Comparison of human alveolar osteoblasts cultured on polymer-ceramic composite scaffolds and tissue culture plates

The effects of medical grade polycaprolactone–tricalcium phosphate (mPCL–TCP) (80:20) scaffolds on primary human alveolar osteoblasts (AOs) were compared with standard tissue-culture plates. Of the seeded AOs, 70% adhered to and proliferated on the scaffold surface and within open and interconnected pores; they formed multi-layered sheets and collagen fibers with uniform distribution within 28 days. Elevation of alkaline phosphatase activity occurred in scaffold–cell constructs independent of osteogenic induction. AO proliferation rate increased and significant decrease in calcium concentration of the medium for both scaffolds and plates under induction conditions were seen. mPCL–TCP scaffolds significantly influenced the AO expression pattern of osterix and osteocalcin (OCN). Osteogenic induction down-regulated OCN at both RNA and protein level on scaffolds (3D) by day 7, and up-regulated OCN in cell-culture plates (2D) by day 14, but OCN levels on scaffolds were higher than on cell-culture plates. Immunocytochemical signals for type I collagen, osteopontin and osteocalcin were detected at the outer parts of scaffold–cell constructs. More mineral nodules were found in induced than in non-induced constructs. Only induced 2D cultures showed nodule formation. mPCL–TCP scaffolds appear to stimulate osteogenesis in vitro by activating a cellular response in AO's to form mineralized tissue. There is a fundamental difference between culturing AOs on 2D and 3D environments that should be considered when studying osteogenesis in vitro.

In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts

The ideal dermal matrix should be able to provide the right biological and physical environment to ensure homogenous cell and extracellular matrix (ECM) distribution, as well as the right size and morphology of the neo-tissue required. Four natural and synthetic 3D matrices were evaluated in vitro as dermal matrices, namely (1) equine collagen foam, TissuFleece®, (2) acellular dermal replacement, Alloderm®, (3) knitted poly(lactic-co-glycolic acid) (10:90)–poly(-caprolactone) (PLGA–PCL) mesh, (4) chitosan scaffold. Human dermal fibroblasts were cultured on the specimens over 3 weeks. Cell morphology, distribution and viability were assessed by electron microscopy, histology and confocal laser microscopy. Metabolic activity and DNA synthesis were analysed via MTS metabolic assay and [3H]-thymidine uptake, while ECM protein expression was determined by immunohistochemistry. TissuFleece®, Alloderm® and PLGA–PCL mesh supported cell attachment, proliferation and neo-tissue formation. However, TissuFleece® contracted to 10% of the original size while Alloderm® supported cell proliferation predominantly on the surface of the material. PLGA–PCL mesh promoted more homogenous cell distribution and tissue formation. Chitosan scaffolds did not support cell attachment and proliferation. These results demonstrated that physical characteristics including porosity and mechanical stability to withstand cell contraction forces are important in determining the success of a dermal matrix material.

Genetic resources in wild and cultured stocks of the Asian mud crab, Scylla paramamosain

