Effect of alternative crosslinking methods on the low strain rate viscoelastic properties of bovine pericardial bioprosthetic material

Early failures of bovine pericardial heart valves have been due to leaflet perforation/tearing and calcification. Since glutaraldehyde fixation has been shown to produce marked changes in leaflet mechanics and has been linked to the development of calcification, alternative crosslinking techniques have been suggested as means to overcome these obstacles. We have examined the low strain rate viscoelastic behavior of bovine pericardium: (1) fresh; (2) chemically treated with glutaraldehyde, cyanimide, or polyglycidyl ether; or (3) physically treated by freeze-drying or heat-drying. Shrinkage temperature tests were conducted to assess intrahelical crosslinking. Polyglycidyl ether and glutaraldehyde both produced substantial crosslinking, with the shrinkage temperature rising above 80 degrees C. Mechanical changes were nearly equivalent, both showing decreased stress relaxation and increased extensibility consistent with intrahelical crosslinking and shrinkage during fixation. Cyanimide, known to crosslink pure collagen materials, showed no evidence of crosslinking intact tissue. Heat-drying, also effective in pure collagen preparations, produced an increase in UTS and tissue modulus, but otherwise left the tissue unchanged. Freeze-drying had no mechanical effect, and therefore provides an attractive means for the storage of connective tissues for later mechanical testing.     

Effect of pretreatment with epoxy compounds on the mechanical properties of bovine pericardial bioprosthetic materials.

Early failures of bovine pericardial heart valves are due to leaflet perforation, tearing and calcification. Since glutaraldehyde fixation has been shown to produce marked changes in leaflet mechanics and has been linked to development of calcification, bovine pericardium fixed with the four hydrophilic epoxy formulations and their mechanical properties are studied in this paper. We measured the thicknesses, shrinkage temperatures, stress relaxations and stress-strain curves of bovine pericardiums after different treatments with (1) non-treatment (fresh), (2) glutaraldehyde (GA), (3) epoxy compounds followed by the posttreatment with GA (EP 1#, EP 2#), and (4) epoxy compounds (EP 3# and EP 4#). Results of this study showed that the hydrophilic epoxy compounds are good crosslinking agents. There are no significant differences of shrinkage temperature and ultimate tensile stress among all tissue samples pretreated with GA, EP 1# and EP 2#. However, the stress relaxations of tissue-samples pretreated with epoxy compounds followed by the posttreatment with GA (EP 1# and EP 2#) are significantly slower than that pretreated with GA, and the strains at fracture of EP 1# and EP 2# are also significantly larger than that of GA or epoxy compounds. These facts show that the bovine pericardium pretreated with the epoxy compound followed by the posttreatment with GA (EP 1# and EP 2#) possesses greater tenacity and potential durability in dynamic stress.     

The bovine pericardial xenograft: II. Effect of tethering or pressurization during fixation on the tensile viscoelastic properties of bovine pericardium

Our previous article suggested that control of the extensibility of aldehyde-fixed pericardium could be achieved by controlling shrinkage during fixation. Therefore, to prevent shrinkage, we have used sandpaper-lined plexiglass plates to clamp circular samples of bovine pericardium during fixation in glutaraldehyde, tethering them at their original dimensions. As well, we have applied transmural pressures of 50 or 100 mm Hg during fixation using a hydraulic column of glutaraldehyde solution. Strips cut at 0 degree, 30 degrees, 60 degrees, and 90 degrees to the base-to-apex cardiac direction have been examined for cyclic stress-strain response, stress relaxation, plastic deformation, and fracture behavior. Under physiological stresses, tethered and pressure-fixed materials were both nearly isotropic. Tethering during fixation produced a material with extensibility nearly identical to that of fresh tissue. Plastic deformation during cyclic loading was reduced below that seen in simple fixation while stress relaxation was unchanged. Pressure-fixation produced reduced extensibility similar to that produced in porcine aortic valve leaflets. Plastic deformation and stress relaxation were both markedly reduced. Pressure-fixation reduced the strain at fracture, but fracture behavior was otherwise unaffected. Tethering and pressure-fixation offer attractive means to control the mechanical behavior of bovine xenograft materials.     

