Deoxyribonucleic Acid Content Research Articles

Comparative studies on the content of deoxyribonucleic acid in four gastric carcinomas and their metastatic lesions.

Comparative studies on the content of deoxyribonucleic acid in four gastric carcinomas and their metastatic lesions.

Use of plasma DNA to predict mortality and need for intensive care in patients with abdominal pain

Background We investigated the value of plasma deoxyribonucleic acid concentrations in patients presenting with acute abdominal pain to predict need for intensive care or mortality. Methods Plasma deoxyribonucleic acid taken from patients with acute abdominal pain was analyzed for the β-globin gene using the quantitative polymerase chain reaction. The primary outcome measure was the combined 28-day mortality or admission to the intensive care unit. Results Of 287 consecutive patients with acute abdominal pain recruited, 12 patients were admitted to the intensive care unit and/or died. Median plasma DNA concentrations were higher in patients with cancer and major organ inflammation. Mean plasma DNA concentrations were three-fold higher in patients with systemic inflammatory response syndrome, five-fold higher in patients who died within 28 days, and eight-fold higher in patients admitted to the intensive care unit. The area under the receiver operator curve for plasma DNA concentrations and intensive care unit admission/mortality was 0.804. At a cut-off of 1100 GE/ml, the sensitivity was 67% (95%CI 35–90) and specificity was 89% (95%CI 84–92). At a cut-off of 175 GE/ml, the sensitivity was 100% (95%CI 73–100) and specificity was 30% (95%CI 25–36). Plasma DNA concentration predicted need for intensive care unit admission or death (adjusted odds ratio 1.4; P < 0.0001). Conclusions Plasma DNA may have a role in patients with acute abdominal pain as a marker for inflammation and cancer, and a predictor of intensive care unit admission/mortality.

Biochemical indication of microbial mass changes using ATP and DNA measurement in biological treatment of thiocyanate

This study was designed to monitor changes in the levels of adenosine 5'-triphosphate (ATP) and deoxyribonucleic acid (DNA) per unit of microbial mass during the autotrophic biodegradation of thiocyanate (SCN(-)). An artificial medium containing trace minerals and 500 mg SCN(-)/L was used as a substrate for bacterial growth. An SCN(-)-degrading bioreactor with a working volume of 6 L, equipped with temperature, pH, and dissolved oxygen controls, was operated in batch mode. During the exponential phase of SCN(-) biodegradation, the ratios of ATP and DNA to microbial dry weight varied from 0.6 to 1.1 microg ATP/mg of volatile suspended solid (VSS), and from 3.5 to 8.8 microg DNA/mg of VSS, respectively. The ATP and DNA concentrations correlated linearly with microbial mass (r (2) > 0.9) within the exponential phase. The linear regression equations were as follows: (1) microbial mass concentration (mg/L) = 0.663 x ATP concentration (microg/L) + 11.1 and (2) microbial concentration (mg/L) = 0.081 x DNA concentration (microg/L) + 10.9. The applicable ranges were 6.8 to 47.4 microg/L for ATP concentration and 41.5 to 395 microg/L for DNA concentration, respectively.

Highly Open Porous Biodegradable Microcarriers: In Vitro Cultivation of Chondrocytes for Injectable Delivery

Injectable cell therapy would provide a patient-friendly procedure for treatment of degenerated or wounded tissue. Biodegradable injectable porous microspheres were fabricated to use as dual-purpose microcarriers for cell culture and injectable scaffold for tissue regeneration. Gas foaming in a water-in-oil-in-water double emulsion was performed for fabricating the well-interconnected porous microcarriers using poly(lactic-co-glycolic acid) (PLGA). The gas foaming conditions were finely tuned to control the structural and morphological characteristics. Porous microcarriers with a mean size of approximately 175 microm and an average pore diameter of approximately 29 microm were produced for cell cultivation and injectable delivery. To promote cell seeding, amine-functionalized porous microcarriers were prepared by blending amine-functionalized PLGA with unreacted PLGA. To assess the porous microcarriers for chondrocyte cultivation, bovine articular chondrocytes were seeded and cultured in vitro in spinner flasks for 4 weeks. Visualization and biochemical analyses of the microcarrier-cell constructs were performed to demonstrate cell proliferation and phenotypic expression. Quantification of deoxyribonucleic acid, glycosaminoglycan, and collagen content showed that much greater cell proliferation and expression of cartilage-specific phenotype were observed for cultures in the following order: amine-functionalized porous microcarriers, porous microcarriers, nonporous microcarriers, and monolayer culture.

