Cell adhesion and mechanical properties of a flexible scaffold for cardiac tissue engineering.

Cardiac tissue engineering is focused on obtaining functional cardiomyocyte constructs to provide an alternative to cellular cardiomyoplasty. Mechanical stimuli have been shown to stimulate protein expression and the differentiation of mammalian cells from contractile tissues. Our aim was to obtain a flexible scaffold which could be used to apply mechanical forces during tissue regeneration. Poly(1,8-octanediol-co-citric acid) (POC) is an elastomer that can be processed into scaffolds for tissue engineering. We investigated the effect of modifying the porosity on the mechanical properties of the POC scaffolds. In addition, the effects of the storage method and strain rate on material integrity were assessed. The maximum elongation of POC porous films varied from 60% to 160% of their original length. A decrease in the porosity caused a rise in this elastic modulus. The attachment of HL-1 cardiomyocytes to POC was assessed on films coated with fibronectin, collagen and laminin. These extracellular matrix proteins promoted cell adhesion in a protein-type- and concentration-dependent manner. Therefore, POC scaffolds can be optimised to meet the mechanical and biological parameters needed for cardiac culture. This porous material has the potential to be used for cardiac tissue engineering as well as for other soft tissue applications.

Apoptosis--a significant cause of bone cell death in osteonecrosis of the femoral head.

Osteonecrosis of the femoral head usually affects young individuals and is responsible for up to 12% of total hip arthroplasties. The underlying pathophysiology of the death of the bone cells remains uncertain. We have investigated nitric oxide mediated apoptosis as a potential mechanism and found that steroid- and alcohol-induced osteonecrosis is accompanied by widespread apoptosis of osteoblasts and osteocytes. Certain drugs or their metabolites may have a direct cytotoxic effect on cancellous bone of the femoral head leading to apoptosis rather than purely necrosis.

Osteogenic differentiation of mouse embryonic stem cells: differential gene expression analysis by cDNA microarray and purification of osteoblasts by cadherin-11 magnetically activated cell sorting.

We have previously shown osteogenic differentiation of mouse embryonic stem (ES) cells and temporal enrichment with osteoblastic cells, by stimulation with serum-containing culture medium supplemented with beta-glycerophosphate, ascorbate, and dexamethasone. In our present study we have used similar culture conditions to further investigate osteogenic differentiation of mouse ES cells. Using reverse transcription-polymerase chain reaction (RT-PCR) we demonstrated the expression of genes associated with osteoblast differentiation including the bone matrix protein osteocalcin and the transcription factor Cbfa-1/runx2. Furthermore, results of cDNA microarray analysis, and subsequent RT-PCR analysis of differentiating ES cells after exposure to osteogenic stimuli, revealed a combination of upregulation of genes involved in osteoblast differentiation including osteopontin, HSP-47, and IGF-II coupled with downregulation of genes involved in differentiation of other phenotypes such as the neuroectoderm factor Stra-13. Finally, we have applied magnetically activated cell-sorting methods to ES cell cultures treated with osteogenic stimuli and, using an antibody to cadherin-11, have purified a subpopulation of cells with osteoblastic characteristics.

Stem cells: sources and applications.

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of function of organs through implantation of cells/tissues grown outside the body, or stimulating cells to grow into implanted matrix. In this short review, some of the most recent developments in the use of stem cells for tissue repair and regeneration will be discussed. There is no doubt that stem cells derived from adult and embryonic sources hold great therapeutic potential but it is clear that there is still much research required before their use is commonplace. There is much debate over adult versus embryonic stem cells and whether both are required. It is probably too early to disregard one or other of these cell sources. With regard to embryonic stem cells, the major concern relates to the ethics of their creation and the proposed practice of therapeutic cloning.

Modulation of hydrogen peroxide induced injury to corneal endothelium by virus mediated catalase gene transfer.

