Fresh Allograft Research Articles

Meniscal transplantation using fresh and cryopreserved allografts. An experimental study in goats.

A comparative study of three subgroups of meniscal transplants was undertaken in the goat model: Group 1 (autograft) involved removal and immediate reimplantation of the meniscus; Group 2, fresh meniscal allografts; and Group 3, cryopreserved (30 days) meniscal allografts. Six months after surgery, tissues were evaluated for gross degenerative changes, proteoglycan concentration (as assessed by uronic acid), water content, vascularity, histology, and cell viability. The contralateral knee served as control for all comparisons. There was no statistical difference in the amount of arthritis present and all transplants demonstrated an essentially normal peripheral vascularity compared to controls. Sections revealed reduced numbers of cells in the central portions of the transplanted menisci and these viable cells demonstrated different behavior in multiplication in tissue culture compared to contralateral controls. Grossly and microscopically, the implanted menisci differed little from the controls. The measurement of proteoglycan concentration and water content of the transplanted meniscal cartilage suggest alterations that may affect the long-term mechanical properties. The autograft specimens showed the water content was very slightly increased (3% to 6%), while the proteoglycan concentration was increased (42% in terms of uronic acid). In contrast, the water content of the fresh allograft group and the cryopreserved group was increased 12% to 24%. Proteoglycan concentration in these groups was decreased up to 56% in portions of some menisci compared to controls. Fresh and cryopreserved meniscal allografts showed peripheral healing, revascularization, cellularity, and incorporation, and grossly appeared good at 6 months in the goat model. The biochemical changes in the extracellular matrix at 6 months raises questions on the long-term function of these transplanted menisci.

Intact Articular Cartilage Cryopreservation In Vivo Evaluation

By varying the parameters that determine the cryopreservation process (freezing rate, storage temperature, cryoprotective agents, storage time), various protocols have been designed for preservation of osteochondral allografts of rabbit femoral condyles. The grafts were implanted orthotopically for three months. After harvesting, the results were evaluated by optical microscopy, histochemistry, and electron microscopy (scanning and transmission). A quantitative study and statistical analysis was performed using a gradation scale of chondral degeneration. Synovial reaction was higher in the fresh allograft than in the cryopreservation groups. Glycerol yielded better results than DMSO as a cryoprotective agent. No cryopreservation protocols produced the results equal to the control groups of either fresh auto- or allografts. Optimal preservation was achieved with either the group with hypothermic storage at 4 degrees for no more than 48 hours or the group with slow freezing to -80 degrees and preparative exposure to 15% glycerol at 4 degrees for 60 minutes. No statistically significant differences were found in these two groups.

Evaluation of cryopreserved allograft venous conduits in dogs

We investigated the effects of cryopreservation, immunosuppression, and antibiotic treatment on the patency and histologic appearance of venous conduits in the arterial circulation. Twenty-eight dogs received arterial replacements with autograft vein, fresh allograft, and two types of cryopreserved allograft vein implanted into both carotid and both femoral arteries. All animals were given aspirin, and half were given cyclosporine. After 3 months the vein grafts were harvested. Patency and light, transmission, and scanning electron microscopic criteria were scored to evaluate quality of preservation of the endothelium, the appearance of rejection, and the effects of cryopreservation with and without antibiotic pretreatment. The results show that patency is not statistically different based on graft type or treatment modality. The histologic appearance among the various vein types was remarkably similar at 3 months, with the exception of a cellular infiltrate present most prominently in the fresh allografts and least in the fresh autografts. Cyclosporine, even at a low dose, decreased the incidence of cellular infiltration. Preservation of endothelium was generally good in the cryopreserved allografts both with and without antibiotic pretreatment. In general, the effects of cryopreservation, cyclosporine, and antibiotics ameliorated the effects of venous allografting into an arterial position.

The hydrodynamic function and leaflet dynamics of aortic and pulmonary roots and valves: an in vitro study.

Fresh homograft aortic and pulmonary roots were tested in a pulsatile flow simulator to assess their hydrodynamic function and leaflet opening characteristics. Simultaneous flow and pressure measurements were obtained for a range of cardiac outputs. The effective orifice area and regurgitant volumes were calculated. The mean pressure difference across the pulmonary roots was obtained under both left and right side pressures. A video recording of valve leaflet function was also obtained for each valve. A comparison was made with four porcine bioprosthetic heart valves (21 and 23 mm). The mean pressure difference with respect to flow for the 21 mm Hancock II and Intact porcine bioprostheses was significantly higher than that for all aortic roots tested. The 24 mm aortic root showed significantly lower pressure drop compared to all porcine valves tested. The mean pressure difference across each pulmonary root at pulmonary pressures was significantly greater than at systemic pressures. At systemic pressures the fully open leaflets had a triangular orifice with low leaflet open bending strains at the commissures. At the lower internal pressures, with reduced dilation of the root, higher bending strains were noted. These were not as severe as seen in porcine valves.

