Characterization of the input material quality for the production of tisagenlecleucel by multiparameter flow cytometry and its relation to the clinical outcome.

Tisagenlecleucel (tisa-cel) is a CD19-specific CAR-T cell product approved for the treatment of relapsed/refractory (r/r) DLBCL or B-ALL. We have followed a group of patients diagnosed with childhood B-ALL (n = 5), adult B-ALL (n = 2), and DLBCL (n = 25) who were treated with tisa-cel under non-clinical trial conditions. The goal was to determine how the intensive pretreatment of patients affects the produced CAR-T cells, their in vivo expansion, and the outcome of the therapy. Multiparametric flow cytometry was used to analyze the material used for manufacturing CAR-T cells (apheresis), the CAR-T cell product itself, and blood samples obtained at three timepoints after administration. We present the analysis of memory phenotype of CD4/CD8 CAR-T lymphocytes (CD45RA, CD62L, CD27, CD28) and the expression of inhibitory receptors (PD-1, TIGIT). In addition, we show its relation to the patients' clinical characteristics, such as tumor burden and sensitivity to prior therapies. Patients who responded to therapy had a higher percentage of CD8+CD45RA+CD27+ T cells in the apheresis, although not in the produced CAR-Ts. Patients with primary refractory aggressive B-cell lymphomas had the poorest outcomes which was characterized by undetectable CAR-T cell expansion in vivo. No clear correlation of the outcome with the immunophenotypes of CAR-Ts was observed. Our results suggest that an important parameter predicting therapy efficacy is CAR-Ts' level of expansion in vivo but not the immunophenotype. After CAR-T cells' administration, measurements at several timepoints accurately detect their proliferation intensity in vivo. The outcome of CAR-T cell therapy largely depends on biological characteristics of the tumors rather than on the immunophenotype of produced CAR-Ts.

A Phase I Trial of Allogeneic γδ T Lymphocytes From Haploidentical Donors in Patients With Refractory or Relapsed Acute Myeloid Leukemia.

Introduction We report the results of a phase I clinical trial NCT03790072 of an adoptive transfer of γδ T lymphocytes from haploidentical donors in patients with refractory/relapsed acute myeloid leukemia after lymphodepletion regimen. Patients and methods Healthy donor mononuclear cells collected by leukapheresis were consistently expanded to generate products of 109 to 1010 γδ T cells. Seven patients received donor-derived T cell product at doses of 106/kg (n = 3), 107/kg (n = 3), and 108/kg (n = 1). Results Four patients had bone marrow evaluation at day 28. One patient had a complete remission, one was classified as morphologic leukemia-free state, one had stable disease and one had no evidence of response. In one patient, there was evidence of disease control with repeat infusions up to 100 days after first dosing. There were no treatment-related serious adverse events or treatment-related Common Terminology Criteria for Adverse Events grade 3 or greater toxicities at any dose level. Allogeneic Vγ9Vδ2 T cell infusion was shown to be safe and feasible up to a cell dose of 108/kg. Discussion In agreement with previously published studies, the infusion of allogeneic Vγ9Vδ2 cells was safe. The contribution of lymphodepleting chemotherapy to responses seen cannot be ruled out. Main limitation of the study is the low number of patients and interruption due to COVID-19 pandemic. Conclusion These positive Phase 1 results support progression to phase II clinical trials.

Hydrogel Tissue Expanders for Stomatology. Part II. Poly(styrene-maleic anhydride) Hydrogels.

Self-inflating soft tissue expanders represent a valuable modality in reconstructive surgery. For this purpose, particularly synthetic hydrogels that increase their volume by swelling in aqueous environment are used. The current challenge in the field is to deliver a material with a suitable protracted swelling response, ideally with an induction period (for sutured wound healing) followed by a linear increase in volume lasting several days for required tissue reconstruction. Here, we report on synthesis, swelling, thermal, mechanical and biological properties of novel hydrogel tissue expanders based on poly(styrene-alt-maleic anhydride) copolymers covalently crosslinked with p-divinylbenzene. The hydrogels exerted hydrolysis-driven swelling response with induction period over the first two days with minimal volume change and gradual volume growth within 30 days in buffered saline solution. Their final swollen volume reached more than 14 times the dry volume with little dependence on the crosslinker content. The mechanical coherence of samples during swelling and in their fully swollen state was excellent, the compression modulus of elasticity being between 750 and 850 kPa. In vitro cell culture experiments and in vivo evaluation in mice models showed excellent biocompatibility and suitable swelling responses meeting thus the application requirements as soft tissue expanders.

