without immunosuppression. (1) This experimental work first evaluated the influence of the age of the pig (young versus adult pigs) on the size of the islets, the collagen and the vascular structures. Adult donor pigs were selected. (Dufrane et al., Pancreas 30(2), 2005). (2) After deciding on the age, all the parameters involved in the removal of the pancreas and the procedures of isolating the pig islets were scrutinized by multivariable analysis. (Dufrane et al., Xenotransplantation 13(3), 2006). (3) Before evaluating the effect of encapsulation on the pig islets in diabetic monkeys, the biocompatibility of an alginate membrane was first evaluated in a non-diabetic primate model without immunosuppression. The long term stability and biocompatibility of pig islets encapsulated in alginate have been confirmed for up to six months after implantation under the renal capsule. A small proportion of these encapsulated pig islets not only survived for six months but remained capable of reacting in vitro to stimulation with glucose + forskolin, thus demonstrating preserved endocrine function. This in vivo experience has enabled us to identify the most important parameters for successful encapsulation. (Dufrane et al., Transplantation 81(9), 2006; Dufrane et al., Biomaterials 27(17), 2005). We then (4) investigated the most appropriate means of inducing irreversible diabetes in primates: a low dose (50 mg/kg) of streptozotocine (STZ) permanently destroys more than 97% of the insulin-producing cells of the pancreas, without any side effects on hepatic or renal function. (Dufrane et al., Transplantation 81(1), 2006 (5) After induction of diabetes by STZ in primates, the capacity of encapsulated pig islets to control diabetes after transplantation was evaluated both under the renal capsule and in the form of a graft of a monolayer of cells in the subcutaneous space. The second technique enabled the diabetic status to be controlled for at least six months without any immunosuppression in four primates. This result is unique since only major immunosuppressant regimes have so far brought comparable results.

Is the expression of Gal-alpha1,3Gal on porcine pancreatic islets modified by isolation procedure?

AIM: The aim of this study was to study the Galactosyl alpha(1,3) Gal expression and the vascular tissue distribution prior to and after isolation of porcine pancreatic islets. METHODS: Biopsies of native pancreas were carried out in young (12-15 weeks; n = 4) and adult Landrace pigs (2 years old; n = 7). These pancreases were then digested (Liberase Porcine Islets [PI]) to obtain isolated pancreatic islets from each pancreas. Alpha Gal-specific biotinylated BS-1 isolectin B4 and von Willebrand's Factor (vWF) staining were performed for Galactosyl and vascular structure analysis, respectively. Quantitative Galactosyl expression as well as location of the vascular structure were determined using image analysis in pig islets of different sizes. RESULTS: Vascular structures and Galactosyl expression varied following the islet sizes but not the pig age. In large islets (>100 microm), capillaries were mainly located within the islets, whereas in small islets (<100 microm), 4-fold more vessels were situated at the periphery of the islets. Galactosyl expression followed a comparable distribution than vascular tissue in small and large islets. After isolation, a significant decrease of Gal staining (-49%) was observed, but Galactosyl expression remained positive within both small and large islets. CONCLUSIONS: Galactosyl expression is maintained within pancreatic islets after isolation procedure. Gal knock-out pigs could represent the solution to this hurdle.

Non-heart-beating donor, renewal of an old procedure: a Belgian experience.

The shortage of donated organs has become a problem in transplantation throughout the world. Transplant teams are looking for other ways to increase and improve the donor pool. Non-heart-beating donation may be a source to increase the number of donors, even if some technical, logistical, and emotional problems are encountered. The results obtained by our team should stimulate other centers to implement this kind of donation in their hospitals. We describe our experience in the policy of non-heart-beating donation and encourage transplant centers to develop such a program.

Physical and chemical processing for a human dura mater substitute.

OBJECT: Allogenic human fascia lata used in neurosurgery, as dura mater substitute, can be associated with a risk of viral and bacterial transmission. Chemical and physical procedures, developed to inactivate virus and bacteria, have been applied to fascia lata. The aim of this study consists in the evaluation of the biological properties of this treated graft. METHODS: Grafts were treated with solvent detergents, freeze-dried for conservation and gamma irradiated (25,000 Gy) for sterilization. The indirect toxicity evaluation was performed by extraction method, according to the International Standard Organization (ISO). First, the cytotoxic effect of each extracts incubated in the presence of human fibroblasts (WI38) was quantitatively assessed by measuring the cell growth, the viability (succinate dehydrogenase activity, MTT), the membrane integrity (uptake of the neutral red by viable cells, NR) as well as the release of lactate dehydrogenase in the culture medium. Second, confocal laser scanning microscopy (CLSM) was used to assess the direct contact between human primary fibroblasts and graft. CLSM was performed at days 3 and 7 after cells loading. RESULTS: No acute cytotoxicity was observed for chemically processed allografts. Cells loaded on the graft have demonstrated a good growth and spreading. CONCLUSIONS: Human fascia lata secured against conventional and non-conventional agents is a fully biocompatible alternative to the available dural graft materials.

