Storage Of Allografts Research Articles

Preservation of Vein Allograft Viability during Long-Term Storage

Background: The aim of this study was to compare the vein allograft viability following cryopreservation with that remaining after prolonged refrigerated storage. Materials and Methods: Great saphenous vein biopsies had been cryopreserved, and the samples were divided into two matched groups and stored in tissue culture medium for 42 days at +4°C, either with or without regular medium replacement. Each vein allograft was biopsied and assayed for viability every third day by the methyltetrazolium reduction assay. Viability indexes of vein allografts harvested from brain-dead multi-organ donors and from cadavers whose warm ischemic periods were maximally 24 h were also compared. Results: Vein allografts stored for 42 days at +4°C showed a similar viability (58.9 ± 1.2%) to that of cryopreserved veins (59.7 ± 2.3%). This was true even when cryopreserved and thawed allografts were subjected to 3 days of post-thaw incubation under presumably favorable conditions (58.7 ± 1.6%). There was no viability index difference between the samples with medium replaced and not replaced and the specimens harvested from two different donor groups. Conclusions: Long-term storage of vein allografts at +4°C is a valuable option for regular banking practice. Sufficient amounts can be procured from cadavers similar to tissue donors.

Combined endothelial and myocardial protection by endothelin antagonism enhances transplant allograft preservation

Endothelin is a potent inflammatory peptide associated with myocardial dysfunction, coronary vasculopathy, and reduced survival after cardiac transplantation. We hypothesized that endothelin antagonism during cardiac allograft storage would limit early endothelial dysfunction and improve myocardial performance after transplantation. Porcine orthotopic transplantations (n = 16) were performed after 6 hours of ischemic storage. Intermittent donor blood perfusion (control, n = 8) was compared with donor blood perfusion enhanced with 100 micromol/L of an endothelin receptor blocker (n = 8). Left ventricular performance was assessed after caval occlusion with a Millar micromanometer and conductance catheter. Coronary endothelial function was assessed in vitro with a macrovascular tissue bath apparatus. Myocardial endothelin, tumor necrosis factor alpha, and transforming growth factor beta protein expression were determined. Oxidative stress was inferred on the basis of 8-isoprostane levels, and myocardial metabolism was inferred on the basis of the extraction or production of oxygen, acid, and lactate by the heart. Endothelial function was diminished 48 hours after transplantation but not earlier. Endothelin receptor blocker treatment during preservation limited coronary endothelial dysfunction 48 hours after reperfusion ( P = .001). Weaning from cardiopulmonary bypass and left ventricular performance after transplantation was improved in endothelin receptor blocker-treated hearts (P = .02). Myocardial endothelin expression was equivalent in both groups and increased during reperfusion after transplantation (P = .001). Tumor necrosis factor alpha levels decreased with endothelin receptor blocker treatment (P = .02), whereas transforming growth factor beta levels did not change (P = .86). 8-Isoprostane, oxygen, acid, and lactate levels were similar, suggesting that oxidative stress and metabolism were not important mechanisms of benefit. Endothelin accumulates during allograft storage and contributes to endothelial and myocardial dysfunction after transplantation. Endothelin blockade during allograft preservation limits endothelial injury and enhances ventricular recovery after transplantation.

Is profound hypothermia required for storage of cardiac allografts?

Background: Improved methods of cardiac allograft protection are required to expand the pool of potentially available organs and to enhance the recovery of grafts subjected to prolonged ischemia. We have previously demonstrated that limited coronary perfusion provided by donor blood harvested at the time of organ procurement can improve both metabolic and functional recovery after transplantation. In this study we evaluated the hypothesis that limited coronary perfusion may enable prolonged cardiac storage while avoiding the potentially detrimental effects of profound hypothermia. Methods: Fourteen orthotopic cardiac transplants were performed in female Yorkshire pigs by using donor blood perfusion during 5 hours of either tepid (25°C) or cold (4°C) storage. Assessments of myocardial metabolism and function were performed at baseline and after 45 minutes of normothermic (37°C) reperfusion. Results: Hearts protected with tepid perfusion displayed improved recovery of myocardial function (89% ± 18% vs 63% ± 25%, P = .05). Diastolic compliance was adversely affected in both groups after transplantation. Aerobic myocardial metabolism was better preserved in the tepid group. Conclusions: Profound hypothermia results in depressed myocardial metabolic and functional recovery after transplantation. Limited coronary perfusion with shed donor blood can permit cardiac allograft storage at tepid temperatures, resulting in improved myocardial performance.

