Allograft transplantation in the knee: tissue regulation,procurement, processing,and sterilization

Use of musculoskeletal allografts has become increasingly popular, with widespread use among knee surgeons. The advantages and disadvantages of their use have been documented. In the knee, allografts are used for ligament reconstruction, meniscal transplantation, and articular surface reconstruction. The purpose of this review is to present issues surrounding the allograft industry, including regulation of tissues and tissue banks and procurement, processing, sterilization, and storage of allograft tissue. Tissue bank regulation is ultimately under the jurisdiction and authority of the Food and Drug Administration; some individual states regulate tissue banks. The American Association of Tissue Banks is a scientific organization that encourages education, research, and voluntary accreditation of tissue banks. It promotes safety and standards for retrieval, processing, storage, and distribution of transplantable human tissue. Allograft tissues are generally harvested and processed aseptically, which may not prevent contamination. Tissue sterilization is difficult and controversial. Tissue banks historically have used one of two methods of sterilization, ethylene oxide or gamma radiation. Both methods have risks and benefits. Newer methods of sterilization are being developed. Allograft tissue that is not transplanted fresh can be freeze-dried or deep frozen for storage. Ultimately, allograft transplantation in the knee facilitates knee form and function and enhances the patient's quality of life. Orthopaedic surgeons who use allograft tissue must understand the tissue banking process to provide safe and effective tissues to their patients.     

Allograft update: the current status of tissue regulation, procurement, processing, and sterilization

Allografts are commonly used during sports medicine surgical procedures in the United States, and their frequency of use is increasing. Based on surgeon reports, it is estimated that more than 60 000 allografts were used in knee surgeries by members of the American Orthopaedic Society for Sports Medicine in 2005. In the United States, there are governmental agencies and other regulatory bodies involved in the oversight of tissue banks. In 2005, the Food and Drug Administration finalized its requirements for current good tissue practice and has mandated new rules regarding the "manufacture" of allogenic tissue. In response to well-publicized infections associated with the implantation of allograft tissue, some tissue banks have developed methods to sterilize allograft tissue. Although many surgeons have significant concerns about the safety of allografts, the majority believe that sterilized allografts are safe but that the sterilization process negatively affects tissue biology and biomechanics. However, most know very little about the principles of sterilization and the proprietary processes currently used in tissue banking. This article will review the current status of allograft tissue regulation, procurement, processing, and sterilization in the United States.     

The effects of chlorhexidine irrigation solution on contaminated bone-tendon allografts

The purpose of this study was to determine an expedient and effective method for disinfecting contaminated human bone-tendon allografts. The first part of this study used beef muscle and cadaveric human tissues to determine the most effective solution and volume to decontaminate tissues inoculated with four different organisms: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Of the solutions tested (benzalkonium chloride, castile soap, castile soap followed by benzalkonium chloride, triple antibiotic, chlorhexidine gluconate, and chlorhexidine gluconate/triple antibiotic), only the 4% chlorhexidine power irrigation solution and 4% chlorhexidine/triple antibiotic bath completely disinfected all tissues. Work in part 2 revealed that a 2% chlorhexidine irrigation solution was equally effective as the 4% solutions. Part 3 of the study involved human Achilles tendon-calcaneus allografts. We found similar results: 3 liters of 2% chlorhexidine power irrigation solution thoroughly removed all microorganisms from the contaminated tissues. All control allografts irrigated with normal saline solution alone revealed positive bacterial growth for all four organisms after 72 hours' growth on sheep blood agar. Total decontamination time was 10 to 12 minutes. Two percent chlorhexidine irrigation solution may be an effective method for decontaminating human bone-tendon allografts challenged with a polymicrobial inoculum. This method of disinfecting bone-tendon allografts is at least five times more expeditious than methods in previously reported studies.     

A biomechanical analysis of solvent-dehydrated and freeze-dried human fascia lata allografts. A preliminary report.

This study compares the basic mechanical properties of two groups of commercially available fascia lata allografts processed by different means (solvent-dehydrated and sterilized via gamma radiation, and freeze-dried without secondary sterilization). The results reveal significantly (P less than 0.05) higher stiffness, higher maximum load to failure, and higher maximum load per unit width of graft with the solvent-dried as opposed to the freeze-dried fascia lata. Subsections of individual solvent-dried specimens were also more uniform in their mechanical properties than those of the freeze-dried allografts. Clinical relevance: Fascia lata is used as a graft material in a variety of orthopaedic procedures. Allograft fascia lata offers an increased cross-sectional area of material and eliminates the morbidity associated with the harvesting of autologous tissues. However, the structural uniformity of such large grafts has been questioned. Processing techniques used in the sterilization and storage of such grafts is varied and represents a potential source of variation in the mechanical properties of allograft specimens. The results of this study suggest that a commercially available solvent-dehydrated form of fascia lata provides a more suitable grafting material than freeze-dried specimens obtained from tissue banks.     