AIM: To examine the effect of catalase gene transfer on survival of corneal endothelial cells (EC) following challenge with hydrogen peroxide (H(2)O(2)) in an ex vivo model of oxidative stress. METHODS: A recombinant adenovirus vector (AdCL) was used to transfer human catalase cDNA into EC of whole thickness rabbit corneas ex vivo. The resulting catalase protein concentration was measured in corneal lysates by ELISA; catalase functional activity in lysates was determined using a H(2)O(2) activity assay. To examine the morphological effects of catalase gene transfer in modulation of H(2)O(2) induced injury, transduced corneas were maintained in ex vivo culture and challenged with H(2)O(2). Laser scanning confocal microscopy was used to image EC injury. Cell density, cell morphology, and ratios of viable to necrotic cells were determined. RESULTS: Following incubation with AdCL, catalase expression reached maximum at 5-7 days. Corneas transduced with AdCL showed increased EC cell survival following challenge with H(2)O(2) on day 3 when compared to null vector control or mock infected corneas. CONCLUSIONS: Ex vivo catalase gene transfer can protect EC from death mediated by H(2)O(2). This gene based approach to the protection of corneal endothelium from oxidative stress may have application in prevention of EC loss in pathological conditions in which H(2)O(2) is involved and in ex vivo donor corneal storage before transplantation.

Gene-expression profiling of human osteoblasts following treatment with the ionic products of Bioglass 45S5 dissolution.

The effect of the ionic products of Bioglass 45S5 dissolution on the gene-expression profile of human osteoblasts was investigated by cDNA microarray analysis of 1,176 genes. Treatment with the ionic products of Bioglass 45S5 dissolution increased the levels of 60 transcripts twofold or more and reduced the levels of five transcripts to one-half or less than in control. Markedly up-regulated genes included RCL, a c-myc responsive growth related gene, cell cycle regulators such as G1/S specific cyclin D1, and apoptosis regulators including calpain and defender against cell death (DAD1). Other significantly up-regulated genes included the cell surface receptors CD44 and integrin beta1, and various extracellular matrix regulators including metalloproteinases-2 and -4 and their inhibitors TIMP-1 and TIMP-2. The identification of differentially expressed genes by cDNA microarray analysis has offered new insights into the mode of action of bioactive glasses and has proven to be an effective tool in evaluating their osteoproductive properties.

Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells.

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.

Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity.

Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. However, the contribution made by specific NO synthase (NOS) enzymes is unclear. Here we show that endothelial NOS gene knockout mice (eNOS-/-) have marked abnormalities in bone formation. Histomorphometric analysis of eNOS-/- femurs showed bone volume and bone formation rate was reduced by up to 45% (P: < 0.01) and 52% (P: < 0.01), respectively. These abnormalities were prevalent in young (6 to 9 weeks old) adults but by 12 to 18 weeks bone phenotype was restored toward wild-type. Dual energy X-ray absorptiometry analysis confirmed the age-related bone abnormalities revealing significant reductions in femoral (P: < 0.05) and spinal bone mineral densities (P: < 0.01) at 8 weeks that were normalized at 12 weeks. Reduction in bone formation and volume was not related to increased osteoclast numbers or activity but rather to dysfunctional osteoblasts. Osteoblast numbers and mineralizing activity were reduced in eNOS-/- mice. In vitro, osteoblasts from calvarial explants showed retarded proliferation and differentiation (alkaline phosphatase activity and mineral deposition) that could be restored by exogenous administration of a NO donor. These cells were also unresponsive to 17ss-estradiol and had an attenuated chemotactic response to transforming growth factor-beta. In conclusion, eNOS is involved in the postnatal regulation of bone mass and lack of eNOS gene results in reduced bone formation and volume and this is related to impaired osteoblast function.

Nitric oxide mediates 17beta-estradiol-stimulated human and rodent osteoblast proliferation and differentiation.

Oestradiol can stimulate osteoblast activity. Osteoblast function is thought to be regulated by nitric oxide (NO). We hypothesised that the effect of 17beta-oestradiol (17beta-E(2)) on osteoblast activity is mediated by NO. This hypothesis was tested using osteoblasts isolated from human trabecular bone, calvariae of rats, endothelial NO synthase (eNOS) gene-deficient mice, and their wild-type counterparts. Our results show that 17beta-E(2) dose-dependently stimulated proliferation and differentiation of primary human, rat and wild-typeosteoblasts. The presence of N(G)-monomethyl-l-arginine (10(-3) M), an inhibitor of NOS activity, blocked the 17beta-E(2)-(10(-7) M)-induced increases in thymidine incorporation (P < 0.01), alkaline phosphatase activity (P < 0.01) and bone nodule formation (P < 0.01) of wild-type, human and rat osteoblasts, respectively. Moreover, 17beta-E(2) did not induce a response in eNOS gene-deficient osteoblasts. 17beta-E(2) also increased total eNOS enzyme expression in rat osteoblasts. These findings indicate 17beta-E(2) modulates osteoblast function by NO-dependent mechanisms mediated via the eNOS isoform.

Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis.

Bioglass 45S5 is an osteoproductive material, which resorbs by releasing its constitutive ions into solution. Treatment with the ionic products of Bioglass 45S5 dissolution in DMEM for 4 days increased human osteoblast proliferation to 155% of control. Two days after treatment, differential gene expression was analyzed by cDNA microarrays. Expression of a potent osteoblast mitogenic growth factor, insulin-like growth factor II (IGF-II), was increased to 290%. Additionally, there was a 168% increase in the concentration of unbound IGF-II protein in the conditioned media of treated osteoblasts. Expression levels of IGFBP-3, an IGF-II carrier protein, metalloproteinase-2 and cathepsin-D were also increased to 200, 340, and 310% of control levels, respectively. Metalloproteinase-2 and cathepsin-D are proteases that cleave IGF-II from its carrier proteins, resulting in the release of the unbound biologically active IGF-II. We suggest that the stimulatory effect of the ionic products of Bioglass 45S5 dissolution on osteoblast proliferation may be mediated by IGF-II.

Bioglass 45S5 stimulates osteoblast turnover and enhances bone formation In vitro: implications and applications for bone tissue engineering.

We investigated the concept of using bioactive substrates as templates for in vitro synthesis of bone tissue for transplantation by assessing the osteogenic potential of a melt-derived bioactive glass ceramic (Bioglass 45S5) in vitro. Bioactive glass ceramic and bioinert (plastic) substrates were seeded with human primary osteoblasts and evaluated after 2, 6, and 12 days. Flow cytometric analysis of the cell cycle suggested that the bioactive glass-ceramic substrate induced osteoblast proliferation, as indicated by increased cell populations in both S (DNA synthesis) and G2/M (mitosis) phases of the cell cycle. Biochemical analysis of the osteoblast differentiation markers alkaline phosphatase (ALP) and osteocalcin indicated that the bioactive glass-ceramic substrate augmented osteoblast commitment and selection of a mature osteoblastic phenotype. Scanning electron microscopic observations of discrete bone nodules over the surface of the bioactive material, from day 6 onward, further supported this notion. A combination of fluorescence, confocal, transmission electron microscopy, and X-ray microprobe (SEM-EDAX) examinations revealed that the nodules were made of cell aggregates which produced mineralized collagenous matrix. Control substrates did not exhibit mineralized nodule formation at any point studied up to 12 days. In conclusion, this study shows that Bioglass 45S5 has the ability to stimulate the growth and osteogenic differentiation of human primary osteoblasts. These findings have potential applications for tissue engineering where this bioactive glass substrate could be used as a template for the formation of bioengineered bone tissue.

Pelvic nerve plexus trauma at radical hysterectomy and simple hysterectomy: the nerve content of the uterine supporting ligaments.