A multicenter, randomized, controlled trial of Celsior for flush and hypothermic storage of cardiac allografts

Background. A multicenter, randomized, controlled, open-label trial was conducted to evaluate the safety and efficacy of Celsior when used for flush and hypothermic storage of donor hearts before transplantation.Methods. Heart transplant recipients were randomized to one of two treatment groups in which donor hearts were flushed and stored in either Celsior or conventional preservation solution(s) (control). Study subjects were followed for 30 days after transplantation.Results. A total of 131 heart transplant recipients were enrolled (Celsior, n = 64; control, n = 67). The treatment groups were evenly distributed in donor and recipient base line characteristics. Graft loss rate was lower in the Celsior group on day 7 (3% versus 9%) and on day 30 (6% versus 13%), but the difference was not statistically significant based on 95% confidence interval analysis. No significant difference was measured between the Celsior and control groups in 7-day patient survival (97% versus 94%) and the proportion of patients with one or more adverse events (Celsior, 88%; control 87%) or serious adverse events (Celsior, 38%; control, 46%). Significantly fewer patients in the Celsior group developed at least one cardiac-related serious adverse event (13% versus 25%).Conclusions. Celsior was demonstrated to be as safe and effective as conventional solutions for flush and cold storage of cardiac allografts before transplantation.

Our clinical experience with the use of allografts and bone banking

Objectives: We evaluated patients in whom allografts were used. The allografts were obtained from the bone bank in the Department of Orthopaedics and Traumatology, Medical Faculty of Ondokuz Mayis University. Methods: Allografts were used in 33 patients between August 1994 and January 1998. Mean age was 33.2 years (range 5-70 years). The patients were clinically and radiologically examined monthly and annually before and after complete union was obtained, respectively. Results: Complete union was achieved in all patients but one (97.1%), who was successfully treated with the reutilization of an allograft. The average consolidation time was 8 weeks (range 4-22 weeks). Only three adverse events were encountered. One patient died from pulmonary thromboembolism on the second postoperative day. Sterile and infected drainages occurred in two patients, respectively, both of which were successfully treated. Conclusion: Provided that thorough serologic and microbiologic examinations of donors are made and appropriate techniques are applied in the removal and storage of allografts, use of bone bank allografts is more reliable and economic and offers many advantages including decreased donor site morbidity, decreased blood loss and operation time during surgery, and high osteogenic potential.

Bangkok biomaterial center: 15 years experience in tissue banking.

Tissue banking is started in Thailand in 1979 at the Department of Orthopaedic Surgery, Siriraj Hospital, Bangkok. At that time tissues produced were freeze-dried bone allografts which were sterilized by ethylene oxide. In 1984, the freeze-dried tissue allograft project received an award from the National Research Council of Thailand. The Bangkok Biomaterial Center was officially inaugurated on December 6, 1984 under the Royal Patronage of H.R.H. Princess Galyanivadhana and is located inside the Siriraj Hospital. The Center is involved in the procurement, processing, storage and development of bone and tissue allografts. A variety of allografts including bone, cartilage, fascia lata, dura mater, cornea and also cardiovascular tissues have been procured and processed. Preservation and long-term storage are accomplished by freeze drying and deep freezing. Grafts prepared by the Center are supplied free of charge at the request of surgeons in hospitals throughout Thailand and in neighboring countries. The Center acts as the National Tissue and Allograft Bank of Thailand. From December 1984 to February 2000, the Center has processed a total of 20 524 allografts: 16 981 freeze-dried bones, 705 deep-frozen bones, 1838 freeze-dried amnion, 559 freeze-dried dura mater, 342 freeze-dried fascia lata, 46 costal cartilage, 18 corneas, 2 skin, 5 trachea, 22 fresh tendon and 6 bone substitutes. The allografts processed were used in 2049 patients by 223 surgeons in 53 hospitals in Thailand and 4 cases in neighboring countries. There have been 413 cadaveric donors, 619 living donors, 16 brain dead donors and 270 graveyard donors. There have been complications in 126 patients (6.14%) due to various clinical conditions. There have been production and application of 4 hydroxyapatite occular implant by the Center. The Center is in the process of establishing a full-fledged Research, Clinical and Cell Culture Laboratory.

