Jin Jia Hu

Time Courses of Growth and Remodeling of Porcine Aortic Media During Hypertension: A Quantitative Immunohistochemical Examination

Arteries undergo marked structural and functional changes in human and experimental hypertension that generally involve smooth muscle cell (SMC) hypertrophy/hyperplasia as well as abnormal extracellular matrix turnover. In this study we examined time courses of changes in SMC activity and matrix protein content in a novel mini-pig aortic coarctation model. Cell proliferation was evaluated by immunostaining of Ki-67, apoptosis was assessed by TUNEL, and phenotypic changes were monitored by immunostaining three SMC contractile markers (caldesmon, calponin, and smoothelin). Changes in medial collagen and elastin were examined by picrosirius red and Verhoeff–van Gieson staining, respectively. LabVIEW-based image analysis routines were developed to objectively and efficiently quantify the (immuno)histochemical results. We found that significant cell proliferation and matrix production occurred in the early stages of this coarctation model and then declined gradually; the SMCs also tended to exhibit a less contractile phenotype following these cellular and extracellular changes. Specifically, different aspects of the phenotypic changes associated with hypertension occurred at different rates: cell proliferation and collagen production occurred early and peaked by 2 weeks, whereas changes in contractile protein expression continued to decrease over the entire 8-week study period. Temporal changes found in this study emphasize the importance of simultaneously tracing time courses of SMC growth and differentiation as well as matrix protein production and content. SMCs are multifunctional, and caution must be used to not overdefine phenotype. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Characterization of Engineered Tissue Development Under Biaxial Stretch Using Nonlinear Optical Microscopy

Little is known about the precise mechanical stimuli that cells sense and respond to as they maintain or refashion the extracellular matrix in multiaxially loaded native or bioengineered tissues. Such information would benefit many areas of research involving soft tissues, including tissue morphogenesis, wound healing, and tissue engineering. A custom tissue culture device has been constructed that can impart well-defined biaxial stretches on cruciform-shaped, fibroblast-seeded collagen gels and be mounted on the stage of a nonlinear optical microscopy (NLOM) system for microscopic characterization of matrix organization. The cruciform geometry permitted direct comparison of matrix (re-)organization within regions of the collagen gel exposed to either uniaxial or biaxial boundary conditions and examination by NLOM for up to 6 days. In addition, sequential NLOM measurements of collagen fiber orientations within gels while stretched, unloaded, or decellularized delineated contributions of applied stretches, cell-mediated tractions, and matrix remodeling on the measured distributions. The integration of intravital NLOM with novel bioreactors enables visualization of dynamic tissue properties in culture.

Construction and characterization of an electrospun tubular scaffold for small-diameter tissue-engineered vascular grafts: a scaffold membrane approach.

Based on a postulate that the microstructure of a scaffold can influence that of the resulting tissue and hence its mechanical behavior, we fabricated a small-diameter tubular scaffold (∼3 mm inner diameter) that has a microstructure similar to the arterial media using a scaffold membrane approach. Scaffold membranes that contain randomly oriented, moderately aligned, or highly aligned fibers were fabricated by collecting electrospun poly([epsilon]-caprolactone) fibers on a grounded rotating drum at three different drum rotation speeds (250, 1000, and 1500 rpm). Membranes of each type were wrapped around a small-diameter mandrel to form the tubular scaffolds. Particularly, the tubular scaffolds with three different off-axis fiber angles (30, 45, and 60 degree) were formed using membranes that contain aligned fibers. These scaffolds were subjected to biaxial mechanical testing to examine the effects of fiber directions as well as the distribution of fiber orientations on their mechanical properties. The circumferential elastic modulus of the tubular scaffold was closely related to the fiber directions; the larger the off-axis fiber angle the greater the circumferential elastic modulus. The distribution of fiber orientations, on the other hand, manifested itself in the mechanical behavior via the Poisson effect. Similar to cell sheet-based vascular tissue engineering, tubular cell-seeded constructs were prepared by wrapping cell-seeded scaffold membranes, alleviating the difficulty associated with cell seeding in electrospun scaffolds. Histology of the construct illustrated that cells were aligned to the fiber directions in the construct, demonstrating the potential to control the microstructure of tissue-engineered vascular grafts using the electrospun scaffold membrane.

