Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability

Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC‐seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell‐seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL‐supplemented media resulted in rMSC death, by a process attributable to heat‐labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd.     

Modulation of human mesenchymal stem cell function in a three‐dimensional matrix promotes attenuation of adverse remodelling after myocardial infarction

The application of tissue engineering (TE) practices for cell delivery offers a unique approach to cellular cardiomyoplasty. We hypothesized that human mesenchymal stem cells (hMSCs) applied to the heart in a collagen matrix would outperform the same cells grown in a monolayer and directly injected for cardiac cell replacement after myocardial infarction in a rat model. When hMSC patches were transplanted to infarcted hearts, several measures for left ventricle (LV) remodelling and function were improved, including fractional area change, wall thickness, –dP/dt and LV end‐diastolic pressure. Neovessel formation throughout the LV infarct wall after hMSC patch treatment increased by 37% when compared to direct injection of hMSCs. This observation was correlated with increased secretion of angiogenic factors, with accompanying evidence that these factors enhanced vessel formation (30% increase) and endothelial cell growth (48% increase) in vitro. These observations may explain the in vivo observations of increased vessel formation and improved cardiac function with patch‐mediated cell delivery. Although culture of hMSC in collagen patches enhanced angiogenic responses, there was no effect on cell potency or viability. Therefore, hMSCs delivered as a cardiac patch showed benefits above those derived from monolayers and directly injected. hMSCs cultured and delivered within TE constructs may represent a good option to maximize the effects of cellular cardiomyoplasty. Copyright © 2011 John Wiley & Sons, Ltd.     

Silk–ionomer and silk–tropoelastin hydrogels as charged three‐dimensional culture platforms for the regulation of hMSC response

The response of human bone marrow‐derived mesenchymal stem cells (hMSCs) encapsulated in three‐dimensional (3D) charged protein hydrogels was studied. Combining silk fibroin (S) with recombinant human tropoelastin (E) or silk ionomers (I) provided protein composite alloys with tunable physicochemical and biological features for regulating the bioactivity of encapsulated hMSCs. The effects of the biomaterial charges on hMSC viability, proliferation and chondrogenic or osteogenic differentiation were assessed. The silk–tropoelastin or silk–ionomers hydrogels supported hMSC viability, proliferation and differentiation. Gene expression of markers for chondrogenesis and osteogenesis, as well as biochemical and histological analysis, showed that hydrogels with different S/E and S/I ratios had different effects on cell fate. The negatively charged hydrogels upregulated hMSC chondrogenesis or osteogenesis, with or without specific differentiation media, and hydrogels with higher tropoelastin content inhibited the differentiation potential even in the presence of the differentiation media. The results provide insight on charge‐tunable features of protein‐based biomaterials to control hMSC differentiation in 3D hydrogels, as well as providing a new set of hydrogels for the compatible encapsulation and utility for cell functions. Copyright © 2016 John Wiley & Sons, Ltd.     

Konjac glucomannan‐based hydrogel with hyaluronic acid as a candidate for a novel scaffold for chondrocyte culture

Chondrocytes were cultured using konjac glucomannan (KGM) and hyaluronic acid (HA) as a scaffold for cartilage regeneration. They were subsequently compared with scaffolds produced using agarose hydrogels. Chondrocytes derived from Japanese white rabbits were cultured: 2.0 × 10⁵ cells were seeded on KGM containing hyaluronic acid (KGM/HA) and agarose and cultured for 5 days. Their viability was assayed using WST‐8 procedures; the ultimate stress and modulus of elasticity of each construct was calculated. After 3 days of cultivation, mRNA in chondrocytes, such as collagen types I and II and aggrecan, were measured using RT‐PCR. Both chondrocyte‐seeded constructs were stained with safranin O/fast green and were evaluated histologically. Chondrocyte viability decreased concomitantly with increasing KGM/HA or agarose concentration and with culture time. Cell viability in 2% agarose was significantly lower than that in 2% KGM/HA on the third and fifth days (p < 0.05). The primary elastic modulus increased concomitantly with increasing polysaccharide concentration. Elastic moduli of 2% KGM/HA with chondrocytes (0.389 ± 0.119 N/mm²) showed little difference from those without chondrocytes (0.283 ± 0.243 N/mm²), although those of 2% agarose with chondrocytes (0.403 ± 0.094 N/mm²) were significantly lower than those without chondrocytes (0.736 ± 0.227 N/mm²; p < 0.05). Collagen type II mRNA expression was higher in KGM/HA and agarose than in monolayer cultures, although KGM/HA had lower aggrecan mRNA expression levels than did agarose. Histological tests of KGM/HA–chondrocyte constructs revealed chondrocyte aggregation and proteoglycan production in the pericellular region. The results show that KGM/HA might be useful for chondrocyte culture. Copyright © 2009 John Wiley & Sons, Ltd.     