Continuous perfusion of donor hearts in the beating state extends preservation time and improves recovery of function

Objectives: Improving methods of donor heart preservation may permit prolonged storage and remote procurement of cardiac allografts. We hypothesized that continuous, sanguineous perfusion of the donor heart in the beating, working state may prolong myocardial preservation. Methods: We developed a portable perfusion apparatus for use in donor heart preservation. Contractile, metabolic, and vasomotor functions were monitored simultaneously in an isolated swine heart. The metabolic state was monitored by myocardial tissue pH. Vasomotor function was assessed in isolated coronary ring chambers. Hearts were randomized into 3 groups: group I (n = 5), cardioplegic arrest, 12-hour storage at 4°C with modified Belzer solution, and 2-hour sanguineous reperfusion in the working state; group II (n = 6), 12-hour continuous perfusion in the beating working state, 30 minutes of arrest (to simulate re-implantation time), and 2 hours of reperfusion, as above; group III (n = 7), coronary ring control hearts. Results: At 2 hours of reperfusion, left ventricular developed pressure in group II was higher than in group I (mean ± standard deviation: 90 ± 6 mm Hg, 53 ± 15 mm Hg, P = .005). Significantly less myocardial edema was observed in group II than in group I (73% ± 4%, 80% ± 1% water content, P = .01). Significantly less myocardial acidosis was noted in group II than in group I during preservation (pH 7.3 ± 0.01, 6.1 ± 0.03, P < .001) and reperfusion (pH 7.3 ± 0.008, 6.8 ± 0.05, P < .001). Coronary endothelial vasomotor function was better preserved in group II than in group I as evidenced by dose-response relaxation of coronary rings to 10 -8 mol/L bradykinin (37%, 55% ▵baseline, P = .01). Conclusion: This new method extends the current preservation limit and avoids time-dependent ischemic injury, thereby allowing for distant procurement of donor organs. (J Thorac Cardiovasc Surg 1998;116:821-30)

Effects of Butanedione Monoxime and Temperature on Prolonged Cardiac Storage

Background. The optimal temperature for cardiac allograft storage remains controversial. We conjectured that supplementation of the potent cardioprotective agent 2,3-butanedione monoxime with calcium may improve allograft storage and make the precise storage temperature less critical.Methods. Hearts were harvested from Sprague-Dawley rats (250 to 350 g), mounted on a Langendorff apparatus, and instrumented with an intraventricular balloon. Hearts were flushed and stored with either unmodified University of Wisconsin solution (UWS) or UWS supplemented with 10 mmol/L of 2,3-butanedione monoxime and calcium 0.1 mmol/L (BDM). Hearts were then subjected to 12 hours of storage at one of five temperatures (0°, 4°, 8°, 12°, or 16°C) in a complete 2 × 5 factorial design (n = 6/group). Data are reported either as a percentage of the prestorage results or as an absolute value (mean ± standard deviation).Results. Recovery of developed pressure (p < 0.0001), coronary flow (p < 0.0001), and diastolic volume (p < 0.001) were significantly enhanced, whereas creatine kinase (p < 0.0001) and lactate dehydrogenase release (p < 0.0001) were reduced in the BDM versus the UWS groups. In both the BDM and UWS storage groups, recovery was better at temperatures of 8°C or less than at 12°C or more. The single preferred temperature was 4°C, significantly better than 0°C with unmodified UWS, while similar to 0° and 8°C with BDM. Adenine nucleotide values were decreased equally in the BDM and UWS hearts, but preservation was enhanced at 0°C compared with all warmer temperatures.Conclusions. We conclude that 4°C is the preferred temperature for prolonged cardiac storage with UWS and that the inclusion of 2,3-butanedione monoxime with calcium 0.1 mmol/L markedly enhances recovery for storage temperatures of 8°C or less.(Ann Thorac Surg 1997;63:388–94)

Intratracheal Surfactant Administration Preserves Airway Compliance During Lung Reperfusion

