Effect of cell density on formation of three-dimensional cartilaginous constructs using fibrin & human osteoarthritic chondrocytes.

Background & objectives: Seeding density is one of the major parameters affecting the quality of tissue-engineered cartilage. The objective of this study was to evaluate different seeding densities of osteoarthritis chondrocytes (OACs) to obtain the highest quality cartilage. Methods: The OACs were expanded from passage 0 (P0) to P3, and cells in each passage were analyzed for gross morphology, growth rate, RNA expression and immunochemistry (IHC). The harvested OACs were assigned into two groups: low (1×10[7] cells/ml) and high (3×10[7] cells/ml) cell density. Three-dimensional (3D) constructs for each group were created using polymerised fibrin and cultured for 7, 14 and 21 days in vitro using chondrocyte growth medium. OAC constructs were analyzed with gross assessments and microscopic evaluation using standard histology, IHC and immunofluorescence staining, in addition to gene expression and biochemical analyses to evaluate tissue development. Results: Constructs with a high seeding density of 3×10[7] cells/ml were associated with better quality cartilage-like tissue than those seeded with 1×10[7] cells/ml based on overall tissue formation, cell association and extracellular matrix distribution. The chondrogenic properties of the constructs were further confirmed by the expression of genes encoding aggrecan core protein and collagen type II. Interpretation & conclusions: Our results confirmed that cell density was a significant factor affecting cell behaviour and aggregate production, and this was important for establishing good quality cartilage.

Long-term evaluation of osteoarthritis sheep knee, treated with TGF-ß3 and BMP-6 induced multipotent stem cells.

BACKGROUND: Hyaline articular cartilage, which protects the bones of diarthrodial joints from forces associated with load bearing, frictions, and impacts has very limited capacities for self-repair. Over the years, the trend of treatments has shifted to regenerations and researchers have been on the quest for a lasting regeneration. We evaluated the treatment of osteoarthritis by chondrogenically induced ADSCs and BMSCs for a long time functional recovery. METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5 mls single doses of 2 × 107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography. RESULTS: Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; p = .001. Immunocytochemistry was positive for these chondrogenic markers. 12 months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; P = .001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance. CONCLUSIONS: Both ADSCs and BMSCs had increased chondrogenic gene expressions using TGF-ß3 and BMP-6. The treated knees had improved cartilage scores; PKH26 can provide elongated tracking, while EMG results revealed improved joint recoveries. These could be suitable therapies for osteoarthritis.

Influencing Factors For Cholera And Diarrhoea: Water Sanitation And Hygiene In Impoverished Rural Villages Of Beluran District, Sabah Malaysia

The lower percentage of water, sanitation and hygiene are the root causes of diarrhoea and cholera. Cholera is a sudden onset of acute watery diarrhoea which can progress to severe dehydration and death if untreated. The current pandemic, Vibrio Cholera O1 started in 1961. This study explores water, sanitation, hygiene and cholera and diarrhoea in three affected villages of Beluran District, Sabah Malaysia to support effective and timely public health intervention. This cross sectional study uses purposive sampling. All (114) households were interviewed and household water samples collected. The study reported lower coverage improved sanitation facilities (35.3% to 52.3%), no latrine at home (37% to 63%), improved water supply (52% to 60%), and prevalence of hand washing after toilet (57% - 74%). For water quality, Ecoli was present in household water (32% to 37%) but Vibrio cholerae was not isolated in any of the water samples tested. Statistically significant associations were found for; 1) occupation−non-agriculture and unimproved sanitation facility and 2) house ownership and correct knowledge of ORS preparation. Predictors for household water quality were: latrine at home, and improved household toilet. Aggressive strategies to improve water supply, sanitation and hygiene−hand washing after toilet−were recommended for future prevention of cholera and diarrhoea in the affected area.

Posterolateral spinal fusion with ostegenesis induced BMSC seeded TCP/HA in a sheep model.