Prevascularization of natural nanofibrous extracellular matrix for engineering completely biological three‐dimensional prevascularized tissues for diverse applications

Self‐sustainability after implantation is one of the critical obstacles facing large engineered tissues. A preformed functional vascular network provides an effective solution for solving the mass transportation problem. With the support of mural cells, endothelial cells (ECs) can form microvessels within engineered tissues. As an important mural cell, human mesenchymal stem cells (hMSCs) not only stabilize the engineered microvessel network, but also preserve their multi‐potency when grown under optimal culture conditions. A prevascularized hMSC/extracellular matrix (ECM) sheet fabricated by the combination of hMSCs, ECs and a naturally derived nanofibrous ECM scaffold offers great opportunity for engineering mechanically strong and completely biological three‐dimensional prevascularized tissues. The objective of this study was to create a prevascularized hMSC/ECM sheet by co‐culturing ECs and hMSCs on a nanofibrous ECM scaffold. Physiologically low oxygen (2% O₂) was introduced during the 7 day hMSC culture to preserve the stemness of hMSCs and thereby their capability to secrete angiogenic factors. The ECs were then included to form microvessels under normal oxygen (20% O₂) for up to 7 days. The results showed that a branched and mature vascular network was formed in the co‐culture condition. Angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin‐1 (Ang‐1) were significantly increased by low‐oxygen culture of hMSCs, which further stabilized and supported the maturation of microvessels. A differentiation assay of the prevascularized ECM scaffold demonstrated a retained hMSC multi‐potency in the hypoxia cultured samples. The prevascularized hMSC/ECM sheet holds great promise for engineering three‐dimensional prevascularized tissues for diverse applications.     

Construction of a vascularized hydrogel for cardiac tissue formation in a porcine model

Replacing cardiac tissues lost to myocardial infarction remains a therapeutic goal for regenerative therapy in recovering cardiac function. We assessed the feasibility of constructing a macrosized human cardiac tissue construct using pluripotent stem cell‐derived cardiomyocytes or control fibroblasts infused fibrin/collagen hydrogel and performed ectopic implantation in peripheral vascular system of a porcine model for 3 weeks. Finally, an optimized vascularized cardiac construct was explanted and grafted onto porcine myocardium for 2 weeks. Myocardial‐grafted human cardiac constructs showed a nascent tissue‐like organization with aligned cardiomyocytes within the remodelled collagen matrix. Nevertheless, no significant changes in intraconstruct density of cardiomyocytes were observed in the myocardial‐grafted constructs (human embryonic stem cell [hESC]‐derived cardiomyocyte [n = 4]: 70.5 ± 22.8 troponin I⁺ cardiomyocytes/high power field [HPF]) as compared to peripherally implanted constructs (hESC‐derived cardiomyocyte [n = 4]: 59.0 ± 19.6 troponin I⁺ cardiomyocytes/HPF; human induced pluripotent stem cell‐derived cardiomyocyte [n = 3]: 50.9 ± 8.5 troponin I⁺ cardiomyocytes/HPF, p = ns). However, the myocardial‐grafted constructs showed an increased in neovascularization (194.4 ± 24.7 microvessels/mm² tissue, p < .05), microvascular maturation (82.8 ± 24.7 mature microvessels/mm², p < .05), and tissue‐like formation whereas the peripherally implanted constructs of hESC‐derived cardiomyocyte (168.3 ± 98.2 microvessels/mm² tissue and 68.1 ± 33.4 mature microvessels/mm²) and human induced pluripotent stem cell‐derived cardiomyocyte (86.8 ± 57.4 microvessels/mm² tissue and 22.0 ± 32.7 mature microvessels/mm²) were not significantly different in vascularized response when compared to the control human fibroblasts (n = 3) constructs (65.6 ± 34.1 microvessels/mm² tissue and 30.7 ± 20.7 mature microvessels/mm²). We presented results on technical feasibility and challenges of grafting vascularized centimetre‐sized human cardiac construct that may spur novel approaches in cardiac tissue replacement strategy.     

