Effects of oxygen on zonal marker expression in human articular chondrocytes

Articular cartilage is organized in depth zones with phenotypically distinct subpopulations of chondrocytes that are exposed to different oxygen tensions. Despite growing evidence of the critical role for oxygen in chondrogenesis, little is known about its effect on chondrocytes from different zones. This study evaluates zonal marker expression of human articular chondrocytes from different zones under various oxygen tensions. Chondrocytes isolated from full-thickness, superficial, and middle/deep cartilage from knee replacement surgeries were expanded and redifferentiated under hypoxic (5% O 2) or normoxic (20% O 2) conditions. Differentiation under hypoxia increased expression of hypoxia-inducible factors 1alpha and 2alpha and accumulation of extracellular matrix, particularly in middle/deep chondrocytes, and favored re-expression of proteoglycan 4 by superficial chondrocytes compared with middle/deep cells. Zone-dependent expression of clusterin varied with culture duration. These results demonstrate that zonal chondrocytes retain important phenotypic differences during in vitro cultivation, and that these characteristics can be improved by altering the oxygen environment. However, transcript levels for pleiotrophin, cartilage intermediate layer protein, and collagen type X were similar between zones, challenging their reliability as zonal markers for tissue-engineered cartilage from osteoarthritis patients. Key factors including oxygen tension and cell source should be considered to prescribe zone-specific properties to tissue-engineered cartilage. © 2012, Mary Ann Liebert, Inc.

Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration.

There is a growing need for successful bone tissue engineering strategies and advanced biomaterials that mimic the structure and function of native tissues carry great promise. Successful bone repair approaches may include an osteoconductive scaffold, osteoinductive growth factors, cells with an osteogenic potential and capacity for graft vascularisation. To increase osteoinductivity of biomaterials, the local combination and delivery of growth factors has been developed. In the present study we investigated the osteogenic effects of calcium phosphate (CaP)-coated nanofiber mesh tube-mediated delivery of BMP-7 from a PRP matrix for the regeneration of critical sized segmental bone defects in a small animal model. Bilateral full-thickness diaphyseal segmental defects were created in twelve male Lewis rats and nanofiber mesh tubes were placed around the defect. Defects received either treatment with a CaP-coated nanofiber mesh tube (n = 6), an un-coated nanofiber mesh tube (n=6) a CaP-coated nanofiber mesh tube with PRP (n=6) or a CaP-coated nanofiber mesh tube in combination with 5 μg BMP-7 and PRP (n = 6). After 12 weeks, bone volume and biomechanical properties were evaluated using radiography, microCT, biomechanical testing and histology. The results demonstrated significantly higher biomechanical properties and bone volume for the BMP group compared to the control groups. These results were supported by the histological evaluations, where BMP group showed the highest rate of bone regeneration within the defect. In conclusion, BMP-7 delivery via PRP enhanced functional bone defect regeneration, and together these data support the use of BMP-7 in the treatment of critical sized defects.

ATF5, a possible regulator of osteogenic differentiation in human adipose-derived stem cells

The regulatory pathways involved in maintaining the pluripotency of embryonic stem cells are partially known, whereas the regulatory pathways governing adult stem cells and their "stem-ness" are characterized to an even lesser extent. We, therefore, screened the transcriptome profiles of 20 osteogenically induced adult human adipose-derived stem cell (ADSC) populations and investigated for putative transcription factors that could regulate the osteogenic differentiation of these ADSC. We studied a subgroup of donors' samples that had a disparate osteogenic response transcriptome from that of induced human fetal osteoblasts and the rest of the induced human ADSC samples. From our statistical analysis, we found activating transcription factor 5 (ATF5) to be significantly and consistently down-regulated in a randomized time-course study of osteogenically differentiated adipose-derived stem cells from human donor samples. Knockdown of ATF5 with siRNA showed an increased sensitivity to osteogenic induction. This evidence suggests a role for ATF5 in the regulation of osteogenic differentiation in adipose-derived stem cells. To our knowledge, this is the first report that indicates a novel role of transcription factors in regulating osteogenic differentiation in adult or tissue specific stem cells. © 2012 Wiley Periodicals, Inc.