The bovine pericardial xenograft: III. Effect of uniaxial and sequential biaxial stress during fixation on the tensile viscoelastic properties of bovine pericardium

Our previous two articles have shown that glutaraldehyde-fixed bovine pericardium is nearly isotropic, whether fixed without constraints, with tethering, or with pressure. In this study, we have used uniaxial stress during fixation to produce bovine pericardial material with marked tensile anisotropy. Rectangular and cruciate pericardial samples have been mechanically examined after one of four treatments: (i) fixation under 88-kPa uniaxial stress, (ii) fixation under 176-kPa uniaxial stress, (iii) 3 h of 176-kPa uniaxial stress in saline followed by 24-h fixation under the same stress, (iv) fixation under 176-kPa uniaxial stress followed by a second fixation under 176-kPa stress in a direction normal to the first. Strips of material were cut at 0 degree, 30 degrees, 60 degrees, and 90 degrees to direction of the initial stress, and tested for response to cyclic loading, stress relaxation, plastic deformation, and fracture properties. Fixation under uniaxial stress produced anistropy similar to that seen in porcine aortic valve leaflets; however, the overall extensibility of the material depended on the applied stress and the aspect ratio of the stressed sample. While loading in saline produced no change, the sequential biaxial stressing produced a reduction in anisotropy, suggesting exposure of additional crosslinking sites. Uniaxial stress during fixation may be a useful method for construction of anisotropic heart valve leaflets.     

Interactions of U937 macrophage-like cells with decellularized pericardial matrix materials: Influence of crosslinking treatment

While macrophages have been implicated in the failure of bioprosthetic heart valves, the macrophage response to crosslinked native pericardial collagen has not been previously investigated. Using decellularized bovine pericardium (DBP) as a model for native collagen, this study investigated the response of macrophage-like cells (U937s) to DBP, either: (i) untreated, or (ii) exogenously crosslinked with glutaraldehyde or 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC). We have previously validated the use of U937 cells as models for the response of human monocyte-derived macrophages to decellularized pericardial materials and, per our previous work, differentiated the U937 cells directly on the three material surfaces. After 72 h in culture, the cells and medium were analyzed for DNA content, acid phosphatase activity, and cytokine and matrix metalloproteinase release. As well, cell/substrate samples were fixed for SEM. Fewer cells attached to or survived on the glutaraldehyde-treated substrate, and some showed an abnormal morphology compared to cells cultured on the other surfaces. Further, cells on glutaraldehyde-treated surfaces released more pro-inflammatory cytokines, more MMP-1 and less MMP-2 and MMP-9. The poor performance of the U937 macrophage-like cells on the glutaraldehyde-treated surfaces appears to be due to surface characteristics rather than to soluble aldehyde or other components leaching from the crosslinked material. These results provide evidence that crosslinking with glutaraldehyde is cytotoxic to macrophage-like cells, and that crosslinking with a zero-length crosslinker like EDC can be an acceptable alternative crosslinking treatment for biomaterials.     

Onchocerca armillata contamination of a bovine pericardial xenograft in a human patient with repaired tetralogy of Fallot

BackgroundBovine pericardial patches are used for many purposes, including facilitating right ventricular outflow tract reconstruction in patients with congenital heart disease. Here we present a case of parasitic contamination of a bovine pericardium used as a transannular patch during repair of tetralogy of Fallot 28 years prior at a hospital in China.MethodsThe patient presented to the University of Washington Medical Center for congestive heart failure and pulmonic regurgitation, and heart tissues including the xenograft pericardial patch were submitted to the Pathology Department and subsequently to the Comparative Pathology Program.ResultsThe pericardial parasitic nodules with intralesional adult nematodes and microfilaria in the bovine tissues were preserved at harvest by (presumed) glutaraldehyde fixation.ConclusionOnchocerca armillata parasitic pericardiopathy was diagnosed in the xenograft tissue based on the characteristic nematode morphology and the presumed geographic location of the donor bovine. This resulted in O. armillata contamination of the pericardial xenograft in a human patient with repaired tetralogy of Fallot.     