Alginate microencapsulation of human mesenchymal stem cells as a strategy to enhance paracrine‐mediated vascular recovery after hindlimb ischaemia

Stem cell‐based therapies hold great promise as a clinically viable approach for vascular regeneration. Preclinical studies have been very encouraging and early clinical trials have suggested favourable outcomes. However, significant challenges remain in terms of optimizing cell retention and maintenance of the paracrine effects of implanted cells. To address these issues, we have proposed the use of a cellular encapsulation approach to enhance vascular regeneration. We contained human mesenchymal stem cells (hMSCs) in biocompatible alginate microcapsules for therapeutic treatment in the setting of murine hindlimb ischaemia. This approach supported the paracrine pro‐angiogenic activity of hMSCs, prevented incorporation of hMSCs into the host tissue and markedly enhanced their therapeutic effect. While injection of non‐encapsulated hMSCs resulted in a 22 ± 10% increase in vascular density and no increase in perfusion, treatment with encapsulated hMSCs resulted in a 70 ± 8% increase in vascular density and 21 ± 7% increase in perfusion. The described cellular encapsulation strategy may help to better define the mechanisms responsible for the beneficial effects of cell‐based therapies and provide a therapeutic strategy for inducing vascular growth in the adult. As hMSCs are relatively easy to isolate from patients, and alginate is biocompatible and already used in clinical applications, therapeutic cell encapsulation for vascular repair represents a highly translatable platform for cell‐based therapy in humans. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of cryopreservation on human mesenchymal stem cells attached to different substrates

There is a need to preserve cell‐seeded scaffolds or cell–matrix constructs for tissue‐engineering and other applications. Cryopreservation is likely to be the most practical method. The aim of this study was to investigate how cryopreservation affects cells attached to different substrates and how they respond differently from those in suspension. Human mesenchymal stem cells (hMSCs) were studied for their close relevance to tissue‐engineering and stem cell therapy applications, in particular how cryopreservation affects cell adherence, cell growth and the viability of hMSCs attached to different substrates, including glass, gelatin, matrigel and a matrigel sandwich. The effects of cryopreservation on F‐actin organization, intracellular pH and mitochondrial localization of the adherent hMSCs were further investigated. It was found that cells attached to a glass surface could hardly survive the common cryopreservation protocol using 10% DMSO and a 1°C/min cooling rate. By contrast, cells attached to gelatin and matrigel could survive to a greater extent. Furthermore, cryopreservation affected the potential of cell attachment and proliferation, resulted in distortion of F‐actin, led to alteration of intracellular pH of the hMSCs for all tested substrates and caused a change in the mitochondrial localization of hMSCs on a matrigel substrate and in a matrigel sandwich. Our results showed that cell attachment and cell viability could be improved by changing the interaction between cell and substrate through modification of the substrate properties, which has implications for scaffold design if cell‐seeded scaffolds or engineered tissues need to be cryopreserved. Copyright © 2012 John Wiley & Sons, Ltd.     

Investigation of different cell types and gel carriers for cell‐based intervertebral disc therapy,in vitro and in vivo studies