Hematopoietic Stem Cell Transplantation From Haploidentical Donors in Aplasia After Cladribine/Cytarabine Chemotherapy for Refractory Acute Myeloid Leukemia or Myelodysplastic Syndrome.

INTRODUCTION: Survival rate of patients with chemorefractory acute myeloid leukemia (AML) or myelodysplastic syndrome with excess blasts (MDS-EB) is poor. Allogeneic hematopoietic cell transplantation (HCT) is the only potentially curative therapy in these patients. PATIENTS AND METHODS: We report a retrospective analysis of outcomes of therapy of 24 patients with AML or MDS-EB refractory to high-dose salvage chemotherapy or who had failed previous HCT, who received T-cell-replete HLA haploidentical HCT in aplasia after cladribine/cytarabine-based chemotherapy followed by reduced intensity or myeloablative conditioning. All patients had active disease before commencement of the treatment. RESULTS: Of the patients, 91.7% achieved complete remission (CR), whereas 2 patients (8.2%) died in aplasia. One-year relapse rate was 49.3%. Cumulative incidence of nonrelapse mortality (NRM) was 25.6%. In a subgroup of patients with HCT-comorbidity index score ≤ 3, NRM was 15.4%. Two-year overall survival and relapse-free survival were 30.6% and 22.6%, respectively. Incidence of grade 3 and 4 acute graft versus host disease was 21.3% and 8.3, respectively. CONCLUSION: We found that sequential therapy with HCT in aplasia after cladribine/cytarabine chemotherapy is feasible, results in high CR rates, and has acceptable toxicity profile; however, posttransplant relapse is common in patients treated with active disease.

A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21.

BACKGROUND AIMS: Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. METHODS: We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. RESULTS: We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. CONCLUSIONS: Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.

Hydrogel tissue expanders for stomatology. Part I. Methacrylate-based polymers.

In order to create a soft tissue surplus, implantable volume expanders are often utilized in dental surgery. Implanted tissue expanders should gradually increase their volume, exerting a constant pressure on the surrounding tissue for weeks. Current tissue expanders are based predominantly on externally inflatable balloons or on osmotically active tissue expanders that use soft hydrogels wrapped in perforated plastic coatings, which limit fluid entry and swelling. We have designed and examined tissue expanders based on the controlled rate expansive hydrogels synthesized from copolymers of selected methacrylates and N-vinylpyrrolidone, cross-linked with a combination of non-degradable (glycol dimethacrylates) and hydrolytically degradable (N,O-dimethacryloylhydroxylamine) cross-linkers. These copolymers have close-to-linear volume expansion rates (up to 6-9 times their original volume) and exert an increasing swelling pressure in vitro. The anesthetic benzocaine has been incorporated into the hydrogels, and kinetic release experiments have shown that most of the drug (90%) was released within 48 h. Our proposed hydrogel expanders are homogeneous and have suitable mechanical properties, thus simplifying the surgical manipulations required. Further studies will be needed to completely evaluate their biocompatibility and tissue response to the implants.

Human multipotent mesenchymal stem cells improve healing after collagenase tendon injury in the rat.

