Experimental microvascular polytetrafluoroethylene grafts: 6-month patency

The efficacy of 1-mm internal-diameter polytetrafluoroethylene as a microvascular prosthesis is unclear. In this study, 8-mm-long segments of this material were implanted into the femoral arteries of 30 rats. Animals were examined every 2 weeks up to 6 months by Doppler ultrasound. Cumulative patency by the life-table method was 86.7 percent at 6 months. There were 26 patent grafts, 2 occlusions, and 2 deaths. Intimal hyperplasia adjacent to the anastomoses was seen in the native arteries. The pseudointima lining the grafts was cellular near the anastomoses but usually acellular in midgraft regions. It is concluded that if early failure does not occur, then good long-term patency is possible with 1-mm polytetrafluoroethylene in this setting and that patency is not dependent on development of a cellular pseudointima. Longer graft segments should be evaluated in future studies.     

Solid-state NMR as a probe of amyloid fibril structure

Amyloid fibrils are intrinsically noncrystalline, insoluble, high-molecular-weight aggregates of peptides and proteins, with considerable biomedical and biophysical significance. Solid-state NMR techniques are uniquely capable of providing high-resolution, site-specific structural constraints for amyloid fibrils, at the level of specific interatomic distances and torsion angles. So far, a relatively small number of solid-state NMR studies of amyloid fibrils have been reported. These have addressed issues about the supramolecular organization of beta-sheets in the fibrils and the peptide conformation in the fibrils, and have concentrated on the beta-amyloid peptide of Alzheimer's disease. Many additional applications of solid-state NMR to amyloid fibrils from a variety of sources are anticipated in the near future, as these systems are ideally suited for the technique and are of widespread current interest.     

Porous hydroxyapatite as a bone graft substitute in cranial reconstruction: a histometric study

To assess the potential of a porous hydroxyapatite matrix to serve as a bone graft substitute, bilateral 15 X 20 mm craniectomy defects were reconstructed in 17 dogs with blocks of implant and split-rib autografts. Specimens were retrieved at 3, 6, 12, 24, and 48 months, and undecalcified sections were prepared for microscopy and histometry. The implant and graft cross-sectional areas did not change with time, documenting their equivalent ability to maintain cranial contour. Bone ingrowth extended across the implant from one cranial shelf to the other in 15 specimens. Little apparent bone ingrowth was seen in most graft specimens. Two implants and three grafts were nonunited, possibly due to lack of fixation or the orientation of the histology sections. The implant specimens were composed of 39.3 percent hydroxyapatite matrix, 17.2 percent bone ingrowth, and 43.5 percent soft-tissue ingrowth. The graft specimens were composed of 43.7 percent bone and 56.3 percent soft tissue. This study supported the thesis that a porous hydroxyapatite matrix may function in part as a bone graft substitute. The brittle hydroxyapatite matrix undoubtedly became stronger with bone ingrowth, but the degree of cranial protection achieved was not measured in this study. The size of the cranial defect used in this study did not permit estimation of the distance over which bone ingrowth may be reliably expected. There remains a need for greater understanding of the causes of nonunion, the extent of predictable ingrowth depth, and the strength of the resultant implant-bone composite.     

Roles of chymase in stenosis occurring after polytetrafluoroethylene graft implantations