Biological treatment options for the repair of intervertebral disc damage have been suggested for patients with chronic low back pain. The aim of this study was to investigate possible cell types and gel carriers for use in the regenerative treatment of degenerative intervertebral discs (IVD). In vitro: human mesenchymal cells (hMSCs), IVD cells (hDCs), and chondrocytes (hCs) were cultivated in three gel types: hyaluronan gel (Durolane®), hydrogel (Puramatrix®), and tissue‐glue gel (TISSEEL®) in chondrogenic differentiation media for 9 days. Cell proliferation and proteoglycan accumulation were evaluated with microscopy and histology. In vivo: hMSCs or hCs and hyaluronan gel were co‐injected into injured IVDs of six minipigs. Animals were sacrificed at 3 or 6 months. Transplanted cells were traced with anti‐human antibodies. IVD appearance was visualized by MRI, immunohistochemistry, and histology. Hyaluronan gel induced the highest cell proliferation in vitro for all cell types. Xenotransplanted hMSCs and hCs survived in porcine IVDs for 6 months and produced collagen II in all six animals. Six months after transplantation of cell/gel, pronounced endplate changes indicating severe IVD degeneration were observed at MRI in 1/3 hC/gel, 1/3 hMSCs/gel and 1/3 gel only injected IVDs at MRI and 1/3 hMSC/gel, 3/3 hC/gel, 2/3 gel and 1/3 injured IVDs showed positive staining for bone mineralization. In 1 of 3 discs receiving hC/gel, in 1 of 3 receiving hMSCs/gel, and in 1 of 3 discs receiving gel alone. Injected IVDs on MRI results in 1 of 3 hMSC/gel, in 3 of 3 hC/gel, in 2 of 3 gel, and in 1 of 3 injured IVDs animals showed positive staining for bone mineralization. The investigated hyaluronan gel carrier is not suitable for use in cell therapy of injured/degenerated IVDs. The high cell proliferation observed in vitro in the hyaluronan could have been a negative factor in vivo, since most cell/gel transplanted IVDs showed degenerative changes at MRI and positive bone mineralization staining. However, this xenotransplantation model is valuable for evaluating possible cell therapy strategies for human degenerated IVDs. Copyright © 2011 John Wiley & Sons, Ltd.     

Increased cell seeding efficiency in bioplotted three‐dimensional PEOT/PBT scaffolds

In regenerative medicine studies, cell seeding efficiency is not only optimized by changing the chemistry of the biomaterials used as cell culture substrates, but also by altering scaffold geometry, culture and seeding conditions. In this study, the importance of seeding parameters, such as initial cell number, seeding volume, seeding concentration and seeding condition is shown. Human mesenchymal stem cells (hMSCs) were seeded into cylindrically shaped 4 × 3 mm polymeric scaffolds, fabricated by fused deposition modelling. The initial cell number ranged from 5 × 10⁴ to 8 × 10⁵ cells, in volumes varying from 50 µl to 400 µl. To study the effect of seeding conditions, a dynamic system, by means of an agitation plate, was compared with static culture for both scaffolds placed in a well plate or in a confined agarose moulded well. Cell seeding efficiency decreased when seeded with high initial cell numbers, whereas 2 × 10⁵ cells seemed to be an optimal initial cell number in the scaffolds used here. The influence of seeding volume was shown to be dependent on the initial cell number used. By optimizing seeding parameters for each specific culture system, a more efficient use of donor cells can be achieved. Copyright © 2013 John Wiley & Sons, Ltd.     

Spatial distribution and survival of human and goat mesenchymal stromal cells on hydroxyapatite and β‐tricalcium phosphate

The combination of scaffolds and mesenchymal stromal cells (MSCs) is a promising approach in bone tissue engineering (BTE). Knowledge on the survival, outgrowth and bone‐forming capacity of MSCs in vivo is limited. Bioluminescence imaging (BLI), histomorphometry and immunohistochemistry were combined to study the fate of gene‐marked goat and human MSCs (gMSCs, hMSCs) on scaffolds with different osteoinductive properties. Luciferase–GFP‐labelled MSCs were seeded on hydroxyapatite (HA) or β‐tricalcium phosphate (TCP), cultured for 7 days in vitro in osteogenic medium, implanted subcutaneously in immunodeficient mice and monitored with BLI for 6 weeks. The constructs were retrieved and processed for histomorphometry and detection of luciferase‐positive cells (LPCs). For gMSCs, BLI revealed doubling of signal after 1 week, declining to 60% of input after 3 weeks and remaining constant until week 6. hMSCs showed a constant decrease of BLI signal to 25% of input, indicating no further expansion. Bone formation of gMSCs was two‐fold higher on TCP than HA. hMSCs and gMSCs control samples produced equal amounts of bone on TCP. Upon transduction, there was a four‐fold reduction in bone formation compared with untransduced hMSCs, and no bone was formed on HA. LPCs were detected at day 14, but were much less frequent at day 42. Striking differences were observed in spatial distribution. MSCs in TCP were found to be aligned and interconnected on the surface but were scattered in an unstructured fashion in HA. In conclusion, the spatial distribution of MSCs on the scaffold is critical for cell–scaffold‐based BTE. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel therapy strategy for bile duct repair using tissue engineering technique: PCL/PLGA bilayered scaffold with hMSCs

