A new approach to completely autologous cardiovascular tissue in humans

In cardiovascular tissue engineering, synthetic or biologic scaffolds serve as templates for tissue development. Currently used scaffolds showing toxic degradation and immunogenic reactions are still far from ideal. We present a new alternative method to develop completely autologous human tissue without using any scaffold materials. Human vascular cells of arterial and venous origin were cultured to form cell sheets over a 4 week period under standard conditions. Thereafter, cell sheets of each origin were folded and cultured in a newly developed frame device for an additional 4 weeks. Controls remained under standard culture conditions. Tissue development was evaluated by morphology and biochemical assays. The formation of multilayered cell sheets and production of extracellular matrix were observed in all groups. Folded and framed neo-tissue showed a solid structure, with increased matrix formation and tissue organization when compared with the control groups. DNA content indicated significantly lower cell proliferation, and hydroxyproline assay indicated significantly higher collagen content in the framed cell sheets. We present a new approach to the engineering of cardiovascular tissue without the use of biodegradable scaffold material. Three-dimensional, completely autologous human tissue may be developed on the basis of this structure, thus avoiding scaffold induced toxic degradation or inflammatory reaction.     

一种新型算法在组织光学参数测量系统中的应用

无创测量确定生物组织的光学特性参数在医学诊断和治疗领域中有着广泛的应用前景.目前确定组织参数的方法多建立在单层模型条件下,而实际的许多生物组织均具有分层结构,比如在肌肉、颅骨等.因此在多层模型条件下反演计算组织参数具有更大的实际意义.近年许多研究者针对以上问题提出了各种解决方法,如最小二乘法、神经网络方法等,但这些方法都存在需要时间过长或者误差较大的缺点.本文在组织参数测量领域引入数据挖掘办法--支持向量机(support vector machines,SVM),对双层模型中四个待定组织光学参数的确定进行了研究.结果 表明,利用SVM方法确定组织光学参数具有很好的准确性和实时性.     

A novel approach to HIV therapy: highly active antiretroviral therapy and autologous hematopoietic cell transplantation

Highly active antiretroviral therapy dramatically decreases in vivo viral replication to levels below the level of clinical detection, but does not eradicate HIV-1 infection on the basis of persistent low-level or cryptic viral replication and latent provirus in resting CD4+ T lymphocytes. Immune activation therapy has begun to be used in attempts to increase the turnover rate of the latent virus reservoir through activation of infected cells that comprise this reservoir, in order to promote cell death and accelerate virus clearance. Recent reports have not demonstrated complete virus ablation. Autologous hematopoietic cell transplantation now appears as a safe, feasible, and reasonable approach for Aids-related lymphoma in patients who meet criteria for transplantation. The hypothesis is based on the possibility that hematopoietic stem cells from a HIV-positive patient could be collected before the patient becomes infected with HIV. Then, the proposed treatment consists of the following assumptions. HAART keeps viral replication below the level of detection, limiting the infection to latent provirus in resting CD4+ T lymphocytes. It is presumed here that myeloablative conditioning regimen can lead to the killing of all the cells that, in theory, harbour the virus. The following transplantation of the autologous hematopoietic stem cells, collected before HIV infection, would allow the complete recovery. The hypothesis is to be tested on a suitable animal model. After the collection of hematopoietic stem cells, the animal is experimentally infected with the immunodeficiency virus. HAART is given after plasma viral RNA becomes detectable. By myeloablative conditioning regimen all the cells harbouring the virus are supposed to be killed. Then, as the viral load is kept undetectable by HAART, the animal undergoes autologous hematopoietic stem cell transplantation. Antiretroviral therapy is interrupted a month after engraftment has taken place. Although hematopoietic stem cells from man before infection with HIV are unlikely to be available, a successful test on the animal would suggest a new approach which could allow the cure of HIV in future.     

Tissue-engineering approach to regenerating the intervertebral disc

In today's world there is an ever increasing incidence of low back pain, which is generally attributed to degeneration of the intervertebral disc (IVD) in those in their second or third decade of life. The most prevalent treatment modalities involve conservative methods (physical therapy and medications) or surgical fusion of the upper and lower vertebral bodies. In the last 10 years, there has been a surge of interest in applying tissue-engineering principles to treat spinal problems associated with the IVD. Tissue engineering provides many promising advantages to treating disc degeneration; it adopts a more biological and reparative approach, whereby the main goal is to restore the properties of the disc to its pre-degenerative state. This review outlines the physiology of the IVD and the etiology of disc degeneration. Much of the research carried out in the field of tissue engineering is based on three predominant constituents: cells, scaffolds, and signals. Thus, specific attention is given to these constituents and their potential use in repairing the IVD. Some of the significant challenges involved in IVD tissue engineering are also identified, and a brief discussion regarding possible future areas of research follows.     

