Regional gene expression analysis of multiple tissues in an experimental animal model of post-traumatic osteoarthritis.

OBJECTIVE: To characterize local disease progression of the medial meniscus transection (MMT) model of post-traumatic osteoarthritis (OA) at the molecular level, in order to establish a baseline for therapeutic testing at the preclinical stage. DESIGN: Weight-matched male Lewis rats underwent MMT or sham surgery on the left limb with the right leg as contralateral control. At 1 and 3 weeks post-surgery, tissues were harvested from different areas of the articular cartilage (medial and lateral tibial plateaus, and medial osteophyte region) and synovium (medial and lateral), and analyzed separately. RNA was extracted and used for microarray (RT-PCR) analysis. RESULTS: Gene expression changes due to surgery were isolated to the medial side of the joint. Gene changes in chondrocyte phenotype of the medial tibial plateau cartilage preceded changes in tissue composition genes. Differences in inflammatory markers were only observed at the osteophyte region at 3 weeks post-surgery. There was surgical noise in the synovium at week 1, which dissipated at week 3. At this later timepoint, meniscal instability resulted in elevated expression of matrix degradation proteins and osteogenic markers in the synovium and cartilage. CONCLUSION: These results suggest feedback interactions between joint tissues during disease progression. Regional tissue expression differences found in MMT joints indicated similar pathophysiology to human OA, and provided novel insights about this degeneration model. The examination of gene expression at a localized level in multiple tissues provides a well-characterized baseline to evaluate mechanistic effects of potential therapeutic agents on OA disease progression in the MMT model.

Influence of scaffold properties on the inter-relationship between human bone marrow derived stromal cells and endothelial cells in pro-osteogenic conditions.

One of the significant challenges in bone tissue engineering is the integration of biomaterials designed to facilitate and stimulate mineralization with a simultaneously rapid rate of angiogenesis and vascularization of the tissue construct, a challenge complicated by our lack of knowledge of the interactions among key cell types and scaffold properties. This study compared functional activity of human bone marrow-derived stromal cells (hMSC) seeded onto a porous salt-leached poly(D,L-lactic acid) (PDLLA) scaffolds, with and without the incorporation of silk fibroin fibers and then further investigated their co-culture with human umbilical vein endothelial cells (HUVECs). Cell viability, proliferation, and alkaline phosphatase activity were measured for a range of time points in culture, with osteogenic and angiogenic marker immunolocalization and gene expression at selected stages. Our findings suggest that, despite similar porosity and pore size distribution exhibited by the PDLLA and PDLLA plus silk fibroin scaffolds, there were marked differences in cell distribution and function. In the absence of fibers, a highly osteogenic response was observed in hMSCs in the scaffolds co-cultured with endothelial cells, greater than that observed with hMSCs alone or in either of the scaffolds with fibers added. However, fiber presence clearly better supported endothelial cell cultures, as determined by greater levels of endothelial marker expression at both the gene and protein level after 3 weeks of culture. The design of composite scaffolds integrating beneficial components of differing structures and materials to facilitate appropriate biological responses appears a promising yet challenging avenue of research. STATEMENT OF SIGNIFICANCE: A significant challenge in bone tissue engineering is to promote a rapid vascularization of the tissue construct in parallel to the extracellular matrix mineralization. The design of composite scaffolds integrating beneficial components of differing structures and materials to facilitate appropriate biological responses appears a promising yet challenging avenue of research. Here we investigated cultures of hMSCs and HUVECs on a silk fibroin enhanced PDLLA scaffold, showing that the final output of this in vitro system is not the linear sum of the effects of the single variables. These results are of interest as they demonstrate how the addition of endothelial cells can affect hMSC phenotype and that the output can be further modulated by the introduction of silk fibroin fibers.

The delayed addition of human mesenchymal stem cells to pre-formed endothelial cell networks results in functional vascularization of a collagen-glycosaminoglycan scaffold in vivo.