The bovine pericardial xenograft: I. Effect of fixation in aldehydes without constraint on the tensile viscoelastic properties of bovine pericardium

Tensile testing of tissue strips has been used to examine the effect of simple fixation in glutaraldehyde and formaldehyde on the viscoelastic properties of bovine pericardium. To assess tissue anisotropy, tissue strips were cut at 0 degree, 30 degrees, 60 degrees, and 90 degrees relative to the base-to-apex direction. Fresh anterior pericardium was modestly anisotropic, being least extensible in the base-to-apex direction; however, fixation removed this anisotropy. Fixation also produced a marked change in the response of the material to initial cyclic loading during preconditioning. Overall extensibility of the fixed material was significantly greater than that for the fresh tissue, consistent with a 10.7% shrinkage in aldehydes calculated from strain at fracture data. Reductions in stress relaxation and creep after fixation were noted as well, consistent with intrafibrillar crosslinking. Cyclic hysteresis and ultimate tensile strength were unaffected. Since the observed changes in the stress-strain response were largely attributable to shrinkage, control of shrinkage by physical means would allow for engineering modification of bovine pericardial mechanics for controlled anisotropy.     

The effect of temperature and glutaraldehyde fixation on the mechanical properties of bovine pericardial tissues

A comparison is made of the elastic response of fresh pericardial tissues at 25, 37, and 42 degrees C, and also of fresh and glutaraldehyde-fixed tissues. Strips of bovine pericardial tissues cut perpendicular to the base-apex axis of the heart were used. An Instron machine was used for uniaxial tensile tests, and the strain-rate used was 666.7% X min-1. No significant differences in tissue mechanical properties were observed for temperature values of 25, 37, and 42 degrees C. However tissues fixed in a glutaraldehyde solution were more extensible than fresh tissues. The elastic responses of tissue preserved for 1 day in glutaraldehyde are not very different from those preserved for up to 10 days.     

Biaxial strain effects on cells from the inner and outer regions of the meniscus

During knee joint loading, the fibrocartilaginous menisci experience significant spatial variations in mechanical stimuli. Meniscus cells also exhibit significant variations in biosynthesis and gene expression depending on their location within the tissue. These metabolic patterns are consistent with a more chondrocytic phenotype for cells located within the avascular inner two-thirds compared with a more fibroblastic phenotype for cells within the vascularized outer periphery. The spatial distribution of cell biosynthesis and gene expression patterns within the meniscus suggest that cells may exhibit intrinsically different responses to mechanical stimuli. The objective of our study was to test for intrinsic differences in the responsiveness of these meniscus cell populations to an equivalent mechanical stimulus. Cellular biosynthesis and gene expression for extracellular matrix proteins in isolated inner and outer meniscus cells in monolayer were quantified following cyclic biaxial stretch. The results demonstrate that inner and outer meniscus cells exhibit significant differences in matrix biosynthesis and gene expression regardless of stretching condition. Both inner and outer meniscus cells responded to stretch with increased nitric oxide production and total protein synthesis. The results suggest that inner and outer meniscus cells may respond similarly to biaxial stretch in vitro with measures of biosynthesis and gene expression.     

Crosslinking characteristics and mechanical properties of a bovine pericardium fixed with a naturally occurring crosslinking agent.

Currently available crosslinking agents used in fixing bioprostheses are all highly (or relatively highly) cytotoxic, which may induce an adverse inflammatory reaction in vivo. It is therefore desirable to provide a crosslinking agent that is of low cytotoxicty and may form stable and biocompatible crosslinked products. To achieve this goal, a naturally occurring crosslinking agent-genipin-was used by our group to fix biological tissues. Genipin may be obtained from its parent compound, geniposide, which may be isolated from the fruits of Gardenia jasminoides Ellis. In our previous studies, it was found that the cytotoxicity of genipin is significantly lower than both glutaraldehyde and an epoxy compound. Also, it was shown that genipin can form stable and biocompatible crosslinked products. The present study further investigates the crosslinking characteristics and mechanical properties of a genipin-fixed bovine pericardium. Fresh and glutaraldehyde- and epoxy-fixed counterparts were used as controls. It was found that the denaturation temperatures of the glutaraldehyde- and genipin-fixed tissues were significantly greater than the epoxy-fixed tissue, although their fixation indices were comparable. The mechanical properties of fresh bovine pericardium are anisotropic. However, fixation tended to eliminate tissue anisotropy. The tendency in the elimination of tissue anisotropy for the genipin-fixed tissue was more remarkable than for the glutaraldehyde- and epoxy-fixed tissues. In addition, the genipin-fixed tissue had the greatest ultimate tensile strength and toughness among all the fixed tissues. Distinct patterns in rupture were observed in the study: The torn collagen fibers of the genipin- and glutaraldehyde-fixed tissues appeared to be bound together, while those of fresh and the epoxy-fixed tissues stayed loose. The results obtained in the study suggests that tissue fixation in glutaraldehyde, epoxy compound, and genipin may produce distinct crosslinking structures. The differences in crosslinking structure may affect the crosslinking characteristics and mechanical properties of the fixed tissues.     