The mud crab (Scylla spp.) aquaculture industry has expanded rapidly in recent years in many countries in the Indo - West Pacific (IWP) region as an alternative to marine shrimp culture because of significant disease outbreaks and associated failures of many shrimp culture industries in the region. Currently, practices used to produce and manage breeding crabs in hatcheries may compromise levels of genetic diversity, ultimately compromising growth rates, disease resistance and stock productivity. Therefore, to avoid “genetic pollution” and its harmful effects and to promote further development of mud crab aquaculture and fisheries in a sustainable way, a greater understanding of the genetic attributes of wild and cultured mud crab stocks is required. Application of these results can provide benefits for managing wild and cultured Asian mud crab populations for multiple purposes including for commercial production, recreation and conservation and to increase profitability and sustainability of newly emerging crab culture industries. Phylogeographic patterns and the genetic structure of Asian mud crab populations across the IWP were assessed to determine if they were concordant with those of other widespread taxa possessing pelagic larvae of relatively long duration. A 597 bp fragment of the mitochondrial DNA COI gene was amplified and screened for variation in a total of 297 individuals of S. paramamosain from six sampling sites across the species’ natural geographical distribution in the IWP and 36 unique haplotypes were identified. Haplotype diversities per site ranged from 0.516 to 0.879. Nucleotide diversity estimates among haplotypes were 0.11% – 0.48%. Maximum divergence observed among S. paramamosain samples was 1.533% and samples formed essentially a single monophyletic group as no obvious clades were related to geographical location of sites. A weak positive relationship was observed however, between genetic distance and geographical distance among sites. Microsatellite markers were then used to assess contemporary gene flow and population structure in Asian mud crab populations sampled across their natural distribution in the IWP. Eight microsatellite loci were screened in sampled S. paramamosain populations and all showed high allelic diversity at all loci in sampled populations. In total, 344 individuals were analysed, and 304 microsatellite alleles were found across the 8 loci. The mean number of alleles per locus at each site ranged from 20.75 to 28.25. Mean allelic richness per site varied from 17.2 to 18.9. All sites showed high levels of heterozygosity as average expected heterozygosities for all loci ranged from 0.917 – 0.953 while mean observed heterozygosity ranged from 0.916 – 0.959. Allele diversities were similar at all sites and across all loci. The results did not show any evidence for major differences in allele frequencies among sites and patterns of allele frequencies were very similar in all populations across all loci. Estimates of population differentiation (FST) were relatively low and most probably largely reflect intra – individual variation for very highly variable loci. Results from nDNA analysis showed evidence for only very limited population genetic structure among sampled S. paramamosain, and a positive and significant association for genetic and geographical distance among sample sites. Microsatellite markers were then employed to determine if adequate levels of genetic diversity has been captured in crab hatcheries for the breeding cycle. The results showed that all microsatellite loci were polymorphic in hatchery samples. Culture populations were in general, highly genetically depauperate, compared with comparable wild populations, with only 3 to 8 alleles recorded for the same loci set per population. In contrast, very high numbers of alleles per locus were found in reference wild S. paramamosain populations, which ranged from 18 to 46 alleles per locus per population. In general, this translates into a 3 to 10 fold decline in mean allelic richness per locus in all culture stocks compared with wild reference counterparts. Furthermore, most loci in all cultured S. paramamosain samples showed departures from HWE equilibrium. Allele frequencies were very different in culture samples from that present in comparable wild reference samples and this in particular, was reflected in a large decline in allele diversity per locus. The pattern observed was best explained by significant impacts of breeding practices employed in hatcheries rather than natural differentiation among wild populations used as the source of brood stock. Recognition of current problems and management strategies for the species both for the medium and long-term development of the new culture industry are discussed. The priority research to be undertaken over the medium term for S. paramamosain should be to close the life cycle fully to allow individuals to be bred on demand and their offspring equalised to control broodstock reproductive contributions. Establishing a broodstock register and pedigree mating system will be required before any selection program is implemented. This will ensure that sufficient genetic variation will be available to allow genetic gains to be sustainably achieved in a future stock improvement program. A fundamental starting point to improve hatchery practices will be to encourage farmers and hatchery managers to spawn more females in their hatcheries as it will increase background genetic diversity in culture stocks. Combining crablet cohorts from multiple hatcheries into a single cohort for supply to farmers or rotation of breeding females regularly in hatcheries will help to address immediate genetic diversity problems in culture stocks. Application of these results can provide benefits for managing wild and cultured Asian mud crab populations more efficiently. Over the long-term, application of data on genetic diversity in wild and cultured stocks of Asian mud crab will contribute to development of sustainable and productive culture industries in Vietnam and other countries in the IWP and can contribute towards conservation of wild genetic resources.

Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds

Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.

Assessing genetic diversity in cultured aquatic species : the Sydney Rock Oyster (Saccostrea glomerata) stock improvement program as a model

Genetic variation is the resource animal breeders exploit in stock improvement programs. Both the process of selection and husbandry practices employed in aquaculture will erode genetic variation levels overtime, hence the critical resource can be lost and this may compromise future genetic gains in breeding programs. The amount of genetic variation in five lines of Sydney Rock Oyster (SRO) that had been selected for QX (Queensland unknown) disease resistance were examined and compared with that in a wild reference population using seven specific SRO microsatellite loci. The five selected lines had significantly lower levels of genetic diversity than did the wild reference population with allelic diversity declining approximately 80%, but impacts on heterozygosity per locus were less severe. Significant deficiencies in heterozygotes were detected at six of the seven loci in both mass selected lines and the wild reference population. Against this trend however, a significant excess of heterozygotes was recorded at three loci Sgo9, Sgo14 and Sgo21 in three QX disease resistant lines (#2, #5 and #13). All populations were significantly genetic differentiated from each other based on pairwise FST values. A neighbour joining tree based on DA genetic distances showed a clear separation between all culture and wild populations. Results of this study show clearly, that the impacts of the stock improvement program for SRO has significantly eroded natural levels of genetic variation in the culture lines. This could compromise long-term genetic gains and affect sustainability of the SRO breeding program over the long-term.