BACKGROUND: Mesenchymal stromal cells attract much interest in tissue regeneration because of their capacity to differentiate into mesodermal origin cells, their paracrine properties and their possible use in autologous transplantations. The aim of this study was to investigate the safety and reparative potential of implanted human mesenchymal stromal cells (hMSCs), prepared under Good Manufacturing Practice (GMP) conditions utilizing human mixed platelet lysate as a culture supplement, in a collagenase Achilles tendon injury model in rats. METHODS: Eighty-one rats with collagenase-induced injury were divided into two groups. The first group received human mesenchymal stromal cells injected into the site of injury 3 days after lesion induction, while the second group received saline. Biomechanical testing, morphometry and semiquantitative immunohistochemistry of collagens I, II and III, versican and aggrecan, neovascularization, and hMSC survival were performed 2, 4, and 6 weeks after injury. RESULTS: Human mesenchymal stromal cell-treated rats had a significantly better extracellular matrix structure and a larger amount of collagen I and collagen III. Neovascularization was also increased in hMSC-treated rats 2 and 4 weeks after tendon injury. MTCO2 (Cytochrome c oxidase subunit II) positivity confirmed the presence of hMSCs 2, 4 and 6 weeks after transplantation. Collagen II deposits and alizarin red staining for bone were found in 6 hMSC- and 2 saline-treated tendons 6 weeks after injury. The intensity of anti-versican and anti-aggrecan staining did not differ between the groups. CONCLUSIONS: hMSCs can support tendon healing through better vascularization as well as through larger deposits and better organization of the extracellular matrix. The treatment procedure was found to be safe; however, cartilage and bone formation at the implantation site should be taken into account when planning subsequent in vivo and clinical trials on tendinopathy as an expected adverse event.

Hepatocyte growth on polycapronolactone and 2-hydroxyethylmethacrylate nanofiber sheets enhanced by bone marrow-derived mesenchymal stromal cells.

BACKGROUND/AIMS: The development of hepatocyte-based Bioartificial Liver Assist Devices, intended for the therapy of chronic and fulminant liver failure, is one of the important tasks in the area of tissue engineering. New advances in the development of semipermeable non-woven nanofiber biomaterials and the co-cultivation of bone marrow mesenchymal stromal cells (BMSC) and hepatocytes could be utilized in order to maintain hepatocyte cultures in these devices. METHODOLOGY: We have compared rat hepatocyte growth on nanofiber biomaterials from different polymers, 2-hydroxyethylmethacrylate (HEMA) and ethoxyethylmethacrylate (EOEMA) copolymers, polyurethane (PUR), chitosan and polycapronolactone (PCL) spun from different solvent mixtures. RESULTS: In all cases the adhesion of hepatocytes to nanofibers was significantly better/stronger than to unstructured polymer surfaces; coating the nanofibers with collagen did not increase cell adhesion. We found the best hepatocyte adhesion on HEMA/EOEMA copolymer nanofibers and PCL nanofibers spun from a mixture of ethylacetate and dimethyl sulphoxide. Using a migration assay, we observed the migration of BMSC towards hepatocytes; hepatocytes cocultivated with BMSC excreted lower amounts of stress enzymes. CONCLUSIONS: The results demonstrate that nonwoven nanofiber layers, particularly those containing BMSC, are a suitable biocompatible support for functional hepatocyte cultures and that they can be used in a laboratory bioreactor or potentially in clinical setting.

The use of dopamine-hyaluronate associate-coated maghemite nanoparticles to label cells.

Sodium hyaluronate (HA) was associated with dopamine (DPA) and introduced as a coating for maghemite (γ-Fe(2)O(3)) nanoparticles obtained by the coprecipitation of iron(II) and iron(III) chlorides and oxidation with sodium hypochlorite. The effects of the DPA anchorage of HA on the γ-Fe(2)O(3) surface on the physicochemical properties of the resulting colloids were investigated. Nanoparticles coated at three different DPA-HA/γ-Fe(2)O(3) and DPA/HA ratios were chosen for experiments with rat bone marrow mesenchymal stem cells and human chondrocytes. The nanoparticles were internalized into rat bone marrow mesenchymal stem cells via endocytosis as confirmed by Prussian Blue staining. The efficiency of mesenchymal stem cell labeling was analyzed. From among the investigated samples, efficient cell labeling was achieved by using DPA-HA-γ-Fe(2)O(3) nanoparticles with DPA-HA/γ-Fe(2)O(3) = 0.45 (weight/ weight) and DPA/HA = 0.038 (weight/weight) ratios. The particles were used as a contrast agent in magnetic resonance imaging for the labeling and visualization of cells.

