1082 Full-thickness cartilage loss in the knee is a difficult clinical problem, especially in young patients. Defects involving loss of subchondral bone are particularly challenging to treat in this population. Osteochondral allograft transplantation is one option currently used to address these lesions (Figure 1). This study focuses on the clinical, radiographic, and histologic outcome of large osteoarticular allografts stored 4 to 6 weeks. Osteochondral allograft transplantation should be considered in the context of a comprehensive treatment algorithm for addressing chondral damage, as well as con-comitant lesions, in the injured knee. Treatment of the Background: Fresh osteoarticular allograft transplantation has a long history of clinical success. These grafts have typically been implanted less than 1 week from donor asystole.

Background: Fresh osteoarticular allograft transplantation has a long history of clinical success. These grafts have typically been implanted less than 1 week from donor asystole.Hypothesis: Osteoarticular allografts stored 4 to 6 weeks represent a viable alternative to treat full-thickness cartilage and osteochondral defects of the distal femur as measured by clinical, histologic, and magnetic resonance imaging (MRI) criteria.Study Design: Case series' Level of evidence, 4.Methods: Osteoarticular allografts were implanted after a mean graft storage time (at 4 degrees C) of 36 days (range, 28-43). Sixty-seven patients received massive hypothermically stored osteoarticular allografts. Ten knees in 8 of these patients underwent second-look arthroscopic evaluation and biopsy at a mean of 40 months (range, 23-60) after implantation. Clinical assessment was performed using multiple outcome measures and sequential MRI evaluations. Biopsy specimens were obtained from the graft as well as from native articular cartilage at the time of second-look arthroscopy for histologic analysis.Results: The mean International Knee Documentation Committee scores were as follows: preoperative, 27 (range, 9-55); postoperative, 79 (range, 56-99); P = .002. The mean Lysholm scores were as follows: preoperative, 37 (range, 12-47); postoperative, 78 (range, 55-90); P = .002. The mean Short Form-36 physical scores were as follows: preoperative, 38 (range, 24-55); postoperative, 51 (range, 39-61); P = .002. The mean Tegner scores were as follows: preoperative, 4.3 (range, 1-9); postoperative, 5.3 (range, 4-7); P = .16. The mean International Cartilage Repair Society score at follow-up was 10 (nearly normal) (range, 7-11). The mean modified Outerbridge scores were as follows: preoperative, 4.3 (range, 3-5); postoperative, 0.6 (range, 0-1); P = .002. The mean graft and native cartilage cellular density and viability were not statistically different.Conclusions: Fresh-stored osteoarticular grafts for full-thickness articular surface defects of the distal femur appear to offer a viable biological method to restore knee function. Our study suggests that osteoarticular grafts stored in cell culture medium at 4 degrees C for 4 to 6 weeks provide successful short-term clinical outcomes.

Historically, immune-mediated degradation and subsequent aneurysm formation have limited the usefulness of cryopreserved arterial allografts. This study tested the hypothesis that modern cryopreserved arterial allografts are protected from immune-mediated dilation. Abdominal aortas were harvested from anesthetized rats (Lewis and Brown-Norway) for immediate implantation or cryopreservation. Subsequently, Lewis rats underwent infrarenal aortic replacement with either an acutely harvested or a cryopreserved graft. There were four experimental groups: (1) acutely harvested isografts (Iso; n = 6), (2) cryopreserved isografts (C-Iso; n = 6), (3) cryopreserved allografts (C-Allo; n = 6), and (4) acutely harvested allografts (Allo; n = 6). All grafts were explanted at 8 weeks. A video camera and edge detection software were used to measure systolic and diastolic in vivo graft diameter (d). Measurement of arterial blood pressure (p) allowed calculation of compliance (Dd/Dp). Tail-cuff plethysmography was used to assess graft patency at 1 week. Graft diameter and blood pressure measurements were repeated at harvest. All harvested grafts were examined histologically. Our results showed that cryopreservation prevented immune-mediated dilation in arterial allografts in our 8-week rat implant model. Furthermore, the compliance of the cryopreserved grafts and was similar to that of controls. Further investigation is needed to delineate the exact mechanism of these potential cliniclly significant findings.