A novel cultured tissue model of rat aorta: VSMC proliferation mechanism in relationship to atherosclerosis

Development of a cultured tissue experimental model of rat aorta was explored in order to study mechanism of vascular smooth muscle (VSMC) proliferation. This particular model has potential with regard to amelioration of atherosclerosis and other vascular diseases in comparison to whole animal and cell culture models. The aorta segments of rats were divided into 4 experimental groups: the injured endothelium, injured endothelium plus BQ123, without injured endothelium and without injured endothelium plus BQ123. Each of group was subdivided into a further 2 subgroups and cultured with 20% serum and with serum-free DMEM. Each group cultured in vitro for 5, 8 and 13 days respectively. The control group was not cultured in vitro. Bromodeoxyuridine (BrDU 8x10(-4) mol/l) was added into the cultured medium of all groups, 24 h prior to harvesting. These segments were fixed in 4% paraformaldehyde for paraffin slice used to HE and immunocytochemical staining and other aorta segments were used to detect the expressions of hypertension-related gene-1 (HRG-1) and smooth muscle 22 alpha (SM22alpha) by RT-PCR. ET-1 content in the supernatant was detected with radioimmunology. Proliferous VSMC can be observed on artery segments cultured in vitro, and conspicuous plaques were developed on model vascular wall cultured for 13 days. Labeled cells increased with an increase in culture time but were not seen in the control group. A greater number of labeled cells were observed in injured endothelium group cultured in 20% serum DMEM. Hyperplasia was inhibited after BQ123 was added into the medium, suggesting that serum and ET-1 are important factors that lead to VSMC proliferation. Expressions of HRG-1 and SM22alpha were decreased while the aorta segments were cultured in vitro, minimum or even absent mRNA expressions of HRG-1 and SM22alpha were detected in injured endothelium cultured in 20% serum DMEM and increased in injured endothelium plus BQ123 group cultured. ET-1 content in the supernatant increased in injured endothelium cultured in 20% serum DMEM. These results show that the phenotypic transform and VSMC proliferation on cultured artery segments were related not only to serum culture, but also to ET-1 secreting. ET-1 and serum may be the main factors of contributing to the proliferation and phenotypic transform. This model provides a favorable experimental platform for research into the mechanism of vascular proliferous diseases as well as its prevention and treatment.     

Bone regeneration by grafting of cultured human bone

A 3-mL sample of bone marrow was collected from the iliac bones of 27 orthopedic patients (8 men and 19 women with a mean age of 56.1 years [range, 17 to 76 years]), followed by culture in standard culture medium (minimal essential medium containing 15% fetal bovine serum). In all 7 patients randomly selected from these 27 patients, significant in vitro osteogenic ability of marrow mesenchymal cells was demonstrated by scanning electron microscopy and biochemical analyses. In all 27 cases, to investigate the in vivo osteogenic potential of this human cultured bone, porous ceramics were impregnated with marrow cells and subcultured in osteogenic culture medium (standard medium supplemented with sodium beta-glycerophosphate, vitamin C phosphate, and dexamethasone). After 3 weeks of subculture, the cultured artificial bones of the cultured bone/porous ceramics were grafted into the abdominal cavity of nude mice. Histological and biochemical (alkaline phosphatase activity and human osteocalcin) examinations indicated that the cultured artificial bone possessed significant ability to regenerate bone. This result suggests that the bone-regenerating ability of human marrow cells may not depend on age, and that cultured artificial bone may be useful for bone regeneration treatment if appropriate cultured marrow cells can be successfully prepared.     

A novel approach to sperm cryopreservation

Human spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been achieved by the standard approaches of cryobiology. Conventional approaches to cryopreservation impose a linear change of temperature with time; however, the stresses that cells encounter during cryopreservation are all non-linear with time. In this paper it is shown that improved methods of cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes instead of imposing a linear temperature reduction. Several treatments were compared: control of solidification to achieve constant ice formation with time was more damaging than the standard linear reduction in temperature. However, treatments which followed a chosen non-linear concentration profile, referred to as 'controlled concentration' allowed recovery of almost all the cells which were motile before freezing. The biophysical basis of these different responses was examined using the cryostage of a scanning electron microscope and freeze substitution and it was found that, surprisingly, all samples of spermatozoa in the frozen state were neither osmotically dehydrated nor had any visible intracellular ice. Viability on thawing did not appear to correlate with conventional theories of cellular freezing injury, which suggests that for human spermatozoa other factors determine viability following freezing and thawing.     