The biomechanical effects of low-dose irradiation on bone-patellar tendon-bone allografts

BACKGROUND: Despite evidence that low-dose irradiation of 2 Mrad (20 kGy) is not virucidal for patellar tendon allografts and reduces tissue strength, many tissue bank protocols include low-dose irradiation. HYPOTHESIS: Maintaining tissue mechanical integrity may be particularly relevant toward accelerated rehabilitation of the injured knee, where the cyclic function of patellar tendon allografts is critical. STUDY DESIGN: Controlled laboratory study. METHODS: The cyclic and failure mechanical properties of paired bone-patellar tendon-bone allografts, with and without current low-dose irradiation of 20 kGy, were evaluated. Specimens were loaded from 50 N to 250 N for 1000 cycles at 0.5 Hz and subsequently loaded to failure at a strain rate of 100% per second. RESULTS: After 1000 cycles, grafts elongated 27% more when irradiated than when not (4.4 +/- 1.5 mm vs 3.4 +/- 1.0 mm; P = .03). Failure load averaged 1965 +/- 512 N for irradiated grafts and 2457 +/- 647 N for nonirradiated grafts (P = .007). CONCLUSIONS: The diminished strength of irradiated grafts may contribute to overt anterior cruciate ligament graft failure, and the increase in cyclic elongation may also be detrimental to graft function. CLINICAL RELEVANCE: These results suggest that one should consider the use of nonirradiated allografts as an alternative to irradiated grafts in anterior cruciate ligament reconstruction.     

Sterilization of bone allografts by microwave and gamma radiation

Abstract Purpose: Bone allografts are used to enhance healing in osteotomies, arthrodesis, fractures and to replace bone loss resulting from tumour or trauma. However, a major concern associated with the bone allografts is the potential for disease transmission. Various sterilization techniques have been developed to prevent infection through allografts. This study was undertaken with the aim of exploring the use of microwave radiation for sterilization of bone allografts and to compare with gamma radiation sterilization. Materials and methods: Bone allografts were processed from femoral heads obtained from living donors. The effect of microwave and gamma radiation on the bacteria isolated from bone allograft was evaluated. The microwave radiation treatment was performed at 2450 MHz (frequency) for varying lengths of time at maximum power 900 Watts (W). Viability of three Gram-positive bacteria - Bacillus subtilis, Corynebacterium, Staphylococcus aureus and three Gram-negative bacteria - Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa was examined after irradiation of bacterial suspensions and contaminated processed bone allografts. The sterility test of microwave and gamma irradiated bone allograft was carried out in accordance with ISO (International Organization for Standardization) 11737-2. Results: Microwave irradiation (2450 MHz and 900 W) of bacterial isolates resulted in complete inactivation within 60 seconds. The contaminated bone samples showed no growth of organisms after 2 minutes of exposure to microwave irradiation. No viable counts were detected in bone grafts inoculated with Gram-negative bacterial species on gamma irradiation to a dose of 15 kGy. Bones contaminated with Gram-positive bacteria required a higher dose of 20 kGy for complete inactivation. Conclusions: The study shows that sterilization of contaminated femoral head bone allografts can be achieved by short exposure of 2 min to 2450 MHz and 900 W microwave radiation.     

Sterilization of bone allografts by microwave and gamma radiation

Abstract

Purpose: Bone allografts are used to enhance healing in osteotomies, arthrodesis, fractures and to replace bone loss resulting from tumour or trauma. However, a major concern associated with the bone allografts is the potential for disease transmission. Various sterilization techniques have been developed to prevent infection through allografts. This study was undertaken with the aim of exploring the use of microwave radiation for sterilization of bone allografts and to compare with gamma radiation sterilization.

Materials and methods: Bone allografts were processed from femoral heads obtained from living donors. The effect of microwave and gamma radiation on the bacteria isolated from bone allograft was evaluated. The microwave radiation treatment was performed at 2450 MHz (frequency) for varying lengths of time at maximum power 900 Watts (W). Viability of three Gram-positive bacteria – Bacillus subtilis, Corynebacterium, Staphylococcus aureus and three Gram-negative bacteria – Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa was examined after irradiation of bacterial suspensions and contaminated processed bone allografts. The sterility test of microwave and gamma irradiated bone allograft was carried out in accordance with ISO (International Organization for Standardization) 11737-2.

Results: Microwave irradiation (2450 MHz and 900 W) of bacterial isolates resulted in complete inactivation within 60 seconds. The contaminated bone samples showed no growth of organisms after 2 minutes of exposure to microwave irradiation. No viable counts were detected in bone grafts inoculated with Gram-negative bacterial species on gamma irradiation to a dose of 15 kGy. Bones contaminated with Gram-positive bacteria required a higher dose of 20 kGy for complete inactivation.