Evaluation of RGD- or EGF-immobilized chitosan scaffolds for chondrogenic activity

Chitosan scaffolds were prepared by freeze-drying method and modified with Arg-Gly-Asp (RGD) sequence of fibronectin or epidermal growth factor (EGF) by covalent immobilization. The results obtained from FTIR-ATR, fluorescence visualization and quantitative measurements showed that biosignal molecules, RGD and EGF, were successfully immobilized on chitosan scaffolds. ATDC5 murine chondrogenic cells were seeded on both type of scaffolds, chitosan-RGD and chitosan-EGF, and cultured for 28 days in stationary conditions. According to the results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) test, considerable increase in cell proliferation was only detected on chitosan-EGF scaffolds. Biochemical analysis of the chondrocyte seeded scaffolds showed that glycosaminoglycan (GAG) and deoxyribonucleic acid (DNA) content of the scaffolds increases with time. In conclusion, EGF-modified chitosan scaffolds (containing 1.83 μg EGF/3 mg dry scaffold) have been proposed to promote chondrogenesis and to have potential for reticular cartilage regeneration.

In vitro–matured rat oocytes have low mitochondrial deoxyribonucleic acid and adenosine triphosphate contents and have abnormal mitochondrial redistribution

To study the development and function of mitochondria in in vitro-matured rat oocytes derived from follicles of different sizes. Experimental animal study. Department of Anatomy at the University of Hong Kong. Immature female Sprague-Dawley rats that were 25 days of age. Immature oocytes were collected from rat ovarian follicles of different sizes and were induced to mature in vitro. The number of copies of mitochondrial DNA, mitochondrial activity, adenosine triphosphate content of matured oocytes, and rates of fertilization and blastulation were determined. The mitochondrial DNA copy number of oocytes increased linearly with the diameter of antral follicles. The mitochondrial DNA copy number, adenosine triphosphate content, and proportion of oocytes with peripheral distribution of mitochondria in in vitro-matured oocytes from small antral follicles were significantly lower than those from preovulatory follicles and in vivo-matured oocytes. Compared with in vitro-matured oocytes from small antral follicles, those from preovulatory follicles and in vivo-matured oocytes also had significantly better fertilization potential and higher blastulation rate. The inferior developmental potential of in vitro-matured oocytes may be attributed partly to a reduced number of mitochondria, resulting in insufficient production of adenosine triphosphate for required developmental events.

Ultrasonic pre-treatment of biosolid

Low-frequency and high-intensity ultrasonic treatment of biosolid is a good method for biosolid disintegration. So, as a pre-treatment method of anaerobic digestion, it may replace the hydrolysis. Thus, we studied the effect of ultrasonic disintegration of biosolid at different ultrasonic power densities. Results showed that the Soluble Chemical Oxygen Demand (SCOD), Total Nitrogen (TN), Total Phosphorus (TP), supernatant protein, polysaccharide and Deoxyribonucleic Acid (DNA) concentrations and biosolid mass reduction increased with the increase of sonication time and energy density. Ultrasonic treatment at 1.6 W/m for 30 min when the degree of biosolid disintegration (DDCOD) was 49.29% increased the SCOD, TN, TP, supernatant protein, polysaccharide and DNA concentrations by 3285, 150.13, 68.50, 1488.85, 531.66 and 123.35 mg/L, respectively, indicating that ultrasonic treatment can effectively disintegrate the biosolid. Higher energy ultrasound was more efficient than lower energy ultrasound for biosolid treatment.