Chymase is an important enzyme for the generation of angiotensin (Ang) II and in the activation of transforming growth factor (TGF)-beta1. Therefore, chymase may be involved in the hemodialysis access dysfunction, which is caused by intimal hyperplasia that occurs after polytetrafluoroethylene (PTFE) graft implantations. Bilateral U-shaped PTFE grafts were placed between the femoral vein and artery in dogs. Chymase inhibitor (NK3201, 1 mg/kg per day, p.o.) treatments were initiated 3 days before the operation. After the implantation, the stenosis by neointima proliferation was most frequently observed in the venous side of the PTFE grafts. In the hyperplastic neointima, myofibroblasts were the main cellular components. On the other hand, fibroblasts only occupied cellular components in a much smaller proportion in the neointima. However, these cells seem to be rich in the properties of proliferation and migration. After PTFE graft implantations, extensive accumulations of chymase-positive mast cells were found mainly in the tissue surrounding the grafts. The Ang II- and TGF-beta-positive cells were found in an adjacent section that was in close proximity to the chymase-positive cells. In contrast, the AT(1) receptors, as well as TGF-beta type II receptors, were expressed either in the neointima or in the outside adventitia of the PTFE grafts. Chymase inhibitor treatment resulted in a reduction of chymase, Ang II and TGF-beta1 expression, leading to a significant inhibition of neointimal formation. These findings indicating that an increase of chymase via promoting Ang II and TGF-beta1 generation plays a pivotal role in the neointimal formation after the implantation of PTFE grafts and also suggesting that chymase inhibition may be a new strategy that can be used to prevent PTFE graft dysfunctions in clinical settings.     

Free microvascular muscle flaps with skin graft reconstruction of extensive defects of the foot: a clinical and gait analysis study

Over the past 4 years at the Massachusetts General Hospital 18 patients have been treated for extensive defects (mean size 130 cm2) of the foot at or below the medial and lateral malleoli. These patients have been treated with free muscle flaps covered with thick split-thickness skin grafts. Full muscle flap survival has been seen in each patient, and all patients are currently ambulatory. A subgroup of nine patients are weight-bearing directly upon their skin grafts covering transferred muscle. All patients are walking without chronic breakdown over a mean follow-up of over 19 months with the exception of a single patient who has had breakdown in a region of redundant improperly tailored muscle flap. None of the skin grafted muscles has significant cutaneous sensibility. Detailed gait analysis of these patients has confirmed the weight-bearing capabilities of free muscle flaps with skin grafts and has proven to be an excellent method of foot reconstruction evaluation. It would appear from this study that cutaneous sensibility may not be necessary for successful reconstruction of the weight-bearing surface of the foot. This method of reconstruction should be considered when local tissues are not suitable for plantar foot reconstruction.     

Inducible overexpression of endothelial proNGF as a mouse model to study microvascular dysfunction

Impaired maturation of nerve growth factor precursor (proNGF) and its accumulation has been reported in several neurodegenerative diseases, myocardial infarction and diabetes. To elucidate the direct impact of proNGF accumulation identified the need to create a transgenic model that can express fully mutated cleavage-resistant proNGF. Using Cre-Lox technology, we developed an inducible endothelial-specific proNGF transgenic mouse (proNGF^Loxp) that overexpresses GFP-conjugated cleavage-resistant proNGF123 when crossed with VE-cadherin-CreERT2 (Cre). Expression of proNGF, inflammatory mediators, NGF and VEGF was evaluated by PCR, Western blot and immunohistochemistry. EC-proNGF overexpression was confirmed using colocalization of anti-proNGF within retinal vasculature. EC-proNGF did not cause retinal neurotoxicity or marked glial activation at 4-weeks. Microvascular preparation from Cre-proNGF mice showed significant imbalance of proNGF/NGF ratio, enhanced expression of TNF-α and p75^NTR, and tendency to impair TrkA phosphorylation compared to controls. EC-proNGF overexpression triggered mRNA expression of p75^NTR and inflammatory mediators in both retina and renal cortex compared to controls. EC-proNGF expression induced vascular permeability including breakdown of BRB and albuminuria in the kidney without affecting VEGF level at 4-weeks. Histopathological changes were assessed after 8-weeks and the results showed that EC-proNGF triggered formation of occluded (acellular) capillaries, hall mark of retinal ischemia. EC-proNGF resulted in glomerular enlargement and kidney fibrosis, hall mark of renal dysfunction. We have successfully created an inducible mouse model that can dissect the contribution of autocrine direct action of cleavage-resistant proNGF on systemic microvascular abnormalities in both retina and kidney, major targets for microvascular complication.     