Survival of cells after intra-articular transplantation of fresh allografts of the patellar and anterior cruciate ligaments. DNA-probe analysis in a goat model.

The fate of donor cells in fresh allografts of the patellar and anterior cruciate ligaments was assessed after transplantation of the allografts as substitutes for the anterior cruciate ligament in goats. DNA-probe analysis was used to distinguish between the DNA of individual goats. Donor DNA was completely replaced by recipient DNA in both the transplanted patellar and anterior cruciate ligaments within a four-week period. Simultaneous full-thickness skin transplants in the same animals were not rejected during the interval of rapid loss of donor DNA from the allografts. The absence of rejection of the skin grafts at the one-week interval suggests that no pre-existing antibody associated with an immune reaction was responsible for the rapid loss of DNA in the allografts.

Immunosuppressive treatment of venous allografts

In order to evaluate whether temporary immunosuppressive therapy is able to improve the results obtained with viable venous allografts and achieve better results than with synthetic grafts, 142 arterial reconstructions were performed in mongrel dogs bypassing their ligated femoral arteries. Histological as well as immunological studies were performed and patency determined by weekly palpation and regular angiography. The 6-month cumulative patency rates were: Group I: synthetic grafts (a) Dacron: 48%, (b) plasma-TFE: 53%. Group II: fresh grafts (a) autografts: 100%, (b) allografts: 37%, (c) allografts treated with cyclosporin 4 mg kg-1 daily for 1 month: 74% (100% after 1 month). Group III: grafts preserved in Hanks' solution with 15% DMSO at -160 degrees C for 1 month (a) autografts: 77%, (b) allografts: 35%, (c) allografts treated with methylprednisolone 1 mg kg-1 daily: 38%, (e) allografts treated with cyclosporin and methylprednisolone: 83%. Group IV: human saphenous veins implanted as xenografts and treated with cyclosporin and methylprednisolone: 18%. Immunosuppressive therapy with cyclosporin seems to be able to prevent early thromboses due to rejection seen after implantation of viable fresh or cryopreserved venous allografts, and the results are significantly better than those obtained with synthetic grafts. Tissue matching might further improve these results. This study suggests that cryopreserved venous allografts could be used for the creation of a vein-bank and their use, in combination with tissue typing and temporary immunosuppressive therapy may be warranted for arterial reconstructions when autologous saphenous vein is not available.

Evaluation of the Damus-Kaye-Stansel operation in infancy

Thirteen patients, 12 of whom younger than 2 years, underwent a Damus-Kaye-Stansel procedure for complete transposition of the great arteries, ventricular septal defect, or double-outlet right ventricle and subpulmonary ventricular septal defect. In 6 patients, associated cardiac anomalies caused systemic flow obstruction. There were six hospital deaths (mortality rate, 42%). In a mean follow-up period of 57 months, 5 of 7 survivors required relief of right ventricular hypertension through conduit replacement or enlargement (4 patients) or conduit valve balloon dilation (1 patient). The aortic valve became regurgitant in 2 patients in whom it had been left in potential connection with the right ventricle. One patient has moderate pulmonary valve regurgitation. The main advantage of the Damus-Kaye-Stansel procedure is that it avoids coronary relocation; also, the spatial relationship of the great arteries and the coronary anatomy do not affect its feasibility. One drawback is the need for a conduit in infancy. Our present indication for Damus-Kaye-Stansel procedure is confined to double-outlet right ventricle with subpulmonary ventricular septal defect; 5 of 6 patients survived repair in this series. Possible indications are for patients with associated subaortic obstruction or unusual coronary arrangements. Fresh or cryopreserved homografts as extracardiac conduits and primary closure of the subaortic area may reduce the need for reoperation after Damus-Kaye-Stansel procedure.

The fate of cancellous and cortical bone after transplantation of fresh and frozen tissue-antigen-matched and mismatched osteochondral allografts in dogs.