The concentration of deoxyribonucleic acid in plasma from 73 patients with colorectal cancer and apparent clinical correlations

Background: Detection of cell-free plasma DNA has considerable potential as a tool for the diagnosis and assessment of the prognosis of many types of cancer. The aim of the present study was to quantify, by spectrophotometry, the cell-free DNA in plasma samples from patients with colorectal cancer at different stages of the disease and to attempt to correlate the resultant values with the clinical picture. Methods: We reviewed the medical reports of 73 patients, who had undergone resection of primary colorectal cancer. Samples of blood had been taken from each patient immediately prior to surgery. DNA was extracted from samples of plasma and quantified, by spectrophotometry, after a storage period of no longer than 2 years in 89% of the cases examined. Results: The mean(±S.D.) concentration of DNA in plasma samples was 108 ± 156 ng/μl. We found a statistically significant correlation between the concentration of DNA and the presence of metastases (mainly liver metastases). Conclusion: The detection and quantitation of cell-free DNA in plasma, using this simple technique, might be of clinical value for the surveillance of colon cancer patients and the detection of metastases.

Oral administration of phytocomponent p-hydroxycinnamic acid prevents bone loss in ovariectomized rats

The preventive effect of phytocomponent p-hydroxycinnamic acid (HCA) on ovariectomy (OVX)-induced bone loss was investigated. HCA (250 or 500 microg/100 g body weight) was orally administered once daily for 30 days to OVX rats. The analysis using a peripheral quantitative computed tomography (pQCT) showed that OVX caused bone loss in the femoral-metaphyseal tissues. This change was significantly restored after the administration of HCA (250 or 500 microg/100 g body weight) to OVX rats. Mineral content, mineral density, and polar strength strain index in the femoral-metaphyseal tissues were significantly decreased in OVX rats. These decreases were significantly restored after the administration of HCA (500 microg/100 g) to OVX rats. Moreover, OVX caused a significant decrease in calcium content or alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. These decreases were significantly restored after the administration of HCA (250 or 500 microg/100 g) to OVX rats. Deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in OVX rats. These increases were significantly restored after oral administration of HCA (500 microg/100 g). This study demonstrates that HCA has preventive effects on OVX-induced bone loss of rats in vivo.

성장 단계에 따른 한국 재래 오골계 근육의 조직학 및 생리학적 특성

본 연구는 한국 재래 오골계의 성장에 따른 조직학적 및 생리학적 특성을 분석하기 위해 수행하였다. 계군들의 체중, 흉근과 가자미근의 무게 측정 및 시료의 채취는 부화 직후 및 3, 5, 15주령에 이루어졌다. 근섬유 특성은 부화 직후에서 15주령까지의 오골계 흉근과 가자미근을 이용하여 분석하였고, DNA, RNA 및 단백질 함량은 왼쪽 흉근에서 측정하였다. 체중의 상대적인 증가율은 부화 직후에서 3주령까지의 기간에 가장 크게 나타났다. 가슴의 무게는 부화 직후에서 3주령까지의 기간에 약 24.46배가 증가했으며, 가슴 중 흉근도 동일한 기간 동안 약 31.14배 증가하였다. 소퇴부 및 가자미근의 무게 역시 부화 직후에서 3주령까지의 시기에 뚜렷한 증가가 나타났다. 모든 근섬유의 단면적은 성장 기간 동안 증가하는데, 15주의 실험 기간 동안 흉근의 근섬유 단면적은 65배 증가(<TEX>$34.06\;{\pm}\;3.08$</TEX>에서 <TEX>$2238\;{\pm}\;177\;{\mu}m^2$</TEX>)하였다. 흉근의 근섬유 단면적은 부화 직후에서 3주령까지의 기간에는 약 18배, 3주령에서 5주령까지의 기간 동안에는 약 1.6배 증가를 보였다. 따라서 흉근의 근섬유 단면적과 무게 역시 3주령 때까지 가장 큰 증가를 보임을 알 수 있다. 핵산 및 단백질의 함량을 분석한 실험에서도 부화 직후에서 3주령 사이에 가장 큰 증가가 나타났는데, 부화부터 3주령까지의 기간 동안 각각 DNA 총량은 13배, RNA 총량은 21배, 단백질 총량은 30배 증가한 것으로 나타났다. This study examined the histochemical and physiological characteristics during Korean native Ogol chickens (KNOC) development. The body weight, Pectoralis major and soleus muscle weights, and muscle samples were taken at hatching as well as at 3, 5, and 15 weeks of age. The fiber characteristics of the Pectoralis major and soleus muscles from the KNOC at hatching to 15 weeks of age were determined, and the deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein concentrations were measured from the left Pectoralis major muscles. A greater increase in body and muscle weights was detected between hatching and 3 weeks of age than during any other period. Moreover, the cross sectional area (CSA) of the fibers, as well as the total concentration of DNA, RNA, and protein also showed a greater increase betweenhatching and 3 weeks of age than during any other period. The KNOC breed is a dual purpose breed, however, the it has lower body and muscle weights than commercial meat type chickens or layer type chickens. Moreover, the KNOC breed has a small muscle fiber CSA of and a low nucleic acid concentration.