Nanofibers prepared by needleless electrospinning technology as scaffolds for wound healing.

Electrospun gelatin and poly-ε-caprolactone (PCL) nanofibers were prepared using needleless technology and their biocompatibility and therapeutic efficacy have been characterized in vitro in cell cultures and in an experimental model of a skin wound. Human dermal fibroblasts, keratinocytes and mesenchymal stem cells seeded on the nanofibers revealed that both nanofibers promoted cell adhesion and proliferation. The effect of nanofibers on wound healing was examined using a full thickness wound model in rats and compared with a standard control treatment with gauze. Significantly faster wound closure was found with gelatin after 5 and 10 days of treatment, but no enhancement with PCL nanofibers was observed. Histological analysis revealed enhanced epithelialisation, increased depth of granulation tissue and increased density of myofibroblasts in the wound area with gelatin nanofibers. The results show that gelatin nanofibers produced by needleless technology accelerate wound healing and may be suitable as a scaffold for cell transfer and skin regeneration.

The use of oligoperoxide-coated magnetic nanoparticles to label stem cells.

Iron oxide nanoparticles obtained by the coprecipitation of Fe(II) and Fe(III) salts and oxidation were coated with a novel poly(vinyl acetate-co-5-tert-(butylperoxy)-5-methylhex-1-en-3-yne-co-butyl acrylate-co-maleic anhydride) (PVBM) oligomer to ensure colloidal stability. The magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. Experiments with rat mesenchymal stem cells (rMSCs) confirmed that PVBM-coated gamma-Fe2O3 nanoparticles were not cytotoxic and that the average efficiency of stem cell labeling was good and comparable to that obtained with commercial agents. The cells labeled with PVBM-coated gamma-Fe2O3 nanoparticles displayed excellent contrast on magnetic resonance (MR) images. Such particles are thus promising for in vivo MR imaging of transplanted cells. Moreover, PVBM offers the possibility of additional modification by grafting compounds that reduce non-specific protein adsorption.

HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury.

Chronic spinal cord injury (SCI) is characterized by tissue loss and a stable functional deficit. While several experimental therapies have proven to be partly successful for the treatment of acute SCI, treatment of chronic SCI is still challenging. We studied whether we can bridge a chronic spinal cord lesion by implantation of our newly developed hydrogel based on 2-hydroxypropyl methacrylamide, either alone or seeded with mesenchymal stem cells (MSCs), and whether this treatment leads to functional improvement. A balloon-induced compression lesion was performed in adult 2-month-old male Wistar rats. Five weeks after injury, HPMA-RGD hydrogels [N-(2-hydroxypropyl)-methacrylamide with attached amino acid sequences--Arg-Gly-Asp] were implanted into the lesion, either with or without seeded MSCs. Animals with chronic SCI served as controls. The animals were behaviorally tested using the Basso­Beattie-Breshnahan (BBB) (motor) and plantar (sensory) tests once a week for 6 months. Behavioral analysis showed a statistically significant improvement in rats with combined treatment, hydrogel and MSCs, compared with the control group (P < 0.05). Although a tendency toward improvement was found in rats treated with hydrogel only, this was not significant. Subsequently, the animals were sacrificed 6 months after SCI, and the spinal cord lesions evaluated histologically. The combined therapy (hydrogel with MSCs) prevented tissue atrophy (P < 0.05), and the hydrogels were infiltrated with axons myelinated with Schwann cells. Blood vessels and astrocytes also grew inside the implant. MSCs were present in the hydrogels even 5 months after implantation. We conclude that 5 weeks after injury, HPMA-RGD hydrogels seeded with MSCs can successfully bridge a spinal cord cavity and provide a scaffold for tissue regeneration. This treatment leads to functional improvement even in chronic SCI.