After implantation, a massive osteochondral allograft cannot be completely protected from the stresses that are produced by weight-bearing, and it is susceptible to collapse during incorporation, revascularization, and substitution. How these processes are affected by disparities between the tissue antigens of the host and the graft remain unclear. To clarify the role of histocompatibility antigen-matching in the incorporation of cancellous and cortical bone, we orthotopically implanted both fresh and cryopreserved dog leukocyte-antigen-matched and mismatched proximal osteochondral radial allografts in beagles. Four groups of beagle dogs were used; they received (1) a dog leukocyte-antigen-mismatched frozen allograft, (2) a dog leukocyte-antigen-mismatched fresh allograft, (3) a dog leukocyte-antigen-matched fresh allograft, or (4) a dog leukocyte-antigen-matched frozen allograft. In twelve dogs, a sham operation was done in the contralateral limb (the first living donor had a sham operation), and in the remaining ten dogs, the proximal part of the contralateral radius was removed and then replaced as an autogenous (control) graft. The animals were given fluorochromes periodically, and they were killed eleven months after the operation. The osseous portion of the grafts was evaluated radiographically, biomechanically, and histomorphometrically. No dog had grossly obvious clinical abnormalities, all host-graft interfaces healed, and no joints dislocated. Radiographic examination of the allografts frequently showed deformation of the radial head and variable peripheral resorption. No significant difference in the modulus of elasticity at the host-graft interface was found among the groups. The repair process of the cortical bone was similar for all grafted segments. New periosteal and endosteal bone formed, and the cortical bone became porotic as vessels penetrated it. The uptake of fluorochrome was the most active in the autogenous grafts and the least active in the fresh antigen-mismatched grafts. The volume of cancellous bone was significantly greater and the trabeculae were thicker in all allografts compared with the bones on which a sham operation had been done and compared with the autogenous grafts. The volume of intertrabecular fibrous connective tissue was directly proportional to the immunogenicity of the allografts, and the percentage of the surface on which bone was forming tended to be inversely proportional to the immunogenicity of the allografts. The grafts were revascularized by the ingrowth of vessels into the intertrabecular spaces; necrotic trabeculae were not penetrated by vessels. This pattern was particularly pronounced in the antigen-mismatched grafts, regardless of whether they were fresh or frozen.(ABSTRACT TRUNCATED AT 400 WORDS)

ALLOGRAFT VEIN PATENCY IN A CANINE MODEL

Autogenous saphenous vein is the preferred conduit for many cardiovascular operations. Attempts to use allograft veins for arterial reconstruction have had poor results. To define circumstances under which allograft veins might prove to be acceptable vascular conduits, dogs underwent femoral artery bypass using reversed saphenous veins. Veins were transplanted fresh or after cryopreservation. Group I dogs received a fresh autograft to replace one femoral artery (group I F) and a cryopreserved (CP) autograft (group I C) to replace the other. Group II dogs received fresh allograft veins, and group III received CP allograft veins, neither group receiving additional treatment. Group IV received fresh allograft veins and Group V received CP allograft veins; both groups received cyclosporine 15 mg/kg. Animals were maintained until grafts occluded or until six months elapsed. Patency was observed in all group I F grafts throughout the observation period. Six-month patency rates in the other groups were: group I C, 9/10 (P = NS vs. group I F); group II, 0/10 (P less than 0.01), group III, 0/10 (P less than 0.01), group IV, 1/10 (P less than 0.01), group V, 7/11 (P = NS). In a separate series of observations 10 cryopreserved allograft veins were implanted in 10 dogs that received CsA for 30 days. CsA was then discontinued. All of these grafts occluded within 30 days of discontinuing the CsA. Long-term patency of saphenous vein allografts was achieved only with the combination of cryopreservation and immunosuppression with continued CsA.

In situ fresh allograft replacement of an infected aortic prosthetic graft: Eighteen months' follow-up

We report a case in which an allogenic aortic graft was used to treat the last one of a series of infectious graft complications. The original operation was a Dacron aortobifemoral bypass for aortoiliac occlusive disease in an institution other than ours. The patient was referred for the treatment of the infection of the prosthetic implant. We inserted a prosthetic bypass from the left axillary to the left deep femoral artery, as well as a crossover autogenous saphenous vein graft from the prosthesis to the right deep femoral artery; in the same operation the infected intraabdominal prosthesis was removed with closure of the proximal aortic stump. Subsequent stenosis of the vein graft made it necessary to insert a prosthetic bypass between the descending thoracic aorta and the two deep femoral arteries. Infection of the last named prosthesis made it necessary to remove it and replace it with a tailored allogenic arterial allograft between the infrarenal abdominal aorta and both deep femoral arteries. The patient had an uneventful recovery as the infection was controlled and both lower limbs were salvaged. Eighteen months later the allograft was patent without any signs of degeneration. Further clinical experience under appropriate conditions may prove the use of allografts effective in the treatment of selected cases of aortic prosthetic infection.