Dynamic compression improves biosynthesis of human zonal chondrocytes from osteoarthritis patients

Objective: We hypothesize that chondrocytes from distinct zones of articular cartilage respond differently to compressive loading, and that zonal chondrocytes from osteoarthritis (OA) patients can benefit from optimized compressive stimulation. Therefore, we aimed to determine the transcriptional response of superficial (S) and middle/deep (MD) zone chondrocytes to varying dynamic compressive strain and loading duration. To confirm effects of compressive stimulation on overall matrix production, we subjected zonal chondrocytes to compression for 2 weeks. Design: Human S and MD chondrocytes from osteoarthritic joints were encapsulated in 2% alginate, pre-cultured, and subjected to compression with varying dynamic strain (5, 15, 50% at 1 Hz) and loading duration (1, 3, 12 h). Temporal changes in cartilage-specific, zonal, and dedifferentiation genes following compression were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The benefits of long-term compression (50% strain, 3 h/day, for 2 weeks) were assessed by measuring construct glycosaminoglycan (GAG) content and compressive moduli, as well as immunostaining. Results: Compressive stimulation significantly induced aggrecan (ACAN), COL2A1, COL1A1, proteoglycan 4 (PRG4), and COL10A1 gene expression after 2 h of unloading, in a zone-dependent manner (P < 0.05). ACAN and PRG4 mRNA levels depended on strain and load duration, with 50% and 3 h loading resulting in highest levels (P < 0.05). Long-term compression increased collagen type II and ACAN immunostaining and total GAG (P < 0.05), but only S constructs showed more PRG4 stain, retained more GAG (P < 0.01), and developed higher compressive moduli than non-loaded controls. Conclusions: The biosynthetic activity of zonal chondrocytes from osteoarthritis joints can be enhanced with selected compression regimes, indicating the potential for cartilage tissue engineering applications. © 2012 Osteoarthritis Research Society International.

The α5 neuronal nicotinic acetylcholine receptor subunits plays an important role in the sedative effects of ethanol but does not modulate consumption in mice

Alcohol use disorders (AUDs) are a major public health problem, and the few treatment options available to those seeking treatment offer only modest success rates. There remains a need to identify novel targets for the treatment of AUDs. The neuronal nicotinic acetylcholine receptors (nAChRs) represent a potential therapeutic target in the brain, as recent human genetic studies have implicated gene variants in the α5 nAChR subunit as high risk factors for developing alcohol dependence. Here, we evaluate the role of 5* nAChR for ethanol-mediated behaviors using α5+/+ and α5-/- mice. We characterized the effect of hypnotic doses of ethanol and investigated drinking behavior using an adapted Drinking-in-the Dark (DID) paradigm that has been shown to induce high ethanol consumption in mice. We found the α5 subunit to be critical in mediating the sedative effects of ethanol. The α5-/- mice showed slower recovery from ethanol-induced sleep, as measured by loss of righting reflex. Additionally the α5-/- mice showed enhanced impairment to ethanol-induced ataxia. We found the initial sensitivity to ethanol and ethanol metabolism to be similar in both α5+/+ and α5-/- mice. Hence the enhanced sedation is likely due to a difference in the acute tolerance of ethanol in mice deficient of the α5 subunit. However the α5 subunit did not play a role in ethanol consumption for ethanol concentrations ranging from 5% to 30% in the DID paradigm. Additionally, varenicline (Chantix®) was effective in reducing ethanol intake in α5-/- mice. Together, our data suggest that the α5 nAChR subunit is important for the sedative hypnotic doses of ethanol but does not play a role in ethanol consumption. Varenicline can be a treatment option even when there is loss of function of the α5 nAChR subunit.

Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: Comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17% total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11% of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means. © 2007 SGM.

More men than women make mucosal IgA antibodies to human papillomavirus type 16 (HPV-16) and HPV-18: A study of oral HPV and oral HPV antibodies in a normal healthy population

Background: We have previously shown the high prevalence of oral anti-human papillomavirus type 16 (HPV-16) antibodies in women with HPV-associated cervical neoplasia. It was postulated that the HPV antibodies were initiated after HPV antigenic stimulation at the cervix via the common mucosal immune system. The present study aimed to further evaluate the effectiveness of oral fluid testing for detecting the mucosal humoral response to HPV infection and to advance our limited understanding of the immune response to HPV. Methods: The prevalence of oral HPV infection and oral antibodies to HPV types 16, 18 and 11 was determined in a normal, healthy population of children, adolescents and adults, both male and female, attending a dental clinic. HPV types in buccal cells were determined by DNA sequencing. Oral fluid was collected from the gingival crevice of the mouth by the OraSure method. HPV-16, HPV-18 and HPV-11 antibodies in oral fluid were detected by virus-like particle-based enzyme-linked immunosorbent assay. As a reference group 44 women with cervical neoplasia were included in the study. Results: Oral HPV infection was h ighest in children (9/114, 7.9%), followed by adolescents (4/78, 5.1%), and lowest in normal adults (4/116, 3.5%). The predominant HPV type found was HPV-13 (7/22, 31.8%) followed by HPV-32 (5/22, 22.7%). The prevalence of oral antibodies to HPV-16, HPV-18 and HPV-11 was low in children and increased substantially in adolescents and normal adults. Oral HPV-16 IgA was significantly more prevalent in women with cervical neoplasia (30/44, 68.2%) than the women from the dental clinic (18/69, 26.1% P = 0.0001). Significantly more adult men than women displayed oral HPV-16 IgA (30/47 compared with 18/69, OR 5.0, 95% CI 2.09-12.1, P < 0.001) and HPV-18 IgA (17/47 compared with 13/69, OR 2.4, 95% CI 0.97-6.2, P = 0.04). Conclusion: The increased prevalence of oral HPV antibodies in adolescent individuals compared with children was attributed to the onset of sexual activity. The increased prevalence of oral anti-HPV IgA in men compared with women was noteworthy considering reportedly fewer men than women make serum antibodies, and warrants further investigation. © 2006 Marais et al; licensee BioMed Central Ltd.

Human papillomavirus prevalence, viral load and pre-cancerous lesions of the cervix in women initiating highly active antiretroviral therapy in South Africa : a cross-sectional study

Background Cervical cancer and infection with human immunodeficiency virus (HIV) are both important public health problems in South Africa (SA). The aim of this study was to determine the prevalence of cervical squamous intraepithelial lesions (SILs), high-risk human papillomavirus (HR-HPV), HPV viral load and HPV genotypes in HIV positive women initiating anti-retroviral (ARV) therapy. Methods A cross-sectional survey was conducted at an anti-retroviral (ARV) treatment clinic in Cape Town, SA in 2007. Cervical specimens were taken for cytological analysis and HPV testing. The Digene Hybrid Capture 2 (HC2) test was used to detect HR-HPV. Relative light units (RLU) were used as a measure of HPV viral load. HPV types were determined using the Roche Linear Array HPV Genotyping test. Crude associations with abnormal cytology were tested and multiple logistic regression was used to determine independent risk factors for abnormal cytology. Results The median age of the 109 participants was 31 years, the median CD4 count was 125/mm3, 66.3% had an abnormal Pap smear, the HR-HPV prevalence was 78.9% (Digene), the median HPV viral load was 181.1 RLU (HC2 positive samples only) and 78.4% had multiple genotypes. Among women with abnormal smears the most prevalent HR-HPV types were HPV types 16, 58 and 51, all with a prevalence of 28.5%. On univariate analysis HR-HPV, multiple HPV types and HPV viral load were significantly associated with the presence of low and high-grade SILs (LSIL/HSIL). The multivariate logistic regression showed that HPV viral load was associated with an increased odds of LSIL/HSIL, odds ratio of 10.7 (95% CI 2.0 – 57.7) for those that were HC2 positive and had a viral load of ≤ 181.1 RLU (the median HPV viral load), and 33.8 (95% CI 6.4 – 178.9) for those that were HC2 positive with a HPV viral load > 181.1 RLU. Conclusion Women initiating ARVs have a high prevalence of abnormal Pap smears and HR-HPV. Our results underscore the need for locally relevant, rigorous screening protocols for the increasing numbers of women accessing ARV therapy so that the benefits of ARVs are not partially offset by an excess risk in cervical cancer.