This paper demonstrates a method to engineer, in vitro, a nascent microvasculature within a collagen-glycosaminoglycan scaffold with a view to overcoming the major issue of graft failure due to avascular necrosis of tissue-engineered constructs. Human umbilical vein endothelial cells (ECs) were cultured alone and in various co-culture combinations with human mesenchymal stem cells (MSCs) to determine their vasculogenic abilities in vitro. Results demonstrated that the delayed addition of MSCs to pre-formed EC networks, whereby MSCs act as pericytes to the nascent vessels, resulted in the best developed vasculature. The results also demonstrate that the crosstalk between ECs and MSCs during microvessel formation occurs in a highly regulated, spatio-temporal fashion, whereby the initial seeding of ECs results in platelet derived growth factor (PDGF) release; the subsequent addition of MSCs 3 days later leads to a cessation in PDGF production, coinciding with increased vascular endothelial cell growth factor expression and enhanced vessel formation. Functional assessment of these pre-engineered constructs in a subcutaneous rat implant model demonstrated anastomosis between the in vitro engineered vessels and the host vasculature, with significantly increased vascularization occurring in the co-culture group. This study has thus provided new information on the process of in vitro vasculogenesis within a three-dimensional porous scaffold for tissue engineering and demonstrates the potential for using these vascularized scaffolds in the repair of critical sized bone defects.

Localized 3D analysis of cartilage composition and morphology in small animal models of joint degeneration.

OBJECTIVE: Current histological scoring methods to evaluate efficacy of potential therapeutics for slowing or preventing joint degeneration are time-consuming and semi-quantitative in nature. Hence, there is a need to develop and standardize quantitative outcome measures to define sensitive metrics for studying potential therapeutics. The objectives of this study were to use equilibrium partitioning of an ionic contrast agent via Equilibrium Partitioning of an Ionic Contrast-Microcomputed tomography (EPIC-µCT) to quantitatively characterize morphological and compositional changes in the tibial articular cartilage in two distinct models of joint degeneration and define localized regions of interest to detect degenerative cartilage changes. MATERIALS AND METHODS: The monosodium iodoacetate (MIA) and medial meniscal transection (MMT) rat models were used in this study. Three weeks post-surgery, tibiae were analyzed using EPIC-µCT and histology. EPIC-µCT allowed measurement of 3D morphological changes in cartilage thickness, volume and composition. RESULTS: Extensive cartilage degeneration was observed throughout the joint in the MIA model after 3 weeks. In contrast, the MMT model showed more localized degeneration with regional thickening of the medial tibial plateau and a decrease in attenuation consistent with proteoglycan (PG) depletion. Focal lesions were also observed and 3D volume calculated as an additional outcome metric. CONCLUSIONS: EPIC-µCT was used to quantitatively assess joint degeneration in two distinct preclinical models. The MMT model showed similar features to human Osteoarthritis (OA), including localized lesion formation and PG loss, while the MIA model displayed extensive cartilage degeneration throughout the joint. EPIC-µCT imaging provides a rapid and quantitative screening tool for preclinical evaluation of OA therapeutics.

Pressure gradients and transport in the murine femur upon hindlimb suspension.

Interstitial fluid flow (IFF) is important in a number of processes, including stimulation of cells and nutrient and waste transport. In bone, it arises from the vascular pressure gradient between the medullary cavity and the lymphatic drainage at the periosteal surface and is enhanced by mechanical loading events. However, little is known about the pressure gradients experienced by bone cells in vivo and the role of the induced IFF in bone adaptation. This study investigated IFF changes in bone, in a disuse model and in ambulatory mice, from pressure gradients measured by telemetry, and by fluorescent tracers. The role of IFF-mediated transport of oxygen was assessed by the levels of hypoxic osteocytes in mouse femur after disuse by hindlimb suspension and with or without femoral vein ligation. Femoral intramedullary pressures in alert mice decreased to 77% upon hindlimb suspension and increased by 25% upon ligation, relative to baseline. To determine relative perfusion of cortical bone by IFF, the localization of intracardiac-injected fluorescent albumin conjugate with osteocytes was monitored. The number of osteocytic lacunae per bone area positive for Texas Red albumin was increased by 31% within 20-40 s, in the ligated femur compared to the contralateral sham femur. This confirmed that interstitial fluid flow was increased by femoral vein ligation and indicated that the increase was proportional to the pressure increase. Unloaded bone osteocytes were not hypoxic when compared to loaded controls and venous ligation did not alter these levels significantly. These results support the hypothesis that disuse by hindlimb suspension leads to decreased pressure gradients, which indicate lower IFF. Similarly, the increased pressure gradients, seen upon venous ligation, increased IFF from marrow to periosteum. While a decrease in intramedullary pressure in disuse suggests a decrease in IFF, this did not lead to hypoxia in osteocytes. We conclude that decreased oxygen convective transport in the mouse hindlimb disuse model does not account for cortical bone loss. This study is important in increasing our understanding of the mechanotransductory pathways involved in bone loading and unloading.

COX-2 is necessary for venous ligation-mediated bone adaptation in mice.