In vitro conservation and sustained production of breadfruit (Artocarpus altilis, Moraceae): modern technologies for a traditional tropical crop

Breadfruit (Artocarpus altilis, Moraceae) is a traditionally cultivated, high-energy, high-yield crop, but widespread use of the plant for food is limited by poor quality and poor storage properties of the fruit. A unique field genebank of breadfruit species and cultivars exists at the National Tropical Botanical Garden in the Hawaiian Islands and is an important global resource for conservation and sustainable use of breadfruit. However, this plant collection could be damaged by a random natural disaster such as a hurricane. We have developed a highly efficient in vitro plant propagation system to maintain, conserve, mass propagate, and distribute elite varieties of this important tree species. Mature axillary shoot buds were collected from three different cultivars of breadfruit and proliferated using a cytokinin-supplemented medium. The multiple shoots were maintained as stock cultures and repeatedly used to develop whole plants after root differentiation on a basal or an auxin-containing medium. The plantlets were successfully grown under greenhouse conditions and were reused to initiate additional shoot cultures for sustained production of plants. Flow cytometry was used to determine the nuclear deoxyribonucleic acid content and the ploidy status of the in vitro grown population. The efficacy of the micropropagation protocols developed in this study represents a significant advancement in the conservation and sustained mass propagation of breadfruit germplasm in a controlled environment free from contamination.

The Potential Use of Enamel Matrix Derivative for In Situ Anterior Cruciate Ligament Tissue Engineering: A Translational In Vitro Investigation

Polyester scaffolds have been used as an alternative to autogenous tissues for the reconstruction of the anterior cruciate ligament (ACL). They are biocompatible and encourage tissue infiltration, leading to neoligament formation. However, rupture can occur, caused by abrasion of the scaffold against the bone tunnels through which it is implanted. Good early tissue induction is therefore considered essential to protect the scaffold from this abrasion. Enamel matrix derivative (EMD) is used clinically in the treatment of periodontal disease. It is a complex mix of proteins with growth factor-like activity, which enhances periodontal ligament fibroblast attachment, proliferation, and differentiation, leading to the regeneration of periodontal bone and ligament tissues. We hypothesized that EMD might, in a similar manner, enhance tissue induction around scaffolds used in ACL reconstruction. This preliminary investigation adopted a translational approach, modelling in vitro 3 possible clinical modes of EMD administration, to ascertain the suitability of each protocol for application in an animal model or clinically. Preliminary investigations in monolayer culture indicated that EMD had a significant dose-dependent stimulatory effect (p < 0.05, n = 6) on the proliferation of bovine primary synovial cells. However, pre-treating culture plates with EMD significantly inhibited cell attachment (p < 0.01, n = 6). EMD's effects on synovial cells, seeded onto ligament scaffolds, were then investigated in several in vitro experiments modelling 3 possible modes for clinical EMD administration (pre-, intra-, and post-operative). In the pre-operative model, EMD was adsorbed onto scaffolds before the addition of cells. In the intra-operative model, EMD and cells were added simultaneously to scaffolds in the culture medium. In the post-operative model, cells were pre-seeded onto scaffolds before EMD was administered. EMD significantly inhibited cell adhesion in the pre-operative model (p < 0.05, n = 6) and had no significant benefit in the intra-operative model. In the post-operative model, the addition of EMD to previously cell-seeded scaffolds significantly increased their total deoxyribonucleic acid content (p < 0.01, n = 5). EMD's stimulative effect on cell proliferation in vitro suggests that it may accelerate scaffold colonization by cells (and in turn tissue induction) in situ. However, its inhibitory effect on synovial cell attachment in vitro implies that it may only be suited to post-operative administration.