The epidermis as a graft

Laboratory epidermal autotransplantation was performed on the surface of a full-thickness skin defect using mongrel female rats. Epidermal graft represented suction blister roofs, formed as the result of the donor skin site treatment with lowered up to -0.6 kg/cm2 pressure. It contained all epidermal cell layers. Following 1, 7 and 28 days after the transplantation recipient bed sites containing grafted epidermis were excised and histological study war performed. It was demonstrated that epidermal graft received by the method described was able to grow as well as to differentiate on the surface of a full-thickness skin defect.     

Elastic anisotropy of bone lamellae as a function of fibril orientation pattern

In this study, the homogenized anisotropic elastic properties of single bone lamellae are computed using a finite element unit cell method. The resulting stiffness tensor is utilized to calculate indentation moduli for multiple indentation directions in the lamella plane which are then related to nanoindentation experiments. The model accounts for different fibril orientation patterns in the lamellae—the twisted and orthogonal plywood pattern, a 5-sublayer pattern and an X-ray diffraction-based pattern. Three-dimensional sectional views of each pattern facilitate the comparison to transmission electron (TEM) images of real lamella cuts. The model results indicate, that the 5-sublayer- and the X-ray diffraction-based patterns cause the lamellae to have a stiffness maximum between 0° and 45° to the osteon axis. Their in-plane stiffness characteristics are qualitatively matching the experimental findings that report a higher stiffness in the osteon axis than in the circumferential direction. In contrast, lamellae owning the orthogonal or twisted plywood fibril orientation patterns have no preferred stiffness alignment. This work shows that the variety of fibril orientation patterns leads to qualitative and quantitative differences in the lamella elastic mechanical behavior. The study is a step toward a deeper understanding of the structure—mechanical function relationship of bone lamellae.     

Determination of optimal graft lengths for posterior cruciate ligament reconstruction--a theoretical analysis

Various graft materials have been used in posterior cruciate ligament (PCL) reconstruction. However, it is unclear if these grafts can reproduce the structural behavior of the PCL. This paper analyzed the effect of graft length on the structural behavior of the graft using a minimal deformation energy method. An analytical solution was obtained to determine the optimal effective graft length that can best reproduce the structural behavior of the PCL. This optimal graft length was determined as a function of the axial rigidity of the graft. Two typical grafts, bone-patella tendon-bone (BPTB) and Achilles tendon, were analyzed. The data demonstrated that in order to reproduce the PCL behavior, the effective length of a BPTB graft (10 mm width) should be 34 mm, while the Achilles tendon graft (with a cross sectional area of 55 mn2) needs to be 48 mm in length. Longer grafts result in less resistance and shorter graft increased the graft resistance. An initial graft tension cannot help recreate the overall structural behavior of the PCL. These results suggest that graft length is an important surgical variable in PCL reconstruction. An optimal reconstruction of the PCL should reproduce the structural properties of the PCL by using a graft with an optimal length.     

Closest packing of two-stranded coiled-coils as a model for the collagen fibril.

We propose that in the collagen fibril, the triple-helical molecules form two-stranded coiled-coils of period 5 × 670A?. Coiled-coils are packed on a tetragonal lattice and are axially staggered with ten in the unit cell (observed side 55A?) so that it carries the 670A?periodicity of the fibril. When nearest neighbours have opposing supercoil hands, the observed tetragonal lattice represents closest packing of two-stranded coiled-coils. This proposal is consistent with the row line spacings measured from the low angle X-ray diffraction pattern of tendon and explains the systematic absences and the two undisputed equatorial reflections. Unlike explanations for the diffraction pattern which invoke a five-stranded microfibril, our interpretation is consistent with its equatorial intensity distribution.     

Quasielastic light scattering study of amyloid beta-protein fibril formation

Quasielastic light scattering spectroscopy (QLS) is an optical method for the determination of diffusion coefficients of particles in solution. Here we discuss the principles of QLS and explain how the distribution of particle sizes can be reconstructed from the measured correlation function of scattered light. Non-invasive observation of the temporal evolution of particle sizes provides a powerful tool for studying protein assembly. We illustrate practical applications of QLS with examples from studies of fibril formation of the amyloid beta-protein.