In osteoblasts, cyclooxygenase 2 (COX-2) is the major isozyme responsible for production of prostaglandins. Prostaglandins are local mediators of bone resorption and formation and are known to be involved in bone's adaptive response to fluid shear stress (FSS). We have previously described a model of trabecular bone loss in hindlimb-suspended mice and rats and demonstrated partial protection from osteopenia by ligation of the femoral vein. The increased FSS resulting from this ligation drove bone adaptation in the absence of mechanical loading. In this study, we investigated the role of COX-2 in this adaptive response to FSS by use of COX-2 knockout mice. COX-2 knockout ("KO"), COX-2 heterozygote ("HET"), and COX-2 wild-type ("WT") animals all lost comparable amounts of trabecular bone from sham-operated limbs as a result of suspension. In WT mice, loss of trabecular BMD in the venous-ligated limb was significantly less than that of the sham-operated limb; this effect, however, was not seen in KO or HET mice. Percentage gain in femoral periosteal circumference was greater in the ligated limb than the sham-operated limb for WT mice. KO and HET mice already possess femora of larger periosteal circumference than their WT littermates and ligation in these bones did not result in an increase in perimeter relative to sham. Histomorphometry on embedded bones revealed thinner cortices and less mineralizing perimeter in KO femora than controls. In conclusion, this is the first in vivo study to show that fluid-flow-mediated bone adaptation, independent of mechanical strain, is COX-2 dependent.

PECAM-1 interacts with nitric oxide synthase in human endothelial cells: implication for flow-induced nitric oxide synthase activation.

OBJECTIVE: We have previously shown that fluid shear stress (FSS) triggers endothelial nitric oxide synthase (eNOS) activity in endothelial cells and that the mechanotransduction mechanisms responsible for activation discriminate between rapid changes in FSS and FSS per se. We hypothesized that the particular sublocalization of eNOS at the cell-cell junction would render it responsive to activation by FSS temporal gradients. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), immunofluorescence revealed strong eNOS membrane staining at the cell-cell junction colocalizing with platelet/endothelial cell adhesion molecule-1 (PECAM-1). In PECAM-1-/- mouse aorta, eNOS junctional localization seen in the wild type was absent. Similarly, junctional staining was lost in wild-type aorta near intercostal artery branches. eNOS/PECAM-1 association in HUVECs was confirmed by coimmunoprecipitation. When HUVECs were subjected to a 0.5s impulse of 12 dynes/cm2, a transient disruption of the eNOS/PECAM-1 complex was observed, accompanied by an increase in eNOS activity (cGMP production). Ramped flow did not trigger complex dissociation or an increase in cGMP production. In a cell-free system, a direct inhibition of eNOS activity by PECAM-1 is shown. CONCLUSIONS: These results suggest that eNOS is complexed with PECAM-1 at the cell-cell junction and is likely involved in the modulation of eNOS activity by FSS temporal gradients but not by FSS itself.

Venous ligation-mediated bone adaptation is NOS 3 dependent.

Interstitial fluid flow (IFF) in bone has been hypothesized to mediate bone modeling in the absence of mechanical strain. The mechanism of this effect has not been clearly defined, though previous studies indicate that nitric oxide (NO) may play an important role in mediating IFF. In the current study, mice with a targeted disruption of the NOS 3 gene were used according to a previously established model of altered interstitial fluid flow in bone. Femoral vein ligation was performed in one limb to increase intramedullary pressure and consequently its IFF; a sham operation was performed on the contralateral limb. The mice were then hindlimb suspended to uncouple the effects of altered flow in the limb from mechanical loading. Differences in radiographic bone density and bone strength were compared for the sham and venous-ligated femurs in wild-type (WT) mice and NOS 3 knockout (KO) mice. Suspension-induced bone loss in the femurs, as evidenced by a loss in radiographic bone mineral density (BMD), was seen in both groups. Differences between sham and venous-ligated femurs were significant only for the WT mice, in which there appeared to be a protective effect of venous ligation against bone loss [-6.69% (ligated) vs. -12.36% (sham), P<0.05]. Furthermore, the difference in bone density between sham and venous-ligated femurs was muted by NOS 3 knockout, suggesting that the protective effect of venous ligation against bone loss observed in the WT group was NO dependent. The differences in relative BMD were mirrored in the mechanical testing experiments, where maximum load to fracture was significantly higher in the venous-ligated limbs relative to the sham limbs of the WT mice, but not in the NOS 3 group. Taken together, these data further support the hypothesis that fluid flow can modulate bone modeling and suggest that IFF-mediated bone adaptation is NOS 3 dependent.