BACKGROUND: A major cause of the pelvic morbidity after a radical hysterectomy (RH) is thought to be damage to the pelvic nerve plexus, but direct evidence is lacking. We set out to determine the nerve content of the uterosacral ligaments (USLs) and cardinal ligaments (CLs) at the level at which they are divided during a radical hysterectomy and a simple hysterectomy. METHODS: Intraoperative cross-sectional biopsies were collected from the lateral third of the uterosacral ligaments (USLs) and cardinal ligaments (CLs) in 20 women undergoing radical hysterectomy (RH) and from the uterine insertion of these ligaments in 11 women undergoing a simple hysterectomy. Quantitative immunocytochemistry was utilized to demonstrate and quantify the nerve content of the uterine supporting ligaments at the level at which they are divided in a RH and in a simple hysterectomy. Indirect immunofluorescence staining of frozen cryostat sections was performed using primary antibodies to PGP 9.5 (a pan-neuronal marker). A computer-assisted image analyzer measured the percentage area of immunoreactivity (PAI) that was used to quantify the nerve density. Confocal microscopy was used to determine the composition and spatial arrangement of nerve fibers in the ligaments. RESULTS: The PAI was significantly greater in the RH biopsies than in the simple hysterectomy biopsies, for both the CLs (P < 0.001) and the USLs (P < 0.001). In the RH biopsies, more nerve tissue was present in the USL than CL (P = 0.01), and compared with the CL more of the nerve fibers in the USL were concentrated in large trunks. Excluding these trunks and autonomic ganglia, the free nerve content of the USL was lower than that of the CL (P < 0.001). The presence of nerve trunks, autonomic ganglia, and free nerve fibers within the lateral third of the USL and CL is consistent with extension of the inferior hypogastric plexus along these ligaments to the pelvic organs. CONCLUSIONS: The uterine supporting ligaments contain autonomic nerves and ganglia, as extensions of the inferior hypogastric plexus. The USLs have a greater nerve density than the CLs. Because RH disrupts more nerve tissue than a simple hysterectomy, these data provide further evidence for the neurogenic etiology of pelvic morbidity after RH.

Growth and adhesion of osteoblast-like cells derived from neonatal rat calvaria on calcium phosphate ceramics.

The effects of biocompatible ceramics on the growth and adhesion of osteoblast-rich rat calvarial cell cultures were investigated. Osteoblast-like cells and mouse fibroblast-like L-929 cells were cultured on composite sinters of hydroxyapatite (HAP) and beta-tricalcium phosphate (TCP) culture carriers, whose Ca/P molar ratios were adjusted to values of 1.50, 1.55, 1.60, 1.64 and 1.67. The growth rates of both cell types were accelerated on the TCP-HAP ceramics as compared to those on polystyrene plastic (LUX) or bioinert zirconia ceramics. The population of osteoblast-like cells reached a density of 2.28 x 10(5) cells/cm2 on 100% HAP (Ca/P ratio 1.67) at 9 d of culture, while the corresponding cell density was 1.66 x 10(5) cells/cm2 on LUX and 1.26 x 10(5) cells/cm2 on zirconia. Adhesion of the osteoblast-like cells on TCP-HAP ceramics was similarly increased as compared with that on LUX or zirconia ceramics. The adhesion of L-929 cells on TCP-HAP ceramics was found to be weaker than that on cultures on LUX or zirconia ceramics. The time-dependent variations in the alkaline phosphatase activity of the osteoblast-like cells showed that the osteoblastic phenotype was potentiated by culturing the cells in calcium-rich media. The surface analyses of the Ca/P ratio and the microstructure by XRD and FTIR suggest that the Ca-rich surface was newly formed by recombination on the surface layer in the culture medium containing fetal bovine serum. These results suggest that the surface of TCP-HAP ceramics, especially that of 100% HAP ceramics, are effective for accelerating growth and differentiation of osteoblast-like cells. This is most probably due to the chemical and physical instability and composition of 100% HAP, which promote the formation of a Ca-rich layer at the cell-material interface and provision of Ca ions to the osteoblast-like cells.

Immunochemical detection of nitric oxide synthase in human tissue.

To date three distinct isoforms of nitric oxide synthase (NOS) have been identified. Two isoforms are considered to be expressed constitutively-neu-ronal NOS (nNOS; type I NOS) and endothelial NOS (eNOS; type III NOS). The third isoform is not generally present in normal cells and tissues but is induced in response to infection, inflammation or trauma-inducible NOS (iNOS; type II NOS). In 1990 Bredt and Synder (1) succeeded in developing antibodies to rat brain NOS (nNOS) and used immuncytochemistry subsequently to furnish one of the first anatomical descriptions of the distribution and localization of nNOS. Today numerous antibodies to all three NOS isoforms isolated from various tissues and different animal species are available and the application of immunocytochemistry is commonplace in the investigation of NOS in healthy and diseased tissues including human (2-6) (Fig. 1 Fig. 2 Fig. 3).

Reciprocal changes in endothelial and inducible nitric oxide synthase expression following carotid angioplasty in the pig.