Autogenous bone graft is the gold standard for fusion procedure. However, pain at donor site and inconsistent outcome have left a surgeon to venture into some other technique for spinal fusion. The objective of this study was to determine whether osteogenesis induced bone marrow stem cells with the combination of ceramics granules (HA or TCP/HA), and fibrin could serve as an alternative to generate spinal fusion. The sheep's bone marrow mesenchymal stem cells (BMSCs) were aspirated form iliac crest and cultured for several passages until confluence. BMSCs were trypsinized and seeded on hydroxyapatite scaffold (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) for further osteogenic differentiation in the osteogenic medium one week before implantation. Six adult sheep underwent three-level, bilateral, posterolateral intertransverse process fusions at L1-L6. Three fusion sites in each animal were assigned to three treatments: (a) HA constructs group/L1-L2, (b) TCP/HA constructs group/L2-L3, and (c) autogenous bone graft group/L5-L6. The spinal fusion segments were evaluated using radiography, manual palpation, histological analysis and scanning electron microscopy (SEM) 12 weeks post implantation. The TCP/HA constructs achieved superior lumbar intertransverse fusion compared to HA construct but autogenous bone graft still produced the best fusion among all.

Island Health Review, Progress And The Way Forward In The Western Pacific Region

Island health differs from other health care systems, particularly in that there are limited resources and referral faculties available. With globalisation and climate change, island populations have become increasingly vulnerable to natural disasters and global pandemics. This study will identify, explore, compare and report on island health issues facing in the western Pacific, before making appropriate recommendations. A review of selected health indicators in Pacific islands was collected from the World Health Organization (WHO) and other publicly available resources. In the Pacific region, 15 islands saw lower health expenditure (

Influencing factors for household water quality improvement in reducing diarrhoea in resource-limited areas

Background and Objectives: Water and sanitation are major public healthissues exacerbated by rapid population growth, limited resources, disasters andenvironmental depletion. This study was undertaken to study the influencing factorsfor household water quality improvement for reducing diarrhoea in resource‑limitedareas.Materials and Methods: Data were collected from articles and reviews from relevantrandomized controlled trials, new articles, systematic reviews and meta‑analysesfrom PubMed, World Health Organization (WHO), United Nations Children’s Fund(UNICEF) and WELL Resource Centre For Water, Sanitation And Environmental Health.Discussion: Water quality on diarrhoea prevention could be affected bycontamination during storage, collection and even at point‑of‑use. Point‑of‑use watertreatment (household‑based) is the most cost‑effective method for prevention ofdiarrhoea. Chemical disinfection, filtration, thermal disinfection, solar disinfectionand flocculation and disinfection are five most promising household water treatmentmethodologies for resource‑limited areas.Conclusion: Promoting household water treatment is most essential for preventingdiarrhoeal disease. In addition, the water should be of acceptable taste, appropriatefor emergency and non‑emergency use

Influencing factors for household water quality improvement in reducing diarrhoea in resource-limited areas.

BACKGROUND AND OBJECTIVES: Water and sanitation are major public health issues exacerbated by rapid population growth, limited resources, disasters and environmental depletion. This study was undertaken to study the influencing factors for household water quality improvement for reducing diarrhoea in resource-limited areas. MATERIALS AND METHODS: Data were collected from articles and reviews from relevant randomized controlled trials, new articles, systematic reviews and meta-analyses from PubMed, World Health Organization (WHO), United Nations Children's Fund (UNICEF) and WELL Resource Centre For Water, Sanitation And Environmental Health. DISCUSSION: Water quality on diarrhoea prevention could be affected by contamination during storage, collection and even at point-of-use. Point-of-use water treatment (household-based) is the most cost-effective method for prevention of diarrhoea. Chemical disinfection, filtration, thermal disinfection, solar disinfection and flocculation and disinfection are five most promising household water treatment methodologies for resource-limited areas. CONCLUSION: Promoting household water treatment is most essential for preventing diarrhoeal disease. In addition, the water should be of acceptable taste, appropriate for emergency and non-emergency use.

Bone marrow and adipose stem cells can be tracked with PKH26 until post staining passage 6 in in vitro and in vivo.