A novel approach to enterovirus typing.

A novel method was developed for typing enteroviruses producing cytopathic effect. Monolayers of primary or secondary rhesus monkey kidney cells were prepared and covered with an agarose overlay. Wells were formed in the agarose, the well bottom being optimally determined to be 3 mm from the monolayer and homotypic enterovirus antiserum, intersecting antiserum pool or antiserum-diluent as control was added. After 2 h at 37 degrees C, the test virus isolate was added to each well. Cultures were incubated at 37 degrees C and were examined daily until cytopathic effect was readily observed in the control well. Monolayers were fixed and stained for macroscopic reading. Enterovirus identity based on the inhibition of cytopathic effect was confirmed with a conventional micro-neutralization method. In all, 51 enterovirus isolates were typed. Included were 21 polioviruses, 9 coxsackieviruses and 21 echoviruses, all of which were correctly identified. This method takes advantage of the ability of enterovirus and antibody to diffuse through agarose. It is simple to perform. It does not require preliminary titration of the test virus isolate and tolerates 1,000 fold fluctuations in virus concentration, thereby offering laboratories a more rapid and efficient means of typing enteroviruses.     

Vaginoplasty using autologous in vitro cultured vaginal tissue in a patient with Mayer-von-Rokitansky-Kuster-Hauser syndrome

Mayer-von-Rokitansky-Küster-Hauser syndrome (MRKHS) is characterized by vaginal agenesis with variable Müllerian duct abnormalities. The Abbè-McIndoe technique is considered a valid treatment option for vaginoplasty but no consensus has been reached on what material should be used for the neovagina canal wall lining. We report the first case of autologous vaginal tissue transplantation in a 28-year-old women with MRKHS. The patient was subjected to a 1 cm2 full-thickness mucosal biopsy from the vaginal vestibule. Following enzymatic dissociation, cells were inoculated onto collagen IV-coated plates and cultured for 2 weeks. The patient was subjected to a vaginoplasty with a modified Abbè-McIndoe vaginoplasty with 314 cm2 autologous in vitro cultured vaginal tissue for the canal lining. At 1 month from surgery, the vagina appeared normal in length and depth and a vaginal biopsy revealed normal vaginal tissue. The use of autologous in vitro cultured vaginal tissue to create a neovagina appears as an easy, minimally invasive and useful method.     

A study of the antigenic properties of regenerating muscle tissue

Summary The precipitation in gel test after Ouchterlony was used to study the antigenic properties of regenerating muscle tissue in albino rats. It was found that antisera against muscle tissue proteins in the rat form with regenerate extracts a large number of precipitation bands on the 7th, 10th, 14th, 21st and 30th day after the operation than with normal muscle extracts. In addition, it is shown that extracts from regenerates obtained at earlier times after the operation forms more precipitation bands than in the case of later regenerate extracts.It is supposed that soluble antigens decrease in their number during regeneration.(Presented by Active Member of Academy of Medical Sciences of USSR N. N Zhukov-Verezhnikov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 61, No. 3, pp. 92–95, March, 1966     

In situ tissue engineering of periodontal tissues by seeding with periodontal ligament-derived cells

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.     

自体成纤维细胞注射移植的存活性初探

为观察自体成纤维细胞移植后在体内成活和分泌胶原情况 ,体外培养兔自体成纤维细胞 ,部分细胞3H TdR掺入 ,标记和未标记的 8× 10 1 0 L- 1 细胞悬液 ,分别注射于兔耳背侧真皮浅、深层交界处 3次。 5月后取材 ,进行放射自显影 ,H E染色 ,天狼猩红染色观察 ,发现3H TdR掺入标记的细胞依然存活 ;注射部位出现大量的幼稚型成纤维细胞 ;真皮厚度增加 ,实验组为 14 0 2 6± 6 31(× 5 μm) ,对照组为 12 7 6 6± 4 78(× 5 μm) (P <0 0 1) ;Ⅲ型胶原含量增加 ,实验组为 2 6 3± 1 4 1(cm2 ) ,对照组为 1 0 5± 0 90 (cm2 ) (P <0 0 5 )。表明自体成纤维细胞注射移植后 ,能够在体内长期存活并分泌新生Ⅲ型胶原 ,为临床应用提供了初步、可靠的理论依据。     