A Comparison of Epithelial Cells, Fibroblasts, and Osteoblasts in Dental Implant Titanium Topographies

The major challenge for dental implants is achieving optimal esthetic appearance and a concept to fulfill this criterion is evaluated. The key to an esthetically pleasing appearance lies in the properly manage the soft tissue profile around dental implants. A novel implant restoration technique on the surface was proposed as a way to augment both soft- and hard-tissue profiles at potential implant sites. Different levels of roughness can be attained by sandblasting and acid etching, and a tetracalcium phosphate was used to supply the ions. In particular, the early stage attaching and repopulating abilities of bone cell osteoblasts (MC3T3-E1), fibroblasts (NIH 3T3), and epithelial cells (XB-2) were evaluated. The results showed that XB-2 cell adhesive qualities of a smooth surface were better than those of the roughened surfaces, the proliferative properties were reversed. The effects of roughness on the characteristics of 3T3 cells were opposite to the result for XB-2 cells. E1 proliferative ability did not differ with any statistical significance. These results suggest that a rougher surface which provided calcium and phosphate ions have the ability to enhance the proliferation of osteoblast and the inhibition of fibroblast growth that enhance implant success ratios.

Transforming growth factor-β signaling in hypertensive remodeling of porcine aorta

A porcine aortic coarctation model was used to examine regulation of gene expression in early hypertensive vascular remodeling. Aortic segments were collected proximal (high pressure) and distal (low pressure) to the coarctation after 2 wk of sustained hypertension (mean arterial pressure>150 mmHg). Porcine 10K oligoarrays used for gene expression profiling of the two regions of aorta revealed downregulation of cytoskeletal and upregulation of extracellular region genes relative to the whole genome. A genomic database search for transforming growth factor-beta (TGF-beta) control elements showed that 19% of the genes that changed expression due to hypertension contained putative TGF-beta control elements. Real-time RT-PCR and microarray analysis showed no change in expression of TGF-beta1, TGF-beta2, TGF-beta3, or bone morphogenetic proteins-2 and -4, yet immunohistochemical staining for phosphorylated SMAD2, an indicator of TGF-beta signaling, and for phosphorylated SMAD1/5/8, an indicator of signaling through the bone morphogenetic proteins, showed the highest percentage of positively stained cells in the proximal aortic segments of occluded animals. For TGF-beta signaling, this increase was significantly different than for sham-operated controls. Western blot analysis showed no difference in total TGF-beta1 protein levels with respect to treatment or aortic segment. Immunohistochemistry showed that the protein levels of latency-associated peptide was decreased in proximal segments of occluded animals. Collectively, these results suggest that activation of TGF-beta, but not altered expression, may be a major mechanism regulating early hypertensive vascular remodeling.

Building a functional artery: issues from the perspective of mechanics.

Despite the many successes of arterial tissue engineering, clinically viable implants may be a decade or more away. Fortunately, there is much more that we can learn from native vessels with regard to designing for optimal structure, function, and properties. Herein, we examine recent observations in vascular biology from the perspective of nonlinear mechanics. Moreover, we use a constrained mixture model to study potential contributions of individual wall constituents. In both cases, the unique biological and mechanical roles of elastin come to the forefront, especially its role in generating and modulating residual stress within the wall, which appears to be key to multiple growth and remodeling responses.

Property evaluation of Bletilla striata /polyvinyl alcohol nano fibers and composite dressings

This study used nonwoven manufacture and electrospinning to create wound dressings with solid mechanical properties and hemostasis function. 10% Polyvinyl alcohol (PVA) and 5% Bletilla striata (BS) were blended into the PVA/BS solution, which can be made into nanomaterial with high specific surface area by electrospinning. The PVA/BS solution was electrospun onto the dressing matrix made of polyester (PET) and absorbent cotton (AC), forming the PVA/BS composite dressings. According to the experiment results, when the volume ratio of PVA to BS was 9 : 1, the resulting dressings had optimal fiber formation, the finest average diameter, and the lowest toxicity.