Background. Decreased airway compliance after lung transplantation has been observed with severe ischemia-reperfusion injury. Further, it has been shown that the surfactant system is impaired after lung preservation and reperfusion. We hypothesized that surfactant replacement after allograft storage could preserve airway compliance during reperfusion. Methods. Rabbit lungs were harvested after flush with 50 mL/kg of cold saline solution. Immediate control lungs were studied with an isolated ventilation/perfusion apparatus using venous rabbit blood recirculated at 40 mL/min, room-air ventilation at 20 breaths/min, and constant airway pressure (n = 8). Twenty-four-hour control lungs were preserved at 4°C for 24 hours and then similarly studied (n = 7). Surfactant lungs underwent similar harvest and preservation for 24 hours, but received 1.5 mL/kg of intratracheal surfactant 5 minutes before reperfusion (n = 10). Airway pressure and flow were recorded continuously during 30 minutes of reperfusion. Tidal volume and airway compliance were calculated at 30 minutes. Results. Tidal volume was 33.67 ± 0.57, 15.75 ± 5.72, and 29.83 ± 1.07 mL in the immediate control, 24-hour control, and surfactant groups, respectively ( p = 0.004, surfactant versus 24-hour control). Airway compliance was 1.94 ± 0.27, 0.70 ± 0.09, and 1.46 ± 0.10 mL/mm Hg in the immediate control, 24-hour control, and surfactant groups, respectively ( p = 0.002, surfactant versus 24-hour control). Conclusions. We conclude that surfactant administration before reperfusion after 24 hours of cold storage preserves tidal volume and airway compliance in the isolated ventilated/perfused rabbit model of lung reperfusion injury.

Allograft Reconstruction for Giant Cell Tumour Close to the Knee

Summary Allograft reconstructive surgery is a justified means of limb preservation in cases of giant cell tumour around the knee joint. Results of studies have shown this method of operative intervention to be highly successful. The purpose of this paper is to present a concise overview of the basic principles of allograft storage, transplantation surgery and the pathophysiology of graft repair. Results of studies pertaining to allograft reconstructive surgery and standard physiotherapy interventions are discussed.

The Effects of Variation of Cryopreservation Protocols on the Immunogenicity of AIIogeneic Skin Grafts

The use of allografts to effect wound closure on full thickness skin injuries is limited by unpredictable graft rejection times. If the period of graft take could be extended, the use of allografts would reduce the morbidity and mortality associated with these wounds. This study evaluated the effects of variation in the cryopreservation protocol on the viability and immunogenicity of skin using a murine model system. lmmunogenicity was assessed by the stimulatory activity of C3H (H-2K) skin-derived epidermal cells (EC) in primary one-way EC/lymphocyte reactions with BALB/c (H-2d) and CBA (H-2K) responder lymphocytes. Viability was determined by measuring tetrazolium reductase activity. The following cryopreservation protocols were assessed: freezing at 1, 30, 64, and > 100°C/min in 10 and 15% (v/v) Me2SO and freezing at 30°C/min in 5 to 20% (v/v) Me2SO or glycerol. A cryopreservation protocol of 30°C/min in 15% (v/v) Me2SO proved optimal for murine skin allograft storage and immunomodulation. The viability of skin treated by this protocol was maintained (78% of fresh skin viability, no significant difference analysis of variance). The stimulatory capacity of treated EC for H-2K and H-2d lymphocytes was 5 ± 4 and 5 ± 9% (±95% confidence limits) of fresh EC (100%) activity. Langerhans cell numbers in epidermal sheets and EC suspensions did not correlate with the stimulatory capacity of fresh and treated EC for allogeneic Iymphocytes. A functional impairment of Langerhans cell immunostimulatory capacity was implied. Thus it may be feasible to provide a single stage storage and immunomodulation treatment for allogeneic skin.

Ischemic-type biliary complications after orthotopic liver transplantation.