The current clinical treatments for complications caused by hepatobiliary surgery still have some inevitable weakness. The aim of the study was to fabricate a tissue‐engineered bile duct that utilized a novel bilayered polymer scaffold combined with human bone marrow‐derived mesenchymal stem cells (hMSCs) for new treatment of biliary disease. The biocompatibility of polycaprolactone (PCL) (PCL)/poly(lactide‐co‐glycolide) (PLGA) scaffold with hMSCs was first examined, and the hMSC–PCL/PLGA constructs (MPPCs) prepared. The MPPCs and blank scaffolds were then transplanted into 18 pigs for evaluation its efficacy on bile duct repairing, respectively. In vitro, the PCL/PLGA scaffold was verified to support the adhesion, proliferation and matrix deposition of hMSCs. There was no sign of bile duct narrowing and cholestasis in all experimental animals. At 6 months, the MPPCs had a superior repairing effect on the bile duct injury, compared with the blank PCL/PLGA scaffolds. Therefore, the implanted scaffolds could not only support the biliary tract and allow free bile flow but also had direct or indirect positive effects on repair of injured bile duct. Copyright © 2015 John Wiley & Sons, Ltd.     

Variance of peptidic nerve innervation in a canine model of atrial fibrillation produced by prolonged atrial pacing

Background: Long‐term rapid atrial pacing may result in nerve sprouting and sympathetic hyperinnervation in atrial fibrillation (AF) in dogs. Whether peptidic nerve is involved in neural remodeling is unclear. Method and Results: We performed rapid left atrial pacing in six dogs to induce sustained AF. Tissues from six healthy dogs were used as controls. Nerve was identified by immunocytochemical techniques. The degree of nerve innervation was quantified by measuring the amount of staining area for each antibody and the heterogeneity of nerve distribution was qualitatively studied. In dogs with AF, the density of growth‐associated protein 43 (GAP43) immunopositivenerve fibers in the left atrium (LA), atrial septum (AS), and right atrium (RA) was significantly (19,454.31 ± 1,592.81 μm²/mm² vs 1,673.41 ± 142.62 μm²/mm²P < 0.001, 3,931.26 ± 361.78 μm²/mm² vs 1,614.20 ± 140. 41 μm²/mm² P < 0.05 and 2,324.15 ± 1,123.77 μm²/mm² vs 1,620.47 ± 189.05 μm²/mm² P < 0.05, respectively) higher than the nerve density in control tissues. The density of (neuropeptide Y) NPY‐positive nerves in the, AS, and RA was (13,547.62 ± 2,983.37 μm²/mm² vs 703.72 ± 287.52 μm²/mm² P < 0.01, 2,689.22 ± 340.93 μm²/mm² vs 651.7 ± 283.02 μm²/mm² P < 0.01 and 1,574.70 ± 424.37 μm²/mm² vs 580.42 ± 188.12 μm²/mm² P < 0.001, respectively) higher than the nerve density in control tissues. At the same time, vasoactive intestinal polypeptide (VIP) positive nerve innervation shrank in dogs with AF. The density of VIP positive in LA, AS, and RA was statistically lower than the nerve density in control tissues, respectively. (110.48 ± 45.63μm²/mm² vs 1679.32 ± 1020.34μm²/mm² P < 0.01, 265.92 ± 52.51 μm²/mm² vs 2602.68 ± 1257.16μm²/mm² P < 0.001 and 609.56 ± 139.75μm²/mm² vs 2771.68 ± 779.08μm²/mm² P < 0.01, respectively) Conclusions: Combined with VIP‐ergic nerve denervation, significant nerve sprouting and NPY‐ergic nerve hyperinnervation are present in a canine model of sustained AF produced by prolonged atrial pacing.     