Early diagenesis and trophic role of extracellular DNA in different benthic ecosystems

To provide new insights into the early diagenesis of extracellular deoxyribonucleic acid (DNA) and its relevance in trophodynamic processes, we collected sediment samples from a variety of coastal and deep‐sea sites. Turnover times of extracellular DNA were approximately threefold shorter in coastal benthic systems than in deep‐sea sediments (0.35 yr and 1.2 yr, respectively). This was due to the higher concentrations of bioavailable extracellular DNA in deep‐sea over coastal sediments (19.8 ± 0.6 and 9.8 ± 2.6 mg DNA g−1, respectively), since deoxyribonuclease (DNase) activities did not show significant differences between sampling sites (135.8 ± 72.7 and 62.8 ± 19.0 ng DNA g−1 d−1, in coastal and deep‐sea sediments, respectively). The coastal benthic systems were characterized by DNase activities per prokaryotic cell significantly lower than those of their deep‐sea counterparts (0.12 ± 0.05 and 0.27 ± 0.06 fg DNA degraded cell−1 d−1, respectively), although carbon production per cell was not significantly different (6.9 ± 4.4 and 2.6 ± 0.9 fg C cell−1 d−1, respectively). These results suggest that DNase activity per cell may be more dependent on the amount of bioavailable substrate rather than on prokaryotic metabolism. Extracellular DNA may supply 20% and 47% of the daily prokaryote anabolic requirements for organic phosphorous in both coastal and deep‐sea sediments, respectively. Overall, our data suggest that bioavailable extracellular DNA might represent an important nutrient source for benthic heterotrophic metabolism and open new perspectives for a better understanding of the factors that influence the functioning of benthic systems.

Monitoring of Microbiological Water Quality by Real-Time PCR

A new method was developed to evaluate the microbiological water quality. Deoxyribonucleic acid of water-borne bacteria was extracted and quantified using real-time polymer chain reaction detection system with a selected universal primer set. Quantification of the deoxyribonucleic acid in environmental water samples is independent of the culture condition, nutrient condition, or the bacterial metabolic states and can reflect the relatively small environmental changes which can not be detected through the other parameters. Therefore, this method can well represent the microbiological water quality. Compared with the conventional plate count method, deoxyribonucleic acid quantification has higher sensitivity, precision and reliability. The relationship between concentration of bacterial deoxyribonucleic acid and other water quality parameters was examined. Concentration of deoxyribonucleic acid was not well correlated with the plate count number, turbidity or other physical and chemical parameters.

Light and low-frequency pulsatile hydrostatic pressure enhances extracellular matrix formation by bone marrow mesenchymal cells: An in-vitro study with special reference to cartilage repair