A novel self-guided approach to alpha activity training

Fifty healthy participants took part in a double-blind placebo-controlled study in which they were either given auditory alpha activity (8-12Hz) training (N=18), random beta training (N=12), or no training at all (N=20). A novel wireless electrode system was used for training without instructions, involving water-based electrodes mounted in an audio headset. Training was applied approximately at central electrodes. Post-training measurement using a conventional full-cap EEG system revealed a 10% increase in alpha activity at posterior sites compared to pre-training levels, when using the conventional index of alpha activity and a non-linear regression fit intended to model individual alpha frequency. This statistically significant increase was present only in the group that received the alpha training, and remained evident at a 3 month follow-up session, especially under eyes open conditions where an additional 10% increase was found. In an exit interview, approximately twice as many participants in the alpha training group (53%) mentioned that the training was relaxing, compared to those in either the beta (20%) or no training (21%) control groups. Behavioural measures of stress and relaxation were indicative of effects of alpha activity training but failed to reach statistical significance. These results are discussed in terms of a lack of statistical power. Overall, results suggest that self-guided alpha activity training using this novel system is feasible and represents a step forward in the ease of instrumental conditioning of brain rhythms.     

A stepwise approach to prescribing novel lipid-lowering medications

The stepwise approach to prescribing novel lipid-lowering medications; AF: Atrial Fibrillation; ASCVD: Atherosclerotic Cardiovascular Disease; CV: Cardiovascular; GI: Gastrointestinal; HDL-C: High Density Lipoprotein-Cholesterol; HoFH: Homozygous Familial Hypercholesterolemia; IPE: Icosapent Ethyl; LDL-C: Low Density Lipoprotein-Cholesterol; LLT: Lipid Lowering Therapy; mAb: Monoclonal Antibody; PCSK9: Proprotein Convertase Subtilisin/Kexin type 9; TG: Triglyceride.

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A novel approach to study adhesion mechanisms by isolation of the interacting system

For decades most investigations into mechanisms of adhesive interactions have examined whole organisms or single cells. Results using whole organisms are often unclear because it may not be known if a probe used in an experiment is directly affecting the cellular interaction under study or if it is an indirect effect resulting from action on some other structure or pathway. Here we develop a novel approach to isolate the structural components of a cellular interaction by dissecting them out of the organism to study them in a pristine environment away from all confounding factors. We used the adhesion between the archenteron and blastocoel roof of the sea urchin gastrula stage embryo as a model that can be replicated in many other developmental and pathological systems. The isolated components of the cellular interaction and those in the whole organism possessed identical cell surface receptors and adhesive affinities.     

A new approach to the validation of tissue microarrays

Although tissue microarrays (TMA) have been widely used for a number of years, it is still not clear how many core biopsies should be taken to determine a reliable value for percentage positivity or how much heterogeneity in marker expression influences this number. The first aim of this study was to validate the human visual semi-quantitative scoring system for positive staining of tumour tissue with the exact values determined from computer-generated images. The second aim was to determine the minimum number of core biopsies needed to estimate percentage positivity reliably when the immunohistochemical staining pattern is heterogeneous and scored in a non-binary way. Tissue sections from ten colorectal cancer specimens were stained for carbonic anhydrase IX (CA IX). The staining patterns were digitized and 400 artificial computer-generated images were generated to test the accuracy of the human scoring system. To determine the minimal number of core biopsies needed to account for tumour heterogeneity, 50 (artificial) core biopsies per section were taken from the tumoural region of the ten digitally recorded full tissue sections. Based on the semi-quantitative scores from the 50 core biopsies per section, 2500 x n (n = 1-10 core biopsies) experimental core biopsies were then generated and scores recorded. After comparison with field-by-field analysis from the tumoural region of the whole tissue section, the number of core biopsies that need to be taken to minimize the influence of heterogeneity could be determined. In conclusion, visual scoring accurately estimated the percentage positivity and the percentage tumour present in a section, as judged by comparison with the artificial images. The exact number of core biopsies that has to be examined to determine tumour marker positivity using TMAs is affected by the degree of heterogeneity in the expression pattern of the protein, but for most purposes at least four is recommended.     

A novel approach to multi-criteria inverse planning for IMRT

Treatment plan optimization is a multi-criteria process. Optimizing solely on one objective or on a sum of a priori weighted objectives may result in inferior treatment plans. Manually adjusting weights or constraints in a trial and error procedure is time consuming. In this paper we introduce a novel multi-criteria optimization approach to automatically optimize treatment constraints (dose-volume and maximum-dose). The algorithm tries to meet these constraints as well as possible, but in the case of conflicts it relaxes lower priority constraints so that higher priority constraints can be met. Afterwards, all constraints are tightened, starting with the highest priority constraints. Applied constraint priority lists can be used as class solutions for patients with similar tumour types. The presented algorithm does iteratively apply an underlying algorithm for beam profile optimization, based on a quadratic objective function with voxel-dependent importance factors. These voxel-dependent importance factors are automatically adjusted to reduce dose-volume and maximum-dose constraint violations.