Impacts of traditional husbandry practices on exploitable levels of genetic diversity in cultured 'Tra' catfish (Pangasianodon hypophthalmus) in the Mekong Delta, Vietnam

Sutchi catfish (Pangasianodon hypophthalmus) – known more universally by the Vietnamese name ‘Tra’ is an economically important freshwater fish in the Mekong Delta in Vietnam that constitutes an important food resource. Artificial propagation technology for Tra catfish has only recently been developed along the main branches of the Mekong River where more than 60% of the local human population participate in fishing or aquaculture. Extensive support for catfish culture in general, and that of Tra (P. hypophthalmus) in particular, has been provided by the Vietnamese government to increase both the scale of production and to develop international export markets. In 2006, total Vietnamese catfish exports reached approximately 286,602 metric tons (MT) and were valued at 736.87 $M with a number of large new export destinations being developed. Total value of production from catfish culture has been predicted to increase to approximately USD 1 billion by 2020. While freshwater catfish culture in Vietnam has a promising future, concerns have been raised about long-term quality of fry and the effectiveness of current brood stock management practices, issues that have been largely neglected to date. In this study, four DNA markers (microsatellite loci: CB4, CB7, CB12 and CB13) that were developed specifically for Tra (P. hypophthalmus) in an earlier study were applied to examine the genetic quality of artificially propagated Tra fry in the Mekong Delta in Vietnam. The goals of the study were to assess: (i) how well available levels of genetic variation in Tra brood stock used for artificial propagation in the Mekong Delta of Vietnam (breeders from three private hatcheries and Research Institute of Aquaculture No2 (RIA2) founders) has been conserved; and (ii) whether or not genetic diversity had declined significantly over time in a stock improvement program for Tra catfish at RIA2. A secondary issue addressed was how genetic markers could best be used to assist industry development. DNA was extracted from fins of catfish collected from the two main branches of the Mekong River inf Vietnam, three private hatcheries and samples from the Tra improvement program at RIA2. Study outcomes: i) Genetic diversity estimates for Tra brood stock samples were similar to, and slightly higher than, wild reference samples. In addition, the relative contribution by breeders to fry in commercial private hatcheries strongly suggest that the true Ne is likely to be significantly less than the breeder numbers used; ii) in a stock improvement program for Tra catfish at RIA2, no significant differences were detected in gene frequencies among generations (FST=0.021, P=0.036>0.002 after Bonferroni correction); and only small differences were observed in alleles frequencies among sample populations. To date, genetic markers have not been applied in the Tra catfish industry, but in the current project they were used to evaluate the levels of genetic variation in the Tra catfish selective breeding program at RIA2 and to undertake genetic correlations between genetic marker and trait variation. While no associations were detected using only four loci, they analysis provided training in the practical applications of the use of molecular markers in aquaculture in general, and in Tra culture, in particular.

Current status and future prospects for cultured limbal tissue transplants in Australia and New Zealand

Cultured limbal tissue transplants have become widely used over the last decade as a treatment for limbal stem cell deficiency (LSCD). While the number of patients afflicted with LSCD in Australia and New Zealand is considered to be relatively low, the impact of this disease on quality of life is so severe that the potential efficacy of cultured transplants has necessitated investigation. We presently review the basic biology and experimental strategies associated with the use of cultured limbal tissue transplants in Australia and New Zealand. In doing so, we aim to encourage informed discussion on the issues required to advance the use of cultured limbal transplants in Australia and New Zealand. Moreover, we propose that a collaborative network could be established to maintain access to the technology in conjunction with a number of other existing and emerging treatments for eye diseases.

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment. © 2012 Sieh et al.

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts

Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOS-II) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-gamma and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-gamma alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.