In situ fresh allograft replacement of an infected aortic prosthetic graft: Eighteen month's follow-up

In situ fresh allograft replacement of an infected aortic prosthetic graft: Eighteen month's follow-up

Experimental study on allogenic bone grafts

The effects of various processing techniques on the biomechanical properties and the histological incorporation of bone grafts were investigated in a rat model over a 6 month period. The stiffness of bone increased after freezing to -80 degrees C and freeze-drying. Decalcified bone was very soft. Visco-elasticity was pronounced at 4 to 12 weeks and the stiffness of the graft increased at 16 weeks. This progressive rise in stiffness was in line with the progressive incorporation of the graft. The bone incorporation ratio is proposed as a histological indicator of osteo-inductive properties. It was found to be best in fresh autografts, followed by processed allografts (frozen, freeze-dried and decalcified) and fresh allografts. The values of these 3 groups showed a statistically significant difference.

Microsurgical free bone transfer in the dog

Bone grafts have been used extensively for the reconstruction of skeletal injury in dogs. Cancellous and corticocancellous chip autografts are used for their osteoinductive and osteoconductive properties. Fresh, fresh-frozen, freeze-dried, and ethylene oxide-sterilized cortical allografts have been recommended for the reconstruction of segmental cortical defects. Donor sites for microsurgical bone transfer have been identified experimentally in the dog, but these remain to be used clinically. This paper reviews the anatomy and structural characteristics of donor sites for microsurgical bone transfer. Potential clinical indications for microsurgical bone transfer in the dog are described.

Experimental studies on half-joint transplantation

This study was undertaken to examine the fate of articular cartilage in isogenic half-joint grafts implanted after storage by two-stage freezing. The antigenicity was evaluated quantitatively with regard to humoral and cellular immunological responses: none was seen in allogenic cartilage grafts. In the half-joint transplantations, the antigenicity of the cartilage need not be considered; only the antigenicity of cortical and cancellous bone must be reduced. Histologically, half-joint grafts stored at -196 degrees C were superior to fresh allografts. Two-stage freezing, therefore, appears to be useful for the storage of articular cartilage.

Factors affecting patency of venous allografts in miniature swine

In immunologically defined National Institutes of Health miniswine, a segment of internal jugular vein was anastomosed to the carotid artery as an interposition graft. Patency of swine major histocompatibility complex matched, one haplotype mismatched, and complete mismatched veins was 9.8, 6.3, and 3.0 weeks respectively (p = 0.009). More than 90% of mismatched and 20% of matched allografts developed a positive crossmatch before occlusion (p = 0.006). The mixed lymphocyte response did not predict graft occlusion. Treatment of 10 swine with cyclosporine (10 mg/kg/day) did not significantly improve patency for one haplotype mismatched grafts. In haplotype mismatched veins, cryopreserved grafts occluded more rapidly than noncryopreserved grafts: mean 2.4 versus 6.3 weeks, respectively (p = 0.002). In all cryopreserved vein grafts, alloantibody appeared at or after graft occlusion rather than before occlusion as seen with fresh allografts (p = 0.046). The mean patency of cryopreserved versus fresh autografts was 3.3 and greater than 32 weeks, respectively (p = 0.004). In summary, these results indicate that (1) allograft patency is related to the degree of swine major histocompatibility complex match and development of cytotoxic alloantibodies; (2) moderate-dose cyclosporine does not prolong allograft patency nor suppress development of antibody; (3) cryopreservation may accelerate graft occlusion through nonimmunologic mechanisms.

Factors affecting patency of venous allografts in miniature swine

In immunologically defined National Institutes of Health miniswine, a segment of internal jugular vein was anastomosed to the carotid artery as an interposition graft. Patency of swine major histocompatibility complex matched, one haplotype mismatched, and complete mismatched veins was 9.8, 6.3, and 3.0 weeks respectively (p = 0.009). More than 90% of mismatched and 20% of matched allografts developed a positive crossmatch before occlusion (p = 0.006). The mixed lymphocyte response did not predict graft occlusion. Treatment of 10 swine with cyclosporine (10 mg/kg/day) did not significantly improve patency for one haplotype mismatched grafts. In haplotype mismatched veins, cryopreserved grafts occluded more rapidly than noncryopreserved grafts: mean 2.4 versus 6.3 weeks, respectively (p = 0.002). In all cryopreserved vein grafts, alloantibody appeared at or after graft occlusion rather than before occlusion as seen with fresh allografts (p = 0.046). The mean patency of cryopreserved versus fresh autografts was 3.3 and >32 weeks, respectively (p = 0.004). In summary, these results indicate that (1) allograft patency is related to the degree of swine major histocompatibility complex match and development of cytotoxic alloantibodies; (2) moderate-dose cyclosporine does not prolong allograft patency nor suppress development of antibody; (3) cryopreservation may accelerate graft occlusion through nonimmunologic mechanisms.