Monocular myopic defocus and daily changes in axial length and choroidal thickness of human eyes

Recent research indicates that brief periods (60 minutes) of monocular defocus lead to small but significant changes in human axial length. However, the effects of longer periods of defocus on the axial length of human eyes are unknown. We examined the influence of a 12 hour period of monocular myopic defocus on the natural daily variations occurring in axial length and choroidal thickness of young adult emmetropes. A series of axial length and choroidal thickness measurements (collected at ~3 hourly intervals, with the first measurement at ~9 am and the final measurement at ~9 pm) were obtained for 13 emmetropic young adults over three consecutive days. The natural daily rhythms (Day 1, baseline day, no defocus), the daily rhythms with monocular myopic defocus (Day 2, defocus day, +1.50 DS spectacle lens over the right eye), and the recovery from any defocus induced changes (Day 3, recovery day, no defocus) were all examined. Significant variations over the course of the day were observed in both axial length and choroidal thickness on each of the three measurement days (p<0.0001). The magnitude and timing of the daily variations in axial length and choroidal thickness were significantly altered with the monocular myopic defocus on day 2 (p<0.0001). Following the introduction of monocular myopic defocus, the daily peak in axial length occurred approximately 6 hours later, and the peak in choroidal thickness approximately 8.5 hours earlier in the day compared to days 1 and 3 (with no defocus). The mean amplitude (peak to trough) of change in axial length (0.030 ± 0.012 on day 1, 0.020 ± 0.010 on day 2 and 0.033 ± 0.012 mm on day 3) and choroidal thickness (0.030 ± 0.007 on day 1, 0.022 ± 0.006 on day 2 and 0.027 ± 0.009 mm on day 3) were also significantly different between the three days (both p<0.05). The introduction of monocular myopic defocus disrupts the daily variations in axial length and choroidal thickness of human eyes (in terms of both amplitude and timing) that return to normal the following day after removal of the defocus.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines : targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3alpha mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Hormone-dependent bacterial growth persistence and biofilm formation - A pilot study investigating human follicular fluid collected during IVF cycles

Human follicular fluid, considered sterile, is aspirated as part of an in vitro fertilization (IVF) cycle. However, it is easily contaminated by the trans-vaginal collection route and little information exists in its potential to support the growth of microorganisms. The objectives of this study were to determine whether human follicular fluid can support bacterial growth over time, whether the steroid hormones estradiol and progesterone (present at high levels within follicular fluid) contribute to the in vitro growth of bacterial species, and whether species isolated from follicular fluid form biofilms. We found that bacteria in follicular fluid could persist for at least 28 weeks in vitro and that the steroid hormones stimulated the growth of some bacterial species, specifically Lactobacillus spp., Bifidobacterium spp. Streptococcus spp. and E. coli. Several species, Lactobacillus spp., Propionibacterium spp., and Streptococcus spp., formed biofilms when incubated in native follicular fluids in vitro (18/24, 75%). We conclude that bacteria aspirated along with follicular fluid during IVF cycles demonstrate a persistent pattern of growth. This discovery is important since it can offer a new avenue for investigation in infertile couples.