Transfusion of banked red blood cells and the effects on hemorrheology and microvascular hemodynamics in anemic hematology outpatients

BACKGROUND: The aim of this study was to investigate the effects of red blood cell (RBC) transfusion on the hemorrheologic properties and microcirculatory hemodynamics in anemic hematology outpatients receiving 2 to 4 RBC units of either “fresh” (leukoreduced storage for less than 1 week) or “aged” (leukoreduced storage for 3‐4 weeks) RBCs. STUDY DESIGN AND METHODS: Measurements were performed before and 30 minutes after RBC transfusion in hematology outpatients. Leukoreduced RBC suspensions were stored in saline‐adenine‐glucose‐mannitol (SAGM) additive solution. Whole blood viscosity was measured using Couette low‐shear viscometry, RBC deformability and aggregability were measured using laser‐assisted optical rotational cell analysis, and microcirculatory density and perfusion were assessed using sidestream dark field imaging. RESULTS: One group of patients (n = 10) received a median (interquartile range) of 3 (2‐3) RBC bags that were stored for 7 (5‐7) days (fresh) and the other group of patients (n = 10) received 3 (3‐3) RBC bags that were stored for 23 (22‐28) days (aged). After transfusion of fresh versus aged RBCs, hematocrit increased to 32 ± 3% versus 31 ± 2% (p < 0.363), whole blood viscosity increased to 4.2 ± 0.4 Pa/sec versus 4.2 ± 0.6 Pa/sec (p < 0.912), RBC deformability index remained unaffected, RBC aggregability index increased to 55 ± 10 versus 55 ± 13 (p = 0.967), microcirculatory flow remained unaffected, and microcirculatory density increased to 19.3 ± 2.5 mm/mm² versus 18.7 ± 1.9 mm/mm² (p = 0.595), respectively. CONCLUSION: Storing leukoreduced SAGM‐suspended RBCs for 3 to 4 weeks did not affect their ability to improve hemorrheologic properties and microcirculatory hemodynamics in our small group of anemic hematology outpatients. Larger studies are needed to confirm this finding.     

Analysis of the therapeutic potential of different administration routes and frequencies of human mesenchymal stromal cells in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Cellular therapy represents a novel option for the treatment of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Its major aim is the generation of a protective environment for degenerating motor neurons. Mesenchymal stromal cells secrete different growth factors and have antiapoptotic and immunomodulatory properties. They can easily and safely be isolated from human bone marrow and are therefore considered promising therapeutic candidates. In the present study, we compared intraventricular application of human mesenchymal stromal cells (hMSCs) versus single and repeated intraspinal injections in the mutant SOD1^G93A transgenic ALS mouse model. We observed significant reduction of lifespan of animals treated by intraventricular hMSC injection compared with the vehicle treated control group, accompanied by changes in weight, general condition, and behavioural assessments. A potential explanation for these rather surprising deleterious effects lies in increased microgliosis detected in the hMSC treated animals. Repeated intraspinal injection at two time points resulted in a slight but not significant increase in survival and significant improvement of motor performance although no hMSC‐induced changes of motor neuron numbers, astrogliosis, and microgliosis were detected. Quantitative real time polymerase chain reaction showed reduced expression of endothelial growth factor in animals having received hMSCs twice compared with the vehicle treated control group. hMSCs were detectable at the injection site at Day 20 after injection into the spinal cord but no longer at Day 70. Intraspinal injection of hMSCs may therefore be a more promising option for the treatment of ALS than intraventricular injection and repeated injections might be necessary to obtain substantial therapeutic benefit.     

高强度聚焦超声肝脏生物学焦域及超声监控

目的探讨在相同声强作用下,高强度聚焦超声经体外辐照肝脏形成的生物学焦域体积大小与辐照深度和辐照时间的关系.同时探讨超声显像法实时监控生物学焦域的图像变化规律.方法采用重庆医科大学医学超声工程研究所自主研制的JC-A型HIFU肿瘤治疗系统(1MHz,5500W*cm-2),对18只山羊的肝脏组织进行体外定点脉冲辐照,辐照深度为距皮肤3cm和4cm,辐照时间为5s、10s、15s和20s.辐照过程中系列测量靶区超声灰度和强回声区面积的变化.辐照后3～5天处死动物,剖腹观察并测量所形成的肝脏HIFU生物学焦域的体积.结果肝脏经体外HIFU辐照后即刻,靶区内出现明显的回声增强并随观测时间延长而逐渐降低.强回声区面积随辐照时间增加而增大.辐照时间为5s、10s、15s和20s,辐照深度为3cm时,形成的生物学焦域体积分别为(85±28.0)mm3、(274±55.0)mm3、(410±90.0)mm3和(694±131.0)mm3;而辐照深度为4cm时,肝脏HIFU生物学焦域体积则分别为(63±7.0)mm3、(167±25.0)mm3、(273±56.0)mm3和(472±104.0)mm3.结论在相同辐照声强作用下,肝脏HIFU生物学焦域体积随辐照时间的增加而增加,随辐照深度的增加而减小.     