Fresh versus cryopreserved cultured allografts for the treatment of chronic skin ulcers

Both fresh and cryopreserved cultured epithelial allografts were used to treat 20 patients with a total of 30 chronic ulcers. A profound stimulation of host epithelialization from the wound edges and epidermal appendages was observed. Our data suggest that allografts, prepared using a simple cryopreservation technique, retain their morphological and functional characteristics, and are as effective as fresh allografts in healing chronic ulceration.

Diaphyseal Allograft: Eight-year Evaluation in a Dog

The fate of a 4 cm fresh diaphyseal cortical allograft, placed in the femur of a dog, is documented by microradiogram, microangiogram and correlated histology, eight years after implantation. Transplanted bone remained. The graft had not vascularized, it remained acellular, active resorption was not evident and new bone had been deposited directly on the transplanted bone.

The fate of articular cartilage after transplantation of fresh and cryopreserved tissue-antigen-matched and mismatched osteochondral allografts in dogs.

The long-term success of massive osteochondral allografts depends not only on the incorporation of the transplanted articular cartilage. Osteochondral allografts are immunogenic, and, once an immune response is stimulated by exposure to donor cellular antigens, the cartilage becomes vulnerable to direct injury by cytotoxic antibodies or by lymphocytes, or to indirect injury by inflammatory mediators and enzymes induced by the immune response. To clarify the role of histocompatibility antigen-matching on the health of transplanted articular cartilage, we orthotopically implanted canine leukocyte antigen-matched and mismatched proximal osteochondral allografts of the radius, both fresh and cryopreserved, in beagles. Four groups of dogs received: (1) canine leukocyte antigen-mismatched frozen allografts, (2) canine leukocyte antigen-mismatched fresh allografts, (3) canine leukocyte antigen-matched fresh allografts, or (4) canine leukocyte antigen-matched frozen allografts. In twelve of the dogs, the contralateral leg was subjected to a sham operation, and in ten of the dogs, the proximal part of the radius was removed and replaced as an autogenous graft control. All animals were followed for eleven months after the operation and then were killed. The cartilage of the grafts was evaluated grossly, histologically, and biochemically. The biochemical analysis consisted of measurement of dry weight, content of glycosaminoglycan and hydroxyproline, and galactosamine-to-glucosamine ratios. Analyses of variance were used to study the effect of tissue antigen-matching and freezing on degradation of cartilage. During the study, no dog had grossly obvious clinical abnormalities, all host-graft interfaces healed, and no joints dislocated. The gross appearance of the cartilage was normal for both the joints that had an autogenous graft and those that were subjected to the sham operation. The cartilage of all allografts was thinned, dull, and roughened. The synovial membrane of all of the joints that had been operated on was mildly fibrotic and hyperplastic, but only that of the dogs that had an allograft was severely fibrotic and hyperplastic and demonstrated an inflammatory response. The inflammatory response was most severe in joints that had received a fresh canine leukocyte antigen-mismatched allograft. Invasive pannus was more frequent in joints that had received a fresh graft, particularly those that had received a canine leukocyte antigen-mismatched allograft, and cartilage was sometimes eroded to subchondral bone. Freezing was harmful to the cartilage. Very few cells survived the freezing procedure, and frozen grafts received s significantly worse histological scores had significantly less glycosaminoglycans and had a lower ratio of galactosamine to glucosamine than fresh grafts.

Allograft incorporation: a biomechanical evaluation in a rat model.

Allogeneic bone from Sprague-Dawley rat femurs was subjected to levels of freezing and/or irradiation that are known to have produced changes in the associated immune responses to these grafts. This bone was transplanted into an experimentally created gap in the femur of Lewis rats. The subsequent healing of the transplants in the Lewis rats was studied at 2, 4, 8, and 16 weeks after transplantation using torsion testing to failure. There was no clear advantageous biologic response in the union of the grafted material accompanying the alterations in immunologic response as measured by biomechanical testing of the proximal osteosynthesis site in torsion. The torsional strength of all of these groups remained lower than that of intact bone. Furthermore, none of the frozen and/or irradiated allografts exceeded the strength of the fresh allograft at a statistically significant level.