The feasibility of estimating and imaging the mechanical behavior of poroelastic materials using axial strain elastography

In this paper, we have investigated the feasibility of imaging the mechanical behavior of poroelastic materials using axial strain elastography. Cylindrical samples obtained from poroelastic materials having different elastic and permeability properties were subjected to a constant compression force (a classical creep experiment), during which poroelastographic data were acquired. For comparison, we also tested a few gelatin phantoms and non-homogeneous poroelastic phantoms constructed by combining different poroelastic materials. From the acquired data, we generated time-dependent sequences of axial strain elastograms and effective Poisson's ratio elastograms, which were then used for generating axial strain and effective Poisson's ratio time-constant elastograms. Thereafter, the various poroelastographic images were analyzed to evaluate the presence of statistically significant differences among the two types of poroelastic samples and for image quality analysis. The results of this study demonstrate that it is technically feasible to use axial strain elastography to distinguish among homogeneous poroelastic materials characterized by different elastic and permeability properties. They also show that the use of axial strain elastography instead of effective Poisson's ratio elastography results in objectively higher quality poroelastograms of the temporal behavior of the poroelastic materials under loading. However, the use of effective Poisson's ratio elastography may in any case be required to verify that the temporal changes occurring in the axial strains of the homogeneous poroelastic samples are also accompanied by temporal changes of the effective Poisson's ratios and are therefore due to poroelastic behavior.     

Measurement of cancellous bone strain during mechanical tests using a new extensometer device

A device for measuring mid-substance strain in bone during uniaxial compression was developed with special attention to subsequent FE modelling. It is based on dual instrumented cantilever arms that measure the difference in tip-to-tip deflection. The so-called extensometer device was compared to two standard methods of measuring strain based on platen measurements. The extensometer device output was highly linear with tip deflection (r(2)>0.99), and contact of the devices with the specimen was optimized by using an axial distribution of three devices fixed in a free-floating jig. Deflection of the extensometer arms was accurate to 4.8 microm, and precision was between 2-5 microm. Tests included measuring rubber test specimens and cylindrical cancellous bone cores extracted from canine femoral condyles. A trend of decreasing apparent modulus with decreasing strain rate was evident with the extensometer technique. Correlation between the extensometer method and the other two methods was r(2)=0.55. The measure of mid-substance strain avoids non-linearities in the compression tests caused by early failure at the specimen ends, and the uniaxial testing conditions result in boundary conditions that are well suited for subsequent finite element analysis.     

Evidence that deformations which occur during mechanical conditioning of bovine pericardium are not permanent

Natural and glutaraldehyde fixed bovine pericardium samples were mechanically conditioned by a cyclic uniaxial load procedure. The samples tested were controlled for both position and direction in the pericardial sac. The natural tissue demonstrated a significant increase in length and a significant decrease in width after mechanical conditioning. The deformations were not permanent. The test specimens had returned to their original length by 10.5 h after the mechanical test. The control samples of natural tissue showed no significant changes in length during this time. The chemically modified tissue showed a significant increase in length but no significant changes in width or thickness after mechanical conditioning. As in the natural tissue, the length changes were not permanent. Twenty four hours after returning the fixed tissue to its normal buffered glutaraldehyde storage medium the test samples had returned to their original length. A subsequent mechanical retest 8 d after the initial test procedure suggested that the history of the original test had been removed. These observations may be important in the interpretation of 'in vitro' hydrodynamic tests for heart valve substitutes.     