Conclusions: The study shows that sterilization of contaminated femoral head bone allografts can be achieved by short exposure of 2 min to 2450 MHz and 900 W microwave radiation.     

Positive culture in allograft ACL-reconstruction: what to do?

The transmission of disease or infection from the donor to the recipient is always a risk with the use of allografts. We carried out a research study on the behavioural pattern of implanted allografts, which were initially stored in perfect conditions (all cultures being negative) but later presented positive cultures at the implantation stage. Because there is no information available on how to deal with this type of situation, our aim was to set guidelines on the course of action which would be required in such a case. We conducted a retrospective study of 181 patients who underwent an ACL reconstruction using BPTB allografts. All previous bone and blood cultures and tests for hepatitis B and C, syphilis and HIV were negative. An allograft sample was taken for culture in the operating theatre just before its implantation. The results of the cultures were obtained 3–5 days after the operation. We had 24 allografts with positive culture (13.25%) after the implantation with no clinical infection in any of these patients. Positive cultures could be caused by undetected contamination while harvesting, storing or during manipulation before implantation. The lack of clinical signs of infection during the follow-up of our patients may indicate that no specific treatment—other than an antibiotic protocol—would be required when facing a case of positive culture of a graft piece after its implantation.     

Aseptically processed and chemically sterilized BTB allografts for anterior cruciate ligament reconstruction: a prospective randomized study

Purpose

To compare the clinical outcomes of bone-patellar tendon-bone (BTB) allografts processed via a novel sterilization system with the traditional aseptically processed BTB allografts for anterior cruciate ligament (ACL) reconstruction.

Methods

A total of 67 patients undergoing ACL reconstruction at 6 independent investigation sites were randomized into one of two intervention groups, BioCleanse-sterilized or aseptic BTB allografts. Inclusion criteria included an acute, isolated, unilateral ACL tear, and exclusion criteria included prior ACL injury, multi-ligament reconstruction, and signs of degenerative joint disease. Post-op examiners and patients were blinded to graft type. Patients were evaluated at 6, 12, and 24 months. Clinical outcomes were compared using the IKDC, a KT-1000 knee arthrometer, level of effusion, and ranges of motion (ROM).

Results

After randomization, 24 patients received aseptic BTB allografts and 43 patients received BioCleanse-sterilized allografts. Significant improvement in IKDC scores (P < 0.0001) as well as KT-1000 results (P < 0.0001) was noted over the 24-month period for both groups. IKDC or KT-1000 results were not significantly different between groups at any time point. Active flexion ROM significantly improved from pre-op to 24-month follow-up (P < 0.0001) with no difference between groups at any time point. Active extension ROM did not differ significantly between the two groups.

Conclusions

These results indicate that the sterilization process, BioCleanse, did not demonstrate a statistical difference in clinical outcomes for the BTB allograft at 2 years. The BioCleanse process may provide surgeons with allografts clinically similar to aseptically processed allograft tissue with the benefit of addressing donor-to-recipient disease.

Level of evidence

II.     

Mobilization of bone marrow-derived mesenchymal stem cells into the injured tissues after intraarticular injection and their contribution to tissue regeneration

The purpose of present study was to evaluate active mobilization effect of mesenchymal stem cells (MSCs) into injured tissues after intraarticular injection of MSCs, and to evaluate their contribution to tissue regeneration. MSCs, which were obtained from green fluorescent protein (GFP) transgenic Sprague–Dawley (SD) rat and cultivated, were injected into normal SD rats in which multiple tissues had been injured including anterior cruciate ligament (ACL), medial meniscus, and articular cartilage of the femoral condyles. At 4 weeks after injection of MSCs, fluorescent microscopic observation, immunohistochemical or histological examinations were performed to evaluate mobilization of MSCs into injured tissue and their contribution to tissue regeneration. In the group of 1 × 10⁶ MSCs injection, GFP positive cells could mobilize into the injured ACL alone in all 8 knees. In the group of 1 × 10⁷ MSCs injection, GFP positive cells were observed in the injured site of ACL in all 8 knees and in the injured site of medial meniscus and cartilage of femoral condyles in 6 of 8 knees. More interestingly, extracellular matrix stained by toluidine blue was present around GFP positive cells in the injured femoral condyles cartilage and medial meniscus, indicating tissue regeneration. Intraarticularly injected MSCs could mobilize into the injured tissues, and probably contributed to tissue regeneration. This study demonstrated the possibility of intraarticular injection of MSCs for the treatment of intraarticular tissue injuries including ACL, meniscus, or cartilage. If this treatment option is established, it can be minimally invasive compared to conventional surgeries for these tissues.     

Meniscal substitutes – human experience

A number of clinical series have described the effect of meniscus allograft replacement in humans. The general indication has