Ovine bone marrow mesenchymal cells (BMMCs) were seeded on to non-woven filamentous plasma-treated polyester scaffolds and cultured in a chondrogenic medium for 4 weeks. Thereafter a pulsatile hydrostatic pressure (PHP) was applied to these cell-scaffolds constructs at an amplitude of 0.1 MPa and frequency of 0.25 Hz, for 30 min a day, over a period of 10 days. Samples (n = 6) were removed 24 h after PHP stimulation at days 1, 4, 7, and 10 for biochemical analysis. Similar analyses were conducted, at the same time points, on control samples that were not subjected to a PHP. The results showed that the glycosaminoglycan (GAG) content did not significantly increase until after the application of a PHP for 7 days. The GAG content was 1.5 and 2.7 times higher in the PHP group than in the control group at days 7 and 10 respectively (p<0.01). The deoxyribonucleic acid (DNA) content was 1.5 times greater in the PHP group than in the control group at day 10 (p<0.01). GAG synthesis amounts, expressed as the total GAG contents per microgram of DNA, were 1.6 and 1.8 times higher in the PHP group than in the control group at days 7 and 10 respectively (p<0.01). The total collagen content in the medium did not change until after PHP application for 10 days, when it was 1.9 times higher than the control (p < 0.05). The results suggest that a light PHP applied at a low frequency has a cumulative stimulatory effect on the BMMCs' metabolic activities including cell proliferation and synthesis of the extracellular matrix.

Large‐scale, Uniform DNA Network on 11‐mercaptoundecanoic Acid Modified Gold (111) Surface: Atomic Force Microscopy Study

Large-scale, uniform plasmid deoxyribonucleic acid (DNA) network has been successfully constructed on 11-mercaptoundecanoic acid modified gold (111) surface using a self-assembly technique. The effect of DNA concentration on the characteristics of the DNA network was investigated by atomic force microscopy. It was found that the size of meshes and the height of fibers in the DNA network could be controlled by varying the concentration of DNA with a constant time of assembly of 24 h.

Traceable Phosphorus Measurements by ICP-OES and HPLC for the Quantitation of DNA

Measurement of the phosphorus content of nucleotides and deoxyribonucleic acid (DNA) offers an approach to the quantitation of nucleic acids that is traceable to the SI. Such measurements can be an alternative to the commonly used spectroscopic tools that are not traceable. Phosphorus measurements of thymidine 5'-monophosphate (TMP) and acid-digested plasmid and genomic DNA preparations were made using high-performance inductively coupled plasma optical emission spectroscopy (HP-ICP-OES) and high-performance liquid chromatography (HPLC) and compared for bias and uncertainty. A prerequisite for quality measurement is the purity of the materials. Quantitation with the two platforms was comparable for the TMP. However, the HPLC values had larger uncertainties and were all statistically different from the gravimetric values at the 95% confidence level. When using ICP-OES, the digestion of the nucleotide monophosphate can be eliminated, thus simplifying the procedure. The differences between the results obtained by using the two platforms, when measuring genomic or plasmid DNA, were dependent on the mass fraction of the digest. ICP-OES measurement of phosphorus provides a highly accurate quantitation for both nucleotide monophosphates and DNA with expanded uncertainties of less than 0.1%. Currently, ICP-OES requires a significant sample size restricting its usefulness for the quantitation of DNA but represents a valuable tool for certification of reference materials. HPLC requires smaller amounts of material to perform the analysis but is less useful for certification of reference materials because of lower accuracy and 10-fold higher expanded uncertainties.

Therapeutic Effects of Herbal Leaves Group (Green Tea &amp; Sage) on Liver Cancer

Tea is a beverage consumable next to water worldwide, made from the leaves of theaceae family. Green tea (Camellia Sinensis) tree, grown in China and India, contains good medical properties. Name of salvia in Latin meaning treatment, made from the leaves of labiatae family. Sage (Salvia officinalis) plant, grown in general in mediterranean sea Country. In order to study the therapeutic effects of leaves group, each one alone, on liver cancer, 0.1 ml/100g of aflatoxin B1 (AFB1) was administrated intra- peritoneal in male Wister Albino rats for a period of 10 days to cause liver cancer. Green tea and sage, each one alone, were given to rats for a period of 30 days. The animals were killed at the end of the study and blood was tested for some biochemical parameters including some key enzymes such as aspatate amino transferase (AST), alanine amino transferase (ALT), gammaglutamyl transferase (GGT), and other parameters which include bilirubin, urea, uric acid, creatinine, cholesterol, triacylglycerols, glucose, hemoglobin also part of the liver samples were taken to determine the content of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and total proteins, the other part was used for histological examination. Those analyses were compared to control animals that given AFB1 but not treated. The results from this study demonstrated that green tea prevented the proliferation of liver cancer at the initiating stage by inhibiting AFB1 effects on DNA and regeneration of hepatocyte cells, in sage the effects were lesser than green tea.