OBJECTIVE: Nitric oxide produced by nitric oxide synthase appears to have an important role in the regulation of arterial tone, platelet adhesion and smooth muscle cell proliferation. Our aim was to investigate the effects of balloon angioplasty on expression of endothelial NO synthase (cNOS) and inducible NO/synthase (iNOS) in the pig carotid artery and to relate any changes in expression to the processes of reendothelialisation and vascular repair. METHODS: Pigs were sacrificed at various time points to follow NOS expression in the neointima, media and regenerated endothelium. Immunocytochemical staining was used to localize cNOS and iNOS expression in the vessel wall. Relative amounts of cNOS were measured using quantitative in vitro alitoradiography. cNOS mRNA and iNOS mRNA was quantified by competitive PCR based on the sequenced cDNA of porcine cNOS and iNOS. RESULTS: Uninjured carotid arteries exhibited dense uniform luminal endothelial staining for cNOS. Balloon angioplasty caused denudation of cNOS immunoreactive cells and a marked reduction of cNOS gene expression but a complete recovery was noted by day 35. In normal uninjured carotid arteries no evidence of iNOS immunoreactivity was demonstrable but 24 h after injury, marked homogeneous iNOS immunoreactivity was detected in medial vascular smooth muscle cells. By 5 days, staining was evident in cells within the forming neointimal layer with no evidence of iNOS immunoreactivity in the media. iNOS immunoreactivity persisted in cells at the luminal surface at 7 days and iNOS gene expression appeared to be sustained in some animals with ruptured internal elastic lamina at 21 days. CONCLUSION: Balloon injury is associated with de-endothelialisation and a marked reduction in cNOS gene expression and activity. iNOS is induced throughout the arterial media within VSMC soon after balloon injury and persists for up to 21 days. These observations imply an important regulatory role for locally generated NO in the pathophysiological response to balloon injury.

Cytokine-induced prostaglandin E2 synthesis and cyclooxygenase-2 activity are regulated both by a nitric oxide-dependent and -independent mechanism in rat osteoblasts in vitro.

Osteoblasts respond to stimulation with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) by production of nitric oxide and prostaglandins (PGs). In this study the relationship between nitric oxide and PG synthesis was investigated after cytokine stimulation of cultured rat osteoblasts. IL-1, TNF-alpha, IFN-gamma, and exogenous sodium nitroprusside, a nitric oxide donor, all stimulated PGE2 production in a dose-dependent manner. PGE2 production was blocked by L-nitro-arginine methyl ester, an inhibitor of nitric oxide production, after IFN-gamma stimulation and was partially blocked after TNF-alpha stimulation. However, IL-1-induced PGE2 was unaffected. Similarly, expression of the cyclooxygenase-2 protein was stimulated by cytokines, and IFN-gamma-induced expression was again blocked by L-nitro-arginine methyl ester. In contrast, all cytokines induced the cyclooxygenase-2 mRNA expression independently of nitric oxide production, although exogenous sodium nitroprusside was able to induce the cyclooxygenase-2 mRNA in the absence of cytokines. The results show that nitric oxide can induce PG synthesis and cyclooxygenase-2 expression and may regulate cyclooxygenase-2 expression at both transcriptional and post-transcriptional levels. In addition, the data show the existence of both nitric oxide-dependent and -independent pathways of PG synthesis after cytokine stimulation of osteoblasts. The results suggest that nitric oxide may be an important mediator of PG production in inflammatory bone diseases.

Localization of Gene Expression in Human Atherosclerotic Lesions by In SituHybridization.

The technique of in situ hybridization was developed in the late 1960s (1) and is based on the ability of a single-stranded sequence of nucleotides to hybridize specifically to a complementary sequence to form stable double-stranded duplexes or hybrids. Incorporating a readily detectable label into the nucleotide sequence permits it to be used as a probe to identify specific complementary target sequences within cells or tissues and thereby derive useful information on the site(s) of expression of a particular gene. Although this technique can be adapted to detect any nucleotide sequence, this chapter is concerned with the detection of mRNA. The advantage of in situ hybridization for detecting mRNA over other methods such as Northern blotting is that this technique allows morphological identification of a specific mRNA species in a particular cell or a subpopulation of cells within a tissue. This can yield useful information on sites of synthesis, turnover, and, when combined with a method such as immunocytochemistry, storage of the transcribed protein.