Nonanastomotic biliary strictures that involve only the biliary tree of the graft occur after orthotopic liver transplantation in patients with hepatic artery thrombosis, chronic ductopenic rejection and ABO blood group incompatibility. This complication may also occur in the absence of these known risk factors. The major focus of our study was to evaluate the risk factors for nonanastomotic biliary stricturing of unknown cause after orthotopic liver transplantation. Results demonstrate that the development of biliary strictures is strongly associated with the duration of cold ischemic storage of allografts in both Euro-Collins solution and University of Wisconsin solution. Results also demonstrate that strictures are not associated with the type of biliary reconstruction, the primary liver disease, cytomegalovirus infection, allograft rejection or the presence of a positive lymphocytotoxic crossmatch. More recently, we have markedly reduced the occurrence of nonanastomotic biliary stricturing by decreasing the ischemia time of our allografts. Thus nonanastomotic biliary strictures appear to be the result of the ischemia/reperfusion-induced tissue injury associated with the harvest and implantation of allografts.

Predictive value of liver tissue flow in assessment of the viability of liver grafts after extended preservation in pigs.

The crucial damage in cold storage of liver allografts is to the hepatic sinusoidal lining (microcirculation). Using different solutions, we studied whether determinations of graft tissue flow were valuable in estimating the viability of liver grafts. Twenty-three pairs of female pigs underwent orthotopic liver transplantation and were assigned to five groups according to the cold preservation time or solutions used: in group I the liver grafts were stored in Euro-Collins solution (EC) for 4 h (n = 3), in group II the grafts were stored in EC for 12 h (n = 5), in group III the donor was pretreated with azathioprine (AZA), 1 mg/kg per day, orally (p.o.) for 3 days before harvesting and the graft was implanted after 12 h cold storage with EC (n = 6), in group IV the graft was stored in modified University of Wisconsin solution (mUW) for 4 h (n = 3), and in group V the graft was stored in mUW for 24 h (n = 6). Liver tissue blood flow (LTBF) was measured, using a laser doppler device, at 60 min after recirculation of the graft. In the case of EC preservation, LTBF (ml/100 g of liver tissue per min) correlated well with 4-day survival: 21.2 +/- 3.0 ml/100 g of tissue per min mean +/- SD, in group I (3/3, 100%); 10.0 +/- 2.8 ml/100 g of tissue per min in group II (0/5, 0%); and 19.1 +/- 3.4 ml/100 g of tissue per min in group III (5/6, 83.3%) (P < 0.05, group II vs I and III). All grafts with LTBF of more than 15 ml/100 g tissue per min functioned well. However, changes in microcirculation of the mUW-stored livers did not correlate with early function of the graft: 23.0 +/- 2.3 ml/100 g of tissue per min in group IV (4-day survival; 3 of 3, 100%) and 23.5 +/- 9.1 ml/100 g of tissue per min in group V (0 of 6, 0%). This was accompanied by graft dehydration during storage and an increased number of erythrocytes in the hepatic sinusoids post-recirculation. We concluded that assessment of liver tissue flow by LDF was very helpful and easy to apply in predicting liver graft failure in the case of preservation with Euro-Collins solution. However, LTBF should be carefully evaluated as a marker of liver graft viability when the liver graft is preserved with mUW.

Prolonged hypothermic cardiac storage with University of Wisconsin solution: An assessment with human cell cultures

Hypothermic storage of cardiac allografts is routinely used for transplantation but is associated with an increased mortality when ischemic times are greater than 4 hours. The ideal storage conditions (solution and temperature) could extend the current limits of cold ischemia. Human endothelial cells and ventricular myocytes were studied to screen various solutions and temperatures for organ preservation. Four solutions (modified Euro-Collins, phosphate-buffered saline, Stanford cardioplegia, and University of Wisconsin) were evaluated. Endothelial cells were evaluated after prolonged hypothermic storage consisting of 0 degree, 4 degrees, and 8 degrees C for 36 hours, and ventricular myocytes were stored at 0 degree and 8 degrees C for 24 hours. Cell viability was determined by morphology (10 dishes per group), and trypan blue exclusion (5 dishes per group) in addition to a cell adhesion assay (endothelial cells 5 dishes per group) and adenine nucleotide analysis with high-performance liquid chromatography techniques (ventricular myocytes 5 dishes per group). Endothelial cell morphology was best preserved by University of Wisconsin solution (p less than 0.001, chi 2) and at 0 degree C (p less than 0.01, chi 2). Endothelial cells stored with University of Wisconsin solution excluded trypan blue better (1.0% +/- 0.5% cells stained, p less than 0.001. Analysis of variance [ANOVA]). Cell adhesion was poorly protected with Stanford cardioplegia (p less than 0.001, ANOVA). Myocyte morphology was preserved best with University of Wisconsin solution at 0 degree C (p less than 0.001, chi 2). According to trypan blue staining, Euro-Collins and University of Wisconsin solutions were superior to Stanford cardioplegia or phosphate-buffered solutions (p less than 0.001, ANOVA). Temperature did not influence the trypan blue results. Adenosine triphosphate was maintained best with University of Wisconsin solution at 0 degree C (p less than 0.01, ANOVA). Myocytes were more sensitive to the effects of prolonged storage compared with endothelial cells by morphologic criteria and trypan blue staining characteristics, irrespective of the shorter preservation times. University of Wisconsin solution was the most effective solution tested. Colder temperatures (0 degree to 4 degrees C) provided better protection than 8 degrees C. Myocytes were more sensitive to prolonged preservation than endothelial cells. Furthermore, the technique used appears helpful as a model of prolonged hypothermic storage and could be expanded to assess other interventions.