Effects of Growth Differentiation Factor-5 on the Intervertebral Disc−In Vitro Bovine Study and In Vivo Rabbit Disc Degeneration Model Study

In vitro studies on the effects of recombinant human growth and differentiation factor-5 (rhGDF-5) on matrix metabolism of bovine intervertebral disc cells and an in vivo study on the effect of rhGDF-5 in the rabbit anular puncture model. To determine the reparative capacity of rhGDF-5 on the intervertebral disc. The in vitro and in vivo effects of rhGDF-5, a crucial protein in the developing musculoskeletal system, on repair of the degenerated intervertebral disc remain unidentified. In vitro, bovine nucleus pulposus and anulus fibrosus cells were cultured with or without rhGDF-5 (100 or 200 ng/mL). On days 7, 14, and 21, the contents of deoxyribonucleic acid and proteoglycan, and the synthesis of proteoglycan and collagen were assessed. In vivo, 16 adolescent New Zealand white rabbits received anular punctures in 2 lumbar discs. Four weeks later, phosphate buffered saline or rhGDF-5 (10 ng, 1 and 100 mug) was injected into the nucleus pulposus. The rabbits were followed up for 16 weeks for disc height, magnetic resonance imaging, and histologic grading. In vitro, rhGDF-5 increased the deoxyribonucleic acid and proteoglycan contents of both cell types significantly after day 14. rhGDF-5 at 200 ng/mL significantly stimulated proteoglycan synthesis (nucleus pulposus: +138%, anulus fibrosus: +24%) and collagen synthesis (nucleus pulposus: +95%, anulus fibrosus: +23%) at day 21. In vivo, the injection of rhGDF-5 resulted in a restoration of disc height, improvement of magnetic resonance imaging scores, and histologic grading scores with statistical significance (P < 0.05-0.001). A single injection of rhGDF-5 has a reparative capacity on intervertebral discs, presumably based on its effects to enhance extracellular matrix production in vitro.

Establishment of a Novel Intervertebral Disc/Endplate Culture Model: Analysis of an Ex Vivo In Vitro Whole-Organ Rabbit Culture System

Ex vivo in vitro study evaluating a novel intervertebral disc/endplate culture system. To establish a whole-organ intervertebral disc culture model for the study of disc degeneration in vitro, including the characterization of basic cell and organ function. With current in vivo models for the study of disc and endplate degeneration, it remains difficult to investigate the complex disc metabolism and signaling cascades. In contrast, more controlled but simplified in vitro systems using isolated cells or disc fragments are difficult to culture due to the unconstrained conditions, with often-observed cell death or cell dedifferentiation. Therefore, there is a demand for a controlled culture model with preserved cell function that offers the possibility to investigate disc and endplate pathologies in a structurally intact organ. Naturally constrained intervertebral disc/endplate units from rabbits were cultured in multi-well plates. Cell viability, metabolic activity, matrix composition, and matrix gene expression profile were monitored using the Live/Dead cell viability test (Invitrogen, Basel, Switzerland), tetrazolium salt reduction (WST-8), proteoglycan and deoxyribonucleic acid quantification assays, and quantitative polymerase chain reaction. Viability and organ integrity were preserved for at least 4 weeks, while proteoglycan and deoxyribonucleic acid content decreased slightly, and matrix genes exhibited a degenerative profile with up-regulation of type I collagen and suppression of collagen type II and aggrecan genes. Additionally, cell metabolic activity was reduced to one third of the initial value. Naturally constrained intervertebral rabbit discs could be cultured for several weeks without losing cell viability. Structural integrity and matrix composition were retained. However, the organ responded to the artificial environment with a degenerative gene expression pattern and decreased metabolic rate. Therefore, the described system serves as a promising in vitro model to study disc degeneration in a whole organ.