Comparison of novel xenograft (bovine fetal growth plate) and allograft effects on experimental bone defect healing in rabbit

Large bone defects resulting from trauma, tumors, osteitis, implant loosening, or corrective osteotomies require surgical therapy because spontaneous regeneration is limited to relatively small defects. Currently, transplantation of autografts or allografts, mineral bone substitutes, and callus distraction are the most commonly used techniques for skeletal reconstruction. Each method has significant limitations, e.g., availability and biological or biomechanical reasons. This study was designed to evaluate allograft and new xenograft (bovine fetal growth plate) effects on the bone healing process. Twenty male New Zealand White rabbits were used in this study. In the allograft group, the defect was filled by fresh allogeneic cortical graft; in the xenograft group, the defect was filled by a segment of bovine fetal growth plate and was fixed by cerclage wire. Radiological, histopathological, and biomechanical evaluations were performed and results were scored and analyzed statistically. Statistical tests did not support significant differences between the two groups radiographically at the 14th postoperative day (P > 0.05). There was a significant difference in bone formation at the 28th, 42nd, and 56th postoperative days. There were significant radiological differences for bone union and remodeling by the 42nd day postoperatively (P < 0.05). The xenograft was superior to the allograft by the 56th postoperative day for radiological bone formation (P < 0.03); histopathological and biomechanical evaluation revealed no significant differences between the two groups. It can be concluded that the superior bone healing process in the xenograft group was due to the presence of some osteoinduction proteins in bovine fetal growth plate.     

The combined effects of crosslinking and high crystallinity on the microstructural and mechanical properties of ultra high molecular weight polyethylene

Ultra high molecular weight polyethylene (PE) has been used for more than forty years as the bearing surface in total joint replacements. In recent years, there have been numerous advances in processing conditions that have improved the wear resistance of this material. In particular, crosslinking has been shown to dramatically improve the wear behavior of this orthopedic polymer in simulator studies. This benefit to wear resistance, however, is accompanied by a decrease in mechanical properties such as ultimate tensile strength, ductility, toughness and fatigue resistance. This degradation to mechanical properties may have serious implications for devices with high stress concentrations or large cyclic contact stresses. Tailoring microstructure for improved structural performance is essential for implant design. In this work we examined the role of crystallinity and crosslinking on the microstructure and mechanical properties of PE. Crystallinity was increased with a high pressure process and crosslinking was obtained with gamma irradiation. Crystallinity was beneficial to fatigue crack propagation resistance and when coupled with crosslinking a polymer with both wear and fatigue resistance was obtained.     

A study of the effects of glutaraldehyde and formaldehyde on the mechanical behaviour of bovine pericardium

The inherent variability in the mechanical behavior of bovine pericardium causes difficulties in establishing the effects of treatment procedures used in the manufacture of bioprosthetic cardiac valves. A new experimental technique has been developed in which the specimen acts as its own control, obviating the need to perform large numbers of experiments to obtain statistically significant results. The procedure used is described in detail as is the equipment employed. The technique was used to assess the effect of glutaraldehyde and formaldehyde on the stress/strain response of bovine pericardium. The results show that the fixing process in glutaraldehyde is virtually complete within two hours and causes significant changes in the mechanical behaviour of the tissue. The tissue becomes progressively stiffer as the treatment period is extended, which is especially pronounced at low levels of stress. Formaldehyde storage subsequent to fixing in glutaraldehyde was found to have no effect on the stress/strain response of the bovine pericardium.     