Endothelial cell viability in the rat aortic wall

Aortic allografts may offer advantages over prosthetic materials for aortic valve replacement or reconstruction with a valved aortic conduit. Cellular viability may partly determine long-term allograft performance. To evaluate endothelial cell viability in a rat model, valved aortic conduits were subjected to collagenase digestion. The resulting endothelial cell suspension was labeled with Griffonia simplicifolia agglutinin-fluorescein isothiocyanate (GSA-FITC), a marker specific for vascular endothelial cells of the rat. The cells were then incubated with propidium iodide, which is excluded by viable cells. Flow cytometry evaluated endothelial cell viability by determination of percentage of GSA-FITC-positive cells that were negative for propidium iodide. Aortas were studied immediately after harvest or after storage at 4 °C in a nutrient medium for 3 to 21 days. Percentage of viable endothelial cells showed a progressive decline with increasing duration of storage. These results demonstrate flow cytometric measurement of endothelial cell viability, a factor of possible importance in assessing allograft storage methods, and show that endothelial viability declines with prolonged storage at 4 °C in a nutrient medium.

A PROSPECTIVE CONTROLLED TRIAL OF COLD-STORAGE VERSUS MACHINE-PERFUSION PRESERVATION IN CADAVERIC RENAL TRANSPLANTATION

A prospective controlled study was carried out in 60 consecutive cadaver renal donors comparing cold storage to pulsatile machine-perfusion preservation. Each donor served as its own control, by allocating one of the kidneys to each of the two preservation methods. There were 51 evaluable pairs of kidneys. Recipient age, panel-reactive antibody level, history of prior renal transplant, and immunosuppressive regimen were similar in the two preservation groups. Almost all recipients were treated with cyclosporine, and over 50% received antilymphoblast globulin. Total cold ischemic time was 1262 +/- 387 min in the machine-perfused group and 1309 +/- 426 min in the cold-storage group (P = NS). Prolonged ischemia (greater than 24 hr) occurred in 31% of machine-perfused and 22% of cold-stored kidneys (P = NS). Post-operative serum creatinine levels at 1, 7, and 30 days posttransplant were similar in both groups. Dialysis requirements were also similar, with 21 recipients of machine-perfused kidneys (41%) requiring at least one dialysis treatment compared to 16 patients (31%) in the cold-stored group (P = NS); the mean number of dialysis treatments required was 3.14 +/- 1.46 and 3.06 +/- 1.29, respectively (P = NS). Long ischemic time (greater than 24 hr) was associated with a higher rate of dialysis requirement in both groups, but in neither case did this achieve statistical significance. The distribution of graft losses within the first 30 days was similar in both groups, and the incidence of preservation-related graft failure was not significantly different. These results demonstrate that, in the cyclosporine era, machine perfusion offers no significant advantages over cold storage for cadaver renal preservation. Because machine perfusion is considerably more expensive and cold storage is simpler and facilitates the logistics of organ sharing, we recommend simple hypothermic storage of renal allografts as the preservation method of choice.

A histological and biochemical assessment of the cartilage matrix obtained from in vitro storage of osteochondral allografts.