Poly(N,N-dimethylacrylamide)-coated maghemite nanoparticles for stem cell labeling.

Maghemite (gamma-Fe2O3) nanoparticles were obtained by the coprecipitation of Fe(II) and Fe (III) salts with ammonium hydroxide followed by oxidation with sodium hypochlorite. Solution radical polymerization of N,N-dimethylacrylamide(DMAAm) in the presence of maghemite nanoparticles yielded poly(N,N-dimethylacrylamide)(PDMAAm)-coated maghemite nanoparticles. The presence of PDMAAm on the maghemite particle surface was confirmed by elemental analysis and ATR FTIR spectroscopy. Other methods of nanoparticle characterization involved scanning and transmission electron microscopy, atomic adsorption spectroscopy (AAS), and dynamic light scattering (DLS). The conversion of DMAAm during polymerization and the molecular weight of PDMAAmbound to maghemite were determined by using gas and size-exclusion chromatography, respectively. The effect of ionic 4,4'-azobis(4-cyanovaleric acid) (ACVA) initiator on nanoparticle morphology was elucidated. The nanoparticles exhibited long-term colloidal stability in water or physiological buffer. Rat and human bone marrow mesenchymal stem cells (MSCs) were labeled with uncoated and PDMAAm-coated maghemite nanoparticles and with Endorem as a control. Uptake of the nanoparticles was evaluated by Prussian Blue staining, transmission electron microscopy, T(2)-MR relaxometry, and iron content analysis. Significant differences in labeling efficiency were found for human and rat cells. PDMAAm-modified nanoparticles demonstrated a higher efficiency of intracellular uptake into human cells in comparison with that of dextran-modified (Endorem) and unmodified nanoparticles. In gelatin, even a small number of labeled cells changed the contrast in MR images. PDMAAmcoatednanoparticles provided the highest T(2) relaxivity of all the investigated particles. In vivo MR imaging ofPDMAAm-modified iron oxide-labeled rMSCs implanted in a rat brain confirmed their better resolution compared with Endorem-labeled cells.

Surface modification of hydrogels based on poly(2-hydroxyethyl methacrylate) with extracellular matrix proteins.

Infrared attenuated total reflection spectroscopy was used for in situ observation of the deposition of collagen I on poly(2-hydroxyethyl methacrylate-co-methacrylic acid, 2.9%) hydrogels and subsequent attachment of laminin or fibronectin on the collagen surface. While there was no adsorption of collagen dissolved in an acid solution on the hydrogel surface, it deposited on the surface at pH 6.5. The collagen layers with attached laminin or fibronectin were stable on hydrogel surface in physiological solution. The modification with collagen and particularly with collagen and laminin or fibronectin allowed the adhesion and growth of mesenchymal stromal cells and astrocytes on the hydrogel surface.

Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.

New surface-modified iron oxide nanoparticles were developed by precipitation of Fe(II) and Fe(III) salts with ammonium hydroxide and oxidation of the resulting magnetite with sodium hypochlorite, followed by the addition of poly( L-lysine) (PLL) solution. PLL of several molecular weights ranging from 146 ( L-lysine) to 579 000 was tested as a coating to boost the intracellular uptake of the nanoparticles. The nanoparticles were characterized by TEM, dynamic light scattering, FTIR, and ultrasonic spectrometry. TEM revealed that the particles were ca. 6 nm in diameter, while FTIR showed that their surfaces were well-coated with PLL. The interaction of PLL-modified iron oxide nanoparticles with DMEM culture medium was verified by UV-vis spectroscopy. Rat bone marrow stromal cells (rMSCs) and human mesenchymal stem cells (hMSC) were labeled with PLL-modified iron oxide nanoparticles or with Endorem (control). Optical microscopy and TEM confirmed the presence of PLL-modified iron oxide nanoparticles inside the cells. Cellular uptake was very high (more than 92%) for PLL-modified nanoparticles that were coated with PLL (molecular weight 388 00) at a concentration of 0.02 mg PLL per milliliter of colloid. The cellular uptake of PLL-modified iron oxide was facilitated by its interaction with the negatively charged cell surface and subsequent endosomolytic uptake. The relaxivity of rMSCs labeled with PLL-modified iron oxide and the amount of iron in the cells were determined. PLL-modified iron oxide-labeled rMSCs were imaged in vitro and in vivo after intracerebral grafting into the contralateral hemisphere of the adult rat brain. The implanted cells were visible on magnetic resonance (MR) images as a hypointense area at the injection site and in the lesion. In comparison with Endorem, nanoparticles modified with PLL of an optimum molecular weight demonstrated a higher efficiency of intracellular uptake by MSC cells.