Comparative effects of posterior eye cup tissues from Myopic and Hyperopic chick eyes on cultured scleral fibroblasts

The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and -smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or -15D lenses (lens-induced myopia, LIM) for 3 days (post hatch day 5 to 8) (n=6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from -15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9±2.6 µg and 8.4±1.1 µg, p=0.12; GAG 11.2±0.6 µg and 10.1±1.0 µg, p=0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2±1.7 µg versus 23.2±1.5 µg, p=0.21; GAG 28.1±1.7 µg versus. 28.7±1.2 µg, p=0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA, LIM 46.9±6.4 versus LIH 51.5±4.7 µg, p=0.31), whereas GAG content was higher for cells in co-culture with choroid from LIH eyes (GAG 32.5±0.7 µg versus 18.9±1.2 µg, F1,6=9.210, p=0.0002). In contrast, fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA content less for co-culture with RPE from LIH eyes (LIM: 72.4±6.3 µg versus Empty basket: 46.03±1.0 µg; F1,6=69.99, p=0.0005 and LIH: 27.9±2.3 µg versus empty basket: 46.03±1.0 µg; p=0.0004). GAG content was higher with RPE from LIH eyes (LIH: 33.7±1.9 µg versus empty basket: 29.5±0.8 µg, F1,6=13.99, p=0.010) and lower with RPE from LIM eyes (LIM: 27.7±0.9 µg versus empty basket: 29.5±0.8 µg, p=0.021). GAG content of cells in co-culture with choroid from LIH eyes was higher compared to co-culture with choroid from LIM eyes (32.5±0.7 µg versus 18.9±1.2 µg respectively, F1,6=9.210, p=0.0002). In conclusion, these experiments provide evidence for a directional growth signal that is present (and remains) in the ex-vivo RPE, but that does not remain in the ex-vivo retina. The identity of this factor(s) that can modify scleral cell DNA and GAG content requires further research.

Stage-specific embryonic antigen-4 is not a marker for chondrogenic and osteogenic potential in cultured chondrocytes and mesenchymal progenitor cells

One important challenge for regenerative medicine is to produce a clinically relevant number of cells with consistent tissue-forming potential. Isolation and expansion of cells from skeletal tissues results in a heterogeneous population of cells with variable regenerative potential. A more consistent tissue formation could be achieved by identification and selection of potent progenitors based on cell surface molecules. In this study, we assessed the expression of stage-specific embryonic antigen-4 (SSEA-4), a classic marker of undifferentiated stem cells, and other surface markers in human articular chondrocytes (hACs), osteoblasts, and bone marrow-derived mesenchymal stromal cells (bmMSCs) and characterized their differentiation potential. Further, we sorted SSEA-4-expressing hACs and followed their potential to proliferate and to form cartilage in vitro. Cells isolated from cartilage and bone exhibited remarkably heterogeneous SSEA-4 expression profiles in expansion cultures. SSEA-4 expression levels increased up to approximately 5 population doublings, but decreased following further expansion and differentiation cultures; levels were not related to the proliferation state of the cells. Although SSEA-4-sorted chondrocytes showed a slightly better chondrogenic potential than their SSEA-4-negative counterparts, differences were insufficient to establish a link between SSEA-4 expression and chondrogenic potential. SSEA-4 levels in bmMSCs also did not correlate to the cells' chondrogenic and osteogenic potential in vitro. SSEA-4 is clearly expressed by subpopulations of proliferating somatic cells with a MSC-like phenotype. However, the predictive value of SSEA-4 as a specific marker of superior differentiation capacity in progenitor cell populations from adult human tissue and even its usefulness as a stem cell marker appears questionable.

Genetic diversity of cultured and wild populations of the giant freshwater prawn Macrobrachium rosenbergii (de Man, 1879) based on microsatellite analysis

Freshwater prawn (Macrobrachium rosenbergii) culture in the Western Hemisphere is primarily, if not entirely, derived from 36 individual prawns originally introduced to Hawaii from Malaysia in 1965 and 1966. Little information is available regarding genetic variation within and among cultured prawn stocks worldwide. The goal of the current study was to characterize genetic diversity in various prawn populations with emphasis on those cultured in North America. Five microsatellite loci were screened to estimate genetic diversity in two wild (Myanmar and India-wild) and seven cultured (Hawaii-1, Hawaii-2, India-cultured, Israel, Kentucky, Mississippi and Texas) populations. Average allelic richness ranged from 3.96 (Israel) to 20.45 (Myanmar). Average expected heterozygosity ranged from 0.580 (Israel) to 0.935 (Myanmar). Many of the cultured populations exhibited reduced genetic diversity when compared with the Myanmar and the India-cultured populations. Significant deficiency in heterozygotes was detected in the India-cultured, Mississippi and Kentucky populations (overall Fis estimated of 0.053, 0.067 and 0.108 respectively) reflecting moderate levels of inbreeding. Overall estimate of fixation index (Fst = 0.1569) revealed moderately high levels of differentiation among the populations. Outcome of this study provide a baseline assessment of genetic diversity in some available strains that will be useful for the development of breeding programmes.