Tracking of transplanted cells has become an important procedure in cell therapy. We studied the in vitro dye retention, survival and in vivo tracking of stem cells with PKH26 dye. Sheep BMSCs and ADSCs were labeled with 2, 4 and 8 µmol of PKH26 and monitored for six passages. Labeled BMSCs and ADSCs acquired mean cumulative population doubling of 12.7±0.4 and 14.6±0.5; unlabeled samples had 13.8±0.5 and 15.4±0.6 respectively. Upon staining with 2, 4 and 8 µmol PKH26, BMSCs had retentions of 40.0±5.8, 60.0±2.9 and 95.0±2.9%, while ADSCs had 92.0±1.2, 95.0±1.2 and 98.0±1.2%. ADSCs retentions were significantly higher at 2 and 4 µmol. On dye retention comparison at 8 µmol and 4 µmol for BMSCs and ADSCs; ADSCs were significantly higher at passages 2 and 3. The viability of BMSCs reduced from 94.0±1.2% to 90.0±0.6% and ADSCs from 94.0±1.2% to 52.0±1.2% (p<0.05) after 24h. BMSCs had significant up regulation of the cartilage genes for both the labeled and the unlabeled samples compared to ADSCs (p<0.05). PKH26 fluorescence was detected on the resected portions of the regenerated neo-cartilage. The recommended concentration of PKH26 for ADSCs is 2 µmol and BMSCs is 8 µmol, and they can be tracked up to 49 days.

Bone graft substitute using hydroxyapatite scaffold seeded with tissue engineered autologous osteoprogenitor cells in spinal fusion: early result in a sheep model.

Spinal fusion using autologous bone graft is performed in an increasing rate for many spinal disorders. However, graft harvesting procedure is associated with prolonged operation time and potential donor site morbidity. We produced an engineered 'bone graft' substitute by using porous hydroxyapatite (HA) scaffold seeded with autologous bone marrow osteoprogenitor cells (OPCs) and fibrin. This obviates bone graft harvesting, thus eliminates donor site morbidity and shortens the operation time. The aim of this study is to evaluate Hydroxyapatite (HA) ceramics as scaffold for autologous tissue engineered bone construct for spinal fusion in a sheep model. The sheep's marrow was aspirated from iliac crest. The bone marrow mesenchymal stem cells (BMMSCs) were cultured for several passages in the presence of growth and differentiation factors to increase the number of OPCs. After the cultures reached confluence, they were trypsinized and seeded on Hydroxyapatite scaffold (HA). Approximately 5 million cells were generated after 3 weeks of culture. Microscopically, very tight Colony Forming Units (CFU-Fs) were seen on monolayer culture. The Von Kossa and Alizarin Red staining of monolayer culture showed positive mineralization areas; indicating the presence of OPCs. Sheep underwent a posterolateral spinal fusion in which scaffolds with or without OPCs seeded were implanted on both sides of the lumbar spine (L1-L2). Intended fusion segments were immobilized using wires. At the end of third month, the fusion constructs were harvested for histological examination. Fibrous tissue infiltration found in the inter-connecting pores of plain HA ceramics indicates inefficient new bone regeneration. New bone was found surrounding the HA ceramics seeded with autologous cells. The new bone is probably formed by the sheep BMMSCs that were initially encapsulating HA while it remained intact. The new bone is naturally fused with the vertebrae. In conclusion, the incorporation of autologous bone marrow cells improved the effectiveness of HA ceramics as 'bone graft' substitute for spinal fusion.

Age and gender effect on the growth of bone marrow stromal cells in vitro.

Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be selectively isolated and induced to express bone phenotype in vitro. The osteoblastic progenitor can be estimated by counting the number of cells attach using the haemacytometer. This study was undertaken to test the hypothesis that human aging is associated with a significant change on the number of osteoblastic progenitors in the bone marrow. Bone marrow aspirates were harvested from 38 patients, 14 men (age 11-70) and 24 women (age 10-70) and cultured in F12: DMEM (1:1). In total 15 bone marrow samples have been isolated from patients above 40 years old (men/women) of age. Fourteen (93.3%) of this samples failed to proliferate. Only one (6.7%) bone marrow sample from a male patient, aged 59 years old was successfully cultured. Seventy percent (16/23) of the samples from patient below than 40 years old were successfully cultured. However, our observation on the survival rate for cells of different gender from patient below 40 years old does not indicate any significant difference. From this study, we conclude that the growth of bone marrow stromal cells possibly for bone engineering is better from bone marrow aspirates of younger patient.