In vitro evaluation of acute cytotoxicity of human chemically treated allografts.

In order to minimize the risk of contamination associated with tissue transplantation, tissue banks commonly chemically treat the tissues whenever possible. As viral inactivation uses agents lethal to microorganisms, it is imperative to assure that chemically inactivated tissue remains biocompatible. In vitro assays can be an effective means to assess the acute cytotoxicity of chemically treated human allografts. We have used different types of cells cultured in the presence of treated tissue extract. A standard cell line, a human fibroblast (WI38), which was the same for all the samples, was chosen. In addition, as the banked tissues (bone and fascia lata) were prepared to be used in bone or as a dura mater substitute, two other cell types were also used: an osteoblastic cell line (SaOS-2) and a neuronal cell line (Neuro 2A). Cytotoxic assessment was performed by qualitative evaluation of cell morphology based on confluence, granulation, vacuolization and swelling analysis. In addition, quantitative methods based on the use of neutral red (NR) and 3- (4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) were assayed. Qualitative and quantitative evaluation of fascia lata and bone extracts did not show deleterious effects on cell cultures. These results show that in vitro methods can be appropriate to select a non-toxic procedure before it is used in the human body and that several strong chemical treatments can result in a tissue suitable for human.

Low molecular weight dextran sulfate: a strong candidate drug to block IBMIR in clinical islet transplantation.

The instant blood-mediated inflammatory reaction (IBMIR) is triggered in clinical islet transplantation when human pancreatic islets come in contact with blood and may explain the initial tissue loss associated with this procedure. Low molecular weight dextran sulfate (LMW-DS; MM 5000), today available for clinical use, inhibits both complement and coagulation activation. In a tubing loop model, LMW-DS at concentrations ranging from 0.01 to 1 g/L showed a dose-dependent inhibition of IBMIR with an inhibition of coagulation and complement activation and less consumption of platelets and other blood cells. In blood or plasma APTT was demonstrated to be an excellent method for monitoring the LMW-DS concentration both in vitro and in vivo in a nonhuman primate model. The toxicity was assessed using a glucose challenge test and the pharmacokinetics was tested in the nonhuman primate model. Here, we present a tentative protocol for using LMW-DS in clinical islet transplantation.

Impact of porcine islet size on cellular structure and engraftment after transplantation: adult versus young pigs.

OBJECTIVES: To study the impact of porcine islet size on structural properties and cellular engraftment. METHODS: The endocrine structure and collagen/vascular localization in pig islets were studied before and after enzymatic isolation on the pancreas from 6 young and 6 adult Landrace pigs. Isolated islets from both pig types were transplanted under the kidney capsula of diabetic nude rats to assess cellular engraftment. RESULTS: In comparison with adult pig pancreata, a significantly greater number of small beta cells (<100 microm) were observed before and after isolation (82% vs. 32%, respectively, P < 0.005) from young pig pancreata. Small islets (<100 microm) showed a peripheral vascular structure, whereas large islets showed a more centralized vascular organization, thereby providing protection during the enzymatic digestion procedure. The islet endocrine structure was not affected by the islet size, but a loss of glucagon cells (-7.9%, P < 0.005) was observed in large isolated islets. The purity of islet preparation was better with pancreata from adult than young donors (86% vs. 64%, respectively, P < 0.05). A lack of engraftment was observed for small islets from young pig donors as compared with large islets from adult donors. CONCLUSIONS: Large and well-structured islets, mainly found in adult pig pancreata, probably possess a better potential for cellular engraftment due to centralized vascularization and collagen distribution.

Indirect cytotoxicity evaluation of pseudowollastonite.

This study aimed to evaluate the cytotoxicity of substances leached by pseudowollastonite (CaSiO(3)). It has been previously shown that calcium (Ca(2+)) and silicate (SiO(3)(-)) ions are released from pseudowollastonite into biological solutions. Both of these ions are known to influence the biological metabolism of osteoblastic cells essential in the mineralization process and bone-bonding mechanism. The indirect toxicity evaluation was performed by extraction method, according to International Standard Organization (ISO). Pseudowollastonite pellets obtained by solid-state reaction were incubated, in culture medium, during 24, 48, 72 or 168 h at different concentrations (5, 10, 15, 50, 100, 200 mg/ml). The cytotoxicity of each extract in presence of human osteoblastic cell line (SaOS-2) was quantitatively assessed by measuring the viability (succinate dehydrogenase activity, MTT), the membrane integrity (the uptake of the neutral red by viable cells, NR) as well as the cell necrosis by measuring the lactate dehydrogenase (LDH) released in the culture medium. No significant alteration of membrane integrity or cell suffering was detectable. However, increased cell metabolism was observed for cells exposed to pseudowollastonite extract with longest extraction time (168 h). In conclusion, mineral elements leached by pseudowollastonite do not significantly affect the metabolism of osteoblastic cells.