Fresh osteochondral allografts were stored at 4 degrees C in tissue culture media at variable time periods (3, 7, 14 and 28 days). Sterilely dissected tibial plateaus with a standardized 1/2 cm subchondral bone "shell" were obtained from canines 1-3 hrs post mortem. X-rays were taken to determine maturity of the animals. Only mature animals (closed epiphyses) were considered for the study. Histologically, safranin 0 (metachromatic stain for glycosaminoglycans) was observed in all experimental specimens. H&E stained sections showed at all time periods of 3, 7, 14 and 28 days that the cell morphology and arrangements were similar in the superficial and deep areas of the cartilage obtained from the stored osteochondral allograft when compared to the control articular cartilage. The cells were in lacunae and arranged in clusters. Biochemically, glycosaminoglycans and collagen content showed no difference at the 95% level of confidence during the duration of the study (28 days) when compared to the 0 day control cartilage. Collagen typing, based on the assessment by HPLC of the CNBr peptides showed the major presence of type II collagen (no evidence of dedifferentiation was observed). No type I was found to be present. Some apparent variations in the proportions of minor collagen components were noted--e.g. at 14 days the cartilage appeared to contain increased amounts of type XI but little or no type IX collagen (HMW, LMW) when compared to the day 0 control. At 28 days a shift to a larger amount of type IX collagen occurs, especially in the LMW component, with a small amount of type XI collagen when compared to normal day 0 articular cartilage. Cell viability, i.e., the ability of the allograft tissue to incorporate 35SO4 in the synthesis of glycosaminoglycans, was intact up to 28 days of storage.

Methods of Banking Bone and Cartilage for Allograft Transplantation

Storage of bone for use as allograft has become an important endeavor because banked bone is becoming increasingly useful and popular as a substitute for, or as a supplement to, autograft bone. Methods have been developed for safe and reliable bone banking, including standard criteria for acceptable donors, proven techniques of retrieval of tissue, and appropriate storage facilities and conditions. In contrast, cartilage banking is still an investigative procedure, but current research may provide more effective approaches to maintaining viable cells during freezer storage of osteochondral allografts.

Banking of Massive Osteoarticular and Intercalary Bone Allografts???12 Years?? Experience

Preparation and banking of massive osteoarticular allografts and intercalary bone allografts have been performed for the past 12 years. Careful selection of donors as well as extensive laboratory studies of the donor and the allograft have virtually eliminated the danger of transmitting disease from the donor to the recipient. The availability of a variety of allografts in the Tissue Bank allows for the selection, on an anatomic basis, of an allograft best suited for a particular recipient. The authors have supplied several hundred allografts to recipients in many institutions on the premise that excision, preparation, banking, and implantation of bone allografts constitute a clinical service. Thus, the surgeon who excises and prepares the allograft assumes a joint responsibility for the care of the recipient with the surgeon who implants the allograft. This establishes a close working relationship, which encourages frequent consultation between the parties concerned. This relationship is of particular importance in the initial evaluation of the patient and in determining which particular allograft will best serve a given patient. The experience at the authors' institution provides a model for a multiinstitutional facility that may serve as a base for discussion of the methodology involved in the excision, preparation, and storage of bone allografts. The costs associated with the operation of such a facility are not inconsiderable, but the cost of individual osteoarticular and intercalary allografts can be brought down by an increase in the efficiency of operation inherent in the processing of allografts from over 100 donors per year. During the past several years, the cost of excising and preparing intercalary allografts has been $600 per implant, while the cost for osteochondral allografts varied between $900 and $1,200. Such a large multiinstitutional facility offers the advantages of readily available allografts and quality control. However, because of the expense and complexity of operation, it may not be suitable for many medical centers.

Experimental Freeze-Preservation of Chondrocytes

Preservation of articular cartilage chondrocytes is currently being investigated in conjunction with storage and transplantation of osteochondral allografts. Studies on isolated chondrocytes have suggested that viability and function may be retained following freeze-preservation. Up to 90% of frozen isolated cells will survive, and these cells are capable of producing proteoglycans in culture. Results of freezing chondrocytes in a matrix have not been as successful, with viability ranging from 0% to 50%. Inconsistent results are thought to be due to the matrix and associated problems such as poor penetration of the cryopreservative, an unequal rate of cooling, and a high water content. Studies are in progress in an attempt to produce a satisfactory method of freeze-preservation that will promote normal functioning of chondrocytes after transplantation in an articular cartilage matrix.