Structural proteins of bovine coronavirus strain L9: effects of the host cell and trypsin treatment

Summary The polypeptide profile of the cell-adapted strain of bovine coronavirus (Mebus BCV-L9) is remarkably affected by the host cell and trypsin. We compared the structural proteins of virus purified from different cell lines and found cell-dependent differences in the virus structure. BCV was purified from four clones of human rectal tumour cells (HRT-18): 3 F3, D 2, 3 E 3, and 4 B 3. The structural profiles of BCV propagated in clones 3 E 3 and 3 F 3 were identical, consisting of proteins with molecular weights of 185, 160, 140, 125, 110, 100, 52, 46, 37, 31–34, and 26–28 kilodaltons (kd). BCV purified from clone D2 lacked the 100 kd species, and clone 4 B 3 yielded virus lacking the 46 kd protein. We compared the structures of BCV propagated in HRT-18 cells [BCV(HRT-18)] and virus raised in bovine fetal spleen cells [BCV(D 2 BFS)]. The concentration of the 185 kd protein was higher in BCV (D 2BFS), and it also contained a 200 kd species. Protein profiles of in vitro trypsin treated and untreated BCV(HRT-18) differed only under reducing conditions, suggesting that trypsin cleavage sites are located within disulfide-linked regions of affected proteins. Propagation of BCV in D 2 BFS cells in the presence of trypsin resulted in cleavage of the 185 kd protein and a concommitant increase of the 100 kd protein. Activation of the fusion function probably depends on this cleavage process because fusion of BCV-infected D 2 BFS cells is trypsin dependent.     

Innate immune responses of calves during transient infection with a noncytopathic strain of bovine viral diarrhea virus

In this study, six immunocompetent calves were experimentally infected with a noncytopathic strain of bovine viral diarrhea virus (BVDV), and the effects of the viral infection on parameters of the innate immune response of the host were analyzed. Clinical and virological data were compared with the temporal activation of the alpha/beta interferon-regulated Mx gene in white blood cells (WBC) and skin as well as the upregulation of the acute-phase serum proteins haptoglobin (Hp) and serum amyloid A (SAA). The viral strain used did provoke transient health impairment, namely, fever and leukopenia that were associated with viremia, viral shedding with nasal secretions, and antiviral seroconversion. Complete recovery was observed within 3 weeks. Elevated levels of SAA and Hp were apparent from days 4 to 13 and 8 to 11, respectively. In WBC, the levels of Mx mRNA and Mx protein were elevated from days 2 to 15. In the context of this study with BVDV, the level of Mx protein expression in WBC provided the most telling diagnostic window to monitor the host's ongoing innate immune response.     

Influence of mechanical and metabolic strain on the oxygen consumption slow component during forward pulled running

The possible influence of increased eccentric mechanical work on the increase in oxygen uptake (O₂) after 3 min of running (O₂) was investigated through forward pulled running. Ten subjects ran at individually predetermined constant velocity on a treadmill, while being pulled forward. Ground reaction forces, expired gas and EMGs from leg muscles were collected after 3 min and at the end of the run. O₂ and mechanical work were then calculated. The amplitude of O₂ was 138 (139) ml·min^–1 [mean (SD)]. Increased ventilation explained only 8% of O₂. Stride frequency slightly decreased, inducing a similar decrease in internal work and total mechanical work (all P<0.01), while integrated EMG showed no modifications. It was concluded that O₂ does not come from either an increase in mechanical work production or an increase in muscular activity. O₂ could come from a lower muscle efficiency that could be due to a modification of fibre type recruitment.     

Tissue engineering of bone: effects of mechanical strain on osteoblastic cells in type I collagen matrices

The aim of the present study was to investigate the effect of cyclic uniaxial mechanical strain on a human osteoblastic precursor cell line (hFOB 1.19) in three-dimensional type I collagen matrices. Cell seeded collagen constructs were mechanically stretched by a daily application of cyclic uniaxial strain using a special motor-driven apparatus and compared to unstretched controls. Expression of genes involved in cell proliferation and osteoblastic differentiation as well as matrix production were investigated by analyzing the mRNA of histone H4, core binding factor 1, alkaline phosphatase, osteopontin, osteocalcin, and collagen type I (Col I) up to a cultivation period of 3 weeks using real-time PCR. Cyclic stretching of cell seeded Col I matrices at a magnitude occurring in healing bone increased cell proliferation and slightly elevated the expression of nearly all investigated genes over unstrained controls at various time points. It was concluded that mechanical load promotes the proliferation and differentiation of osteoblastic precursor cells in a Col I matrix and that the application of mechanical stimuli may have a beneficial effect on in vitro tissue formation.