Background. Variable performance of allograft tissues in children and some adults may be linked to an immune response and could be mitigated by reducing implant antigenicity. Methods. As endothelial and fibroblast cells are the likely source of valve antigenicity, human (CryoValve SG) and sheep pulmonary valves were decellularized using the SynerGraft treatment process. Treated valves were evaluated in vitro using histochemical, biomechanical, and hydrodynamic methods, and compared with standard cryopreserved valves. Four SynerGraft-treated and two cryopreserved sheep pulmonary valves were implanted as root replacements in the right ventricular outflow tract of growing sheep and monitored echocardiographically and histologically at 3 and 6 months. CryoValve SG human pulmonary valves were implanted in 36 patients. Results. SynerGraft treatment reduced tissue antigen expression but did not alter human valve biomechanics or strength. Decellularized sheep allograft valves were functional during the implantation period, and, they became progressively recellularized with recipient cells. In humans, CryoValve SG pulmonary valves did not provoke a panel reactive antibody response. Conclusions. SynerGraft decellularization leaves the physical properties of valves unaltered and substantially diminishes antigen content. Reduction in implant cellularity enables host recellularization of the matrix, which should favorably impact long-term graft durability.

The purpose of this study was to determine the effects of varying heart cold ischemia times on post-cryopreservation valve leaflet cellular functions. Cellular viability and function were assessed through measurement of protein synthesis, ribonucleic acid synthesis, and glucose phosphorylation. Cold ischemia times varied from 24 to 113 h before processing and cryopreservation. The results show that valve leaflet cellular functions could be detected for at least 42 h of post-procurement cold ischemia. This information can be used as a guide in further defining the acceptable limits for heart valve donation and processing.

The endothelial cell is vital in the regulation of blood vessel wall structure, vasomotor tone, and thrombogenicity. Hypothermic temperatures alter both the physiological and biochemical dynamics of endothelial cells. However, there has been no systematic investigation of the influence of cold temperatures upon endothelial cell biology. This review summarizes the current clinical areas of interests, identifies the problems, and addresses the fundamental requirement for further research in endothelial cell cryobiology.

The cytotoxicity of amphotericin B (Fungizone, containing deoxycholate) was investigated for human heart valve leaflet fibroblasts. Leaflets were obtained from human aortic and pulmonic valves and incubated in culture medium containing amphotericin B. Upon completion of incubation, some leaflet sets were analyzed immediately, and others were cryopreserved and stored below -135°C. Quantitative fibroblast viability assays were performed. The results can be summarized by consideration of the data obtained from autoradiographic analysis of [3H]proline incorporation into collagen. Incubation with 10 μg/ml amphotericin B at 37°C resulted in approximately 11% loss of fibroblast viability. After cryopreservation, the leaflets incubated with amphotericin B experienced an additional 42% loss of fibroblast viability. These results indicate that use of amphotericin B, in this form, is ill advised for treatment of human heart valves prior to cryopreservation.

Long-term in vivo success of bioprosthetic allografts is dependent upon retention of cellular functions, such as protein synthesis. The purpose of the experiments presented in this report was to determine the storage conditions necessary for retention of protein synthetic functions in human allograft heart valve leaflets. Tissue viability was assessed by measurement of tritiated-glycine incorporation into proteins. Comparison of short-term (<3 month)- and long-term (1 and 2 years)-cryopreserved heart valve leaflet storage in a liquid nitrogen freezer below − 135 °C demonstrated preservation of fibroblast protein synthesis. In contrast, storage in a mechanical freezer at − 80 °C resulted in a time-dependent loss of fibroblast protein synthesis. There was no statistically significant effect on protein synthesis in leaflets stored for 1 week at 4 °C compared to control cryopreserved liquid nitrogen-stored leaflets. After 2 weeks of 4 °C storage leaflet protein synthesis declined significantly to 15% that of cryopreserved controls. These results demonstrate that liquid nitrogen storage of valve bioprostheses is required for long-term preservation of cellular functions.