The effect of in vivo mechanical loading on estrogen receptor alpha expression in rat ulnar osteocytes.

The presence of estrogen receptor alpha (ER alpha) in osteocytes was identified immunocytochemically in transverse sections from 560 to 860 microm distal to the midshaft of normal neonatal and adult male and female rat ulnas (n = 3 of each) and from adult male rat ulnas that had been exposed to 10 days of in vivo daily 10-minute periods of cyclic loading producing peak strains of either -3000 (n = 3) or -4000 microstrain (n = 5). Each animal ambulated normally between loading periods, and its contralateral ulna was used as a control. In animals in which limbs were subject to normal locomotor loading alone, 14 +/- 1.2% SEM of all osteocytes in each bone section were ER alpha positive. There was no influence of either gender (p = 0.725) or age (p = 0.577) and no interaction between them (p = 0.658). In bones in which normal locomotion was supplemented by short periods of artificial loading, fewer osteocytes expressed ER alpha (7.5 +/- 0.91% SEM) than in contralateral control limbs, which received locomotor loading alone (14 +/- 1.68% SEM; p = 0.01; median difference, 6.43; 95% CI, 2.60, 10.25). The distribution of osteocytes expressing ER alpha was uniform across all sections and thus did not reflect local peak strain magnitude. This suggests that osteocytes respond to strain as a population, rather than as individual strain-responsive cells. These data are consistent with the hypothesis that ER alpha is involved in bone cells' responses to mechanical strain. High strains appear to decrease ER alpha expression. In osteoporotic bone, the high strains assumed to accompany postmenopausal bone loss may reduce ER alpha levels and therefore impair the capacity for appropriate adaptive remodeling.

New fluorescent probes for the measurement of cell membrane viscosity.

BACKGROUND: Molecular rotors are fluorescent molecules that exhibit viscosity-dependent fluorescence quantum yield, potentially allowing direct measurements of cell membrane viscosity in cultured cells. Commercially available rotors, however, stain not only the cell membrane, but also bind to tubulin and migrate into the cytoplasm. We synthesized molecules related to 9-(dicyanovinyl)-julolidine (DCVJ), which featured hydrocarbon chains of different length to increase membrane compatibility. RESULTS: Longer hydrocarbon chains attached to the fluorescent rotor reduce the migration of the dye into the cytoplasm and internal compartments of the cell. The amplitude of the fluorescence response to fluid shear stress, known to decrease membrane viscosity, is significantly higher than the response obtained from DCVJ. Notably a farnesyl chain showed a more than 20-fold amplitude over DCVJ and allowed detection of membrane viscosity changes at markedly lower shear stresses. CONCLUSIONS: The modification of molecular rotors towards increased cell membrane association provides a new research tool for membrane viscosity measurements. The use of these rotors complements established methods such as fluorescence recovery after photobleaching with its limited spatial and temporal resolution and fluorescence anisotropy, which has low sensitivity and may be subject to other effects such as deformation.

Bcl-2, tissue transglutaminase and p53 protein expression in the apoptotic cascade in ribs of premature infants.

Apoptotic cells of the human growth plate have not previously been demonstrated in situ. We have investigated the distribution of apoptotic cells in costosternal growth plates and bone of premature infants aged 4-11 d with a gestational age of approximately 26 wk. In addition, we investigated the immunolocalisation of apoptosis-related proteins within the growth plates and associated bone. A proportion of late hypertrophic chondrocytes and osteocytes within newly formed primary spongiosa showed evidence of highly fragmented DNA. The incidence of osteocyte apoptosis decreased as the distance from the chondroosseous junction increased. Tissue transglutaminase (tTG) expression was associated with apoptosis of osteocytes and hypertrophic chondrocytes. In contrast the presence of tTG was demonstrated in osteoblasts and bone lining cells but it did not colocalise with evidence of apoptosis. The anti-apoptotic gene product Bcl-2 was absent from the growth plate but was present in osteocytes. Visual assessment indicated a greater occurrence of the protein in cells occupying regions of low apoptosis. P53 was not demonstrated in the growth plate or bone. These findings would indicate that human growth plate chondrocytes appear to show little provision for ensuring cell longevity. In contrast osteocyte apoptosis appears negatively correlated with the skeletal distribution of Bcl-2 protein in the human infant, implying a potential selective vulnerability in individual cells. Lack of Bcl-2 and the high incidence of osteocyte apoptosis in the more rapidly remodelling bone of the human infant suggest a potential role of osteocyte apoptosis in the remodelling process.