The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with ferucarbotran using various protocols: cells were labeled with 100 µg Fe/ml for 4 and 18 h and additional samples were cultured for 6 or 12 days after the 18 h labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP‐spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared with unlabeled controls by staining with Alcian blue and Hematoxylin and Eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3 T was investigated on days 1 and 14 of chondrogenic differentiation by measuring signal‐to‐noise ratios on T₂‐SE and T₂*‐GE sequences. Iron uptake was significant for all labeling protocols (p < 0.05). The uptake was highest after transfection with protamine sulfate (25.65 ± 3.96 pg/cell) and lowest at an incubation time of 4 h without transfection (3.21 ± 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4 h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose‐dependent. MR imaging showed markedly lower SNR values of all labeled cells compared with the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose‐dependent manner when using simple incubation techniques. When decreasing the incubation time to 4 h, inhibition of chondrogenesis was not significant. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of cell sheet technology to bone marrow stromal cell transplantation for rat brain infarct

Bone marrow stromal cells (BMSC) transplantation enhances functional recovery after cerebral infarct, but the optimal delivery route is undetermined. This study was aimed to assess whether a novel cell‐sheet technology non‐invasively serves therapeutic benefits to ischemic stroke. First, the monolayered cell sheet was engineered by culturing rat BMSCs on a temperature‐responsive dish. The cell sheet was analysed histologically and then transplanted onto the ipsilateral neocortex of rats subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their behaviours and histology were compared with those in the animals treated with direct injection of BMSCs or vehicle over 4 weeks post‐transplantation. The cell sheet was 27.9 ± 8.0 μm thick and was composed of 9.8 ± 2.4 × 10⁵ cells. Cell sheet transplantation significantly improved motor function when compared with the vehicle‐injected animals. Histological analysis revealed that the BMSCs were densely distributed to the neocortex adjacent to the cerebral infarct and expressed neuronal phenotype in the cell sheet‐transplanted animals. These findings were almost equal to those for the animals treated with direct BMSC injection. The attachment of the BMSC sheet to the brain surface did not induce reactive astrocytes in the adjacent neocortex, although direct injection of BMSCs profoundly induced reactive astrocytes around the injection site. These findings suggest that the BMSCs in cell sheets preserve their biological capacity of migration and neural differentiation. Cell‐sheet technology may enhance functional recovery after ischaemic stroke, using a less invasive method. Copyright © 2014 John Wiley & Sons, Ltd.     

大鼠种植型肝癌电化学治疗的实验研究

目的　探讨电化学疗法 (ECHT)对大鼠种植型肝癌的治疗效果。方法　制作大鼠肝癌动物模型 ,应用电化学疗法进行治疗 ,1周后影像学、病理光镜观察肿瘤大小、坏死、凋亡变化 ,与对照组比较 ,并观察生存期的长短。结果　电化学治疗前 ,大鼠肝肿瘤平均体积为 ( 10 0± 6)mm3 ,电化学治疗后 1周 ,大鼠肝肿瘤平均体积为 ( 12 5± 10 )mm3 ,而荷瘤对照组肝肿瘤平均体积为 ( 190± 11)mm3 ;治疗组肿瘤生长率为 2 5 % ,而荷瘤对照组肝肿瘤生长率为 90 % ,P <0 .0 5。治疗组病理光镜下肿瘤明显坏死 ,并可诱发细胞凋亡 ;生存期明显延长。结论　电化学疗法可明显抑制肿瘤生长 ,促进肿瘤坏死 ,诱发细胞凋亡 ,延长大鼠生存期。