Acute and delayed implantation of positively charged 2-hydroxyethyl methacrylate scaffolds in spinal cord injury in the rat.

OBJECT: Hydrogels are nontoxic, chemically inert synthetic polymers with a high water content and large surface area that provide mechanical support for cells and axons when implanted into spinal cord tissue. METHODS: Macroporous hydrogels based on 2-hydroxyethyl methacrylate (HEMA) were prepared by radical copolymerization of monomers in the presence of fractionated NaCl particles. Male Wistar rats underwent complete spinal cord transection at the T-9 level. To bridge the lesion, positively charged HEMA hydrogels were implanted either immediately or 1 week after spinal cord transection; control animals were left untreated. Histological evaluation was performed 3 months after spinal cord transection to measure the volume of the pseudocyst cavities and the ingrowth of tissue elements into the hydrogels. RESULTS: The hydrogel implants adhered well to the spinal cord tissue. Histological evaluation showed ingrowth of connective tissue elements, blood vessels, neurofilaments, and Schwann cells into the hydrogels. Morphometric analysis of lesions showed a statistically significant reduction in pseudocyst volume in the treated animals compared with controls and in the delayed treatment group compared with the immediate treatment group (p < 0.001 and p < 0.05, respectively). CONCLUSIONS: Positively charged HEMA hydrogels can bridge a posttraumatic spinal cord cavity and provide a scaffold for the ingrowth of regenerating axons. The results indicate that delayed implantation can be more effective than immediate reconstructive surgery.

Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part 5: hydrolytically degradable materials.

Macroporous hydrogels based on 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and N-(2-hydroxypropyl)methacrylamide, methacrylic acid and [2-(methacryloyloxy)ethyl]trimethylammonium chloride crosslinked with N,O-dimethacryloylhydroxylamine were prepared. Hydrogels were degraded in a buffer of pH 7.4. Completely water-soluble polymers were obtained over time periods ranging from 2 to 40 days. The process of degradation was followed gravimetrically and by optical and electron microscopy. In vivo biological tests with hydrogels based on copolymers of 2-ethoxyethyl methacrylate/N-(2-hydroxypropyl)methacrylamide were performed.

Grid empowered sharing of medical expertise.

Today, applications for Grids emerge in various scientific fields, each with specific requirements. We present concept and architecture which enables biomedical experts to collaborate and share resources by encapsulating their knowledge and expertise as grid services, with (semi-)formally described semantics. Grid Services allow machine processing of the encapsulated knowledge, while their semantic description provides means for their automated discovery and interaction. This brings new possibilities of building biomedical systems offering machine-driven assistance to the biomedical experts.

Bone marrow stem cells and polymer hydrogels--two strategies for spinal cord injury repair.