We have developed a rapid and simple method to determine the level of dimethyl sulfoxide (Me2SO) in both solutions and tissue samples. For analysis of Me2SO in a cryopreservation medium, the solution is simply diluted in 10% (vol/vol) methanol and centrifuged. Then an aliquot of the supernatant is assayed by high-performance liquid chromatography. For tissue samples, the wet weight is measured and the intact sample is extracted with 10% (vol/vol) methanol (e.g., 10 ml/g wet wt) in a sealed vial. The extract is then diluted and centrifuged, and an aliquot of the supernatant is assayed. The dry weight of the tissue is measured after the methanol-extracted sample is placed into either for 2 h and air-dried overnight. The water content of the tissue is calculated as the difference between the wet and the dry weights. The concentration of Me2SO in the aqueous compartment of the tissue can then be calculated by taking into account the concentration of Me2SO in the extract and the dilution factor, based on the tissue water volume and the volume of methanol used to extract the Me2SO. The calculated values for porcine myocardium samples correlated 1:1 with the actual Me2SO concentrations in the solutions in which the tissue samples were equilibrated. Finally, we present results documenting the usefulness of this assay by following the time course of Me2SO penetration into core versus peripheral regions of 1-cm3 samples of porcine myocardium.

ABSTRACT Two genetically variant forms of rat "acid" β-galactosidase were found to differ in isoelectric point and pH dependence, but not in thermostability or sensitivity to inhibition by p-mercuribenzoate (PMB). The results of two backcrosses and an intercross indicated that the isoelectric focusing phenotypes are controlled by two codominant alleles at a single autosomal locus, for which we propose the name Glb-1. No significant linkage between Glb-1 and albino (LG I), brown (LG II), or hooded (LG VI) was observed. Strain-specific differences in total levels of kidney β-galactosidase were detected, but it is not yet known whether the variation is controlled by genes linked to Glb-1. Experiments in which organ homogenates were incubated with neuraminidase indicated that the genetically variant forms do not result from differences in sialylation, though sialylation does appear to be largely responsible for the presence of multiple bands within each phenotype and for differences in the banding patterns of β-galactosidases derived from different organs. The β-galactosidase present in the bands used for Glb-1 typing resembles human GM1 gangliosidase (GLB1) with respect to pH optimum, substrate specificity, and susceptibility to inhibition by PMB. It also appears that Glb-1 is homologous with the Bgl-e locus of the mouse. In rats as in mice the genetically variant bands of β-galactosidase are active at acid pH and have relatively high isoelectric points. In both species these bands are readily detectable in kidney homogenates, and can be revealed in homogenates of liver or spleen following treatment with neuraminidase. The presence of the same β-galactosidase bands in homogenates of rat kidney and small intestine as well as in neuraminidase-treated homogenates of liver and spleen suggests that the Glb-1 variants differ by one or more point mutations in the structural gene for "acid" β-galactosidase.

Two genetically variant forms of rat "acid" P-galactosidase were found to differ in isoelectric point and pH dependence, but not in thermostability or sensitivity to inhibition by p-mercuribenzoate (PMB) . The results of two backcrosses and an intercross indicated that the isoelectric focusing pheno- types are controlled by two codominant alleles at a single autosomal locus, for which we propose the name Glb-1. No significant linkage between Glb-l and albino (LG I), brown (LG 11), or hooded (LG VI) was observed. Strain-specific differences in total levels of kidney P-galactosidase were de- tected, but it is not yet known whether the variation is controlled by genes linked to Glb-1. Experiments in which organ homogenates were incubated with neuraminidase indicated that the genetically variant forms do not re- sult from differences in sialylation, though sialylation does appear to be largely responsible for the presence of multiple bands within each phenotype and for differences in the banding patterns of p-galactosidases derived from dif- ferent organs. The P-galactosidase present in the bands used for Glb-l typing resembles human GM1 gangliosidase (GLB1) with respect to pH optimum, substrate specificity, and susceptibility to inhibition by PMB. It also appears that Glb-1 is homologous with the Bgl-e locus of the mouse. In rats as in mice the genetically variant bands of P-galactosidase are active at acid pH and have relatively high isoelectric points. In both species these bands are readily detectable in kidney homogenates, and can be revealed in homogen- ates of liver or spleen following treatment with neuraminidase. The pres- ence of the same ,&galactosidase bands in homogenates of rat kidney and small intestine as well as in neuraminidase-treated homogenates of liver and spleen suggests that the Glb-l variants differ by one or more point mu- tations in the structural gene for "acid" ,&galactosidase.