Differential progressive remodeling of coronary and cerebral arteries and arterioles in an aortic coarctation model of hypertension

Objectives: Effects of hypertension on arteries and arterioles often manifest first as a thickened wall, with associated changes in passive material properties (e.g., stiffness) or function (e.g., cellular phenotype, synthesis and removal rates, and vasomotor responsiveness). Less is known, however, regarding the relative evolution of such changes in vessels from different vascular beds. Methods: We used an aortic coarctation model of hypertension in the mini-pig to elucidate spatiotemporal changes in geometry and wall composition (including layer-specific thicknesses as well as presence of collagen, elastin, smooth muscle, endothelial, macrophage, and hematopoietic cells) in three different arterial beds, specifically aortic, cerebral, and coronary, and vasodilator function in two different arteriolar beds, the cerebral and coronary. Results: Marked geometric and structural changes occurred in the thoracic aorta and left anterior descending coronary artery within 2 weeks of the establishment of hypertension and continued to increase over the 8-week study period. In contrast, no significant changes were observed in the middle cerebral arteries from the same animals. Consistent with these differential findings at the arterial level, we also found a diminished nitric oxide-mediated dilation to adenosine at 8 weeks of hypertension in coronary arterioles, but not cerebral arterioles. Conclusion: These findings, coupled with the observation that temporal changes in wall constituents and the presence of macrophages differed significantly between the thoracic aorta and coronary arteries, confirm a strong differential progressive remodeling within different vascular beds. Taken together, these results suggest a spatiotemporal progression of vascular remodeling, beginning first in large elastic arteries and delayed in distal vessels.

Fabrication of poly(glycerol sebacate) fibrous membranes by coaxial electrospinning: Influence of shell and core solutions.

Although poly(glycerol sebacate) (PGS) has enjoyed great success in soft tissue engineering, it remains challenging to fabricate PGS fibers. In this study, coaxial electrospinning, in which polylactide (PLA) was used to confine and draw PGS prepolymer, was used to fabricate PGS fibrous membranes. Specifically, effects of adding poly(ethylene oxide) (PEO), which was removed prior to curing, in the shell were investigated. Transmission and scanning electron microscopy were used to confirm core-shell structure and morphology of fibers, respectively. Both the removal of PEO or PLA in the shell and the efficacy of PGS curing were verified by Fourier transform infrared spectroscopy and differential scanning calorimetry. Mechanical properties of the membranes with different shell and core contents were examined. We found that the addition of PEO to the shell reduced Young׳s modulus of the resulting cured membrane and increased its elongation at break significantly, the latter indicating better PGS curing. Moreover, with the addition of PEO, increasing PGS prepolymer concentration further increased the elongation at break and appeared to enhance the structural integrity of fibers; PGS fibrous membranes (with no PLA shell) were thus successfully fabricated after the removal of PLA. The Young׳s modulus of the PGS fibrous membrane was ~0.47MPa, which is similar to that of PGS solid sheets and some soft tissues. Finally, the cytocompatibility of the electrospun membranes was validated by Alamar blue and LDH assays.

Comparison of three calcium phosphate bone graft substitutes from biomechanical, histological, and crystallographic perspectives using a rat posterolateral lumbar fusion model.

This study evaluated the effectiveness of three calcium phosphate bone graft substitutes with different chemical compositions on spinal fusion using a rat posterolateral lumbar fusion model. Specifically, two recently developed non-dispersive tetracalcium phosphate/dicalcium phosphate anhydrous-based calcium phosphate cements (CPCs), namely a CPC consisting of equimolar amounts of the two compounds (nd-CPC) and a CPC consisting of a two-fold greater amount of dicalcium phosphate anhydrous (DCP-rich CPC), were compared with a commercial calcium phosphate bone graft (c-CPG) consisting of hydroxyapatite (60%) and β-tricalcium phosphate (40%). Single-level posterolateral lumbar fusion was performed at the L4-L5 vertebrae in fifteen adult rats (n=5 for each group). Spinal fusion was evaluated with radiographs, manual palpation, mechanical testing, micro-CT, and histology 8 weeks post-surgery. In particular, the crystallographic phases in the three substitutes were identified before and 8 weeks after their implantation. Manual palpation revealed stable constructs in nearly all of the spine specimens. The stiffness and bending load of fused spines in the two CPC groups were comparable to those in the c-CPG group. The radiographs specifically revealed implant resorption and bone remodeling in the DCP-rich CPC group. Analysis of 3D micro-CT images revealed that the bone volume ratio in the DCP-rich CPC group was significantly greater than those in the nd-CPC and c-CPG groups. Histology showed that the DCP-rich CPC group exhibited the highest degree of bone regeneration and osseointegration. Notably, DCP-rich CPC led to a pronounced phase transformation, generating the greatest amount of poorly crystalline apatite among the three groups, which together with adequate resorption may explain the aforementioned positive findings. We therefore conclude that of the bone graft substitutes considered, DCP-rich CPC has the greatest potential to be used in spinal fusion.