1. Emerging clinical studies of treating brain and spinal cord injury (SCI) led us to examine the effect of autologous adult stem cell transplantation as well as the use of polymer scaffolds in spinal cord regeneration. We compared an intravenous injection of mesenchymal stem cells (MSCs) or the injection of a freshly prepared mononuclear fraction of bone marrow cells (BMCs) on the treatment of an acute or chronic balloon-induced spinal cord compression lesion in rats. Based on our experimental studies, autologous BMC implantation has been used in a Phase I/II clinical trial in patients (n=20) with a transversal spinal cord lesion. 2. MSCs were isolated from rat bone marrow by their adherence to plastic, labeled with iron-oxide nanoparticles and expanded in vitro. Macroporous hydrogels based on derivatives of 2-hydroxyethyl methacrylate (HEMA) or 2-hydroxypropyl methacrylamide (HPMA) were prepared, then modified by their copolymerization with a hydrolytically degradable crosslinker, N,O-dimethacryloylhydroxylamine, or by different surface electric charges. Hydrogels or hydrogels seeded with MSCs were implanted into rats with hemisected spinal cords. 3. Lesioned animals grafted with MSCs or BMCs had smaller lesions 35 days postgrafting and higher scores in BBB testing than did control animals and also showed a faster recovery of sensitivity in their hind limbs using the plantar test. The functional improvement was more pronounced in MSC-treated rats. In MR images, the lesion populated by grafted cells appeared as a dark hypointense area and was considerably smaller than in control animals. Morphometric measurements showed an increase in the volume of spared white matter in cell-treated animals. In the clinical trial, we compared intraarterial (via a. vertebralis, n=6) versus intravenous administration of BMCs (n=14) in a group of subacute (10-33 days post-SCI, n=8) and chronic patients (2-18 months, n=12). For patient follow-up we used MEP, SEP, MRI, and the ASIA score. Our clinical study revealed that the implantation of BMCs into patients is safe, as there were no complications following cell administration. Partial improvement in the ASIA score and partial recovery of MEP or SEP have been observed in all subacute patients who received cells via a. vertebralis (n=4) and in one out of four subacute patients who received cells intravenously. Improvement was also found in one chronic patient who received cells via a. vertebralis. A much larger population of patients is needed before any conclusions can be drawn. The implantation of hydrogels into hemisected rat spinal cords showed that cellular ingrowth was most pronounced in copolymers of HEMA with a positive surface electric charge. Although most of the cells had the morphological properties of connective tissue elements, we found NF-160-positive axons invading all the implanted hydrogels from both the proximal and distal stumps. The biodegradable hydrogels degraded from the border that was in direct contact with the spinal cord tissue. They were resorbed by macrophages and replaced by newly formed tissue containing connective tissue elements, blood vessels, GFAP-positive astrocytic processes, and NF-160-positive neurofilaments. Additionally, we implanted hydrogels seeded with nanoparticle-labeled MSCs into hemisected rat spinal cords. Hydrogels seeded with MSCs were visible on MR images as hypointense areas, and subsequent Prussian blue histological staining confirmed positively stained cells within the hydrogels. 4. We conclude that treatment with different bone marrow cell populations had a positive effect on behavioral outcome and histopathological assessment after SCI in rats; this positive effect was most pronounced following MSC treatment. Our clinical study suggests a possible positive effect in patients with SCI. Bridging the lesion cavity can be an approach for further improving regeneration. Our preclinical studies showed that macroporous polymer hydrogels based on derivatives of HEMA or HPMA are suitable materials for bridging cavities after SCI; their chemical and physical properties can be modified to a specific use, and 3D implants seeded with different cell types may facilitate the ingrowth of axons.

Corrosive injury of the oesophagus in children.

OBJECTIVE: This study examined a cohort of pediatric patients treated for suspected corrosive injury of the oesophagus in the ENT department between 1994 and 2003. METHODS: During the study period we examined 337 patients. All patients were treated according to an individual diagnostico-therapeutic protocol that included: foremost, early rigid oesophagoscopy; intensive medical therapy; nasogastric tube placement; and/or surgical intervention. RESULTS: Our proposed system of grading on a scale from 0 to 4 was used to classify the findings upon oesophagoscopy. One hundred eighty eight patients (55.7%) had a negative exam; 1st degree corrosive injury was present in 58 patients (17.4%); 2nd and 3rd degree injury in 81 patients (24%); and 4th degree was found in 10 patients (3%). CONCLUSIONS: Because the endoscopic findings were positive in 44% of patients with suspected corrosive injury, early rigid oesophagoscopy is indicated in all patients suspected of having corrosive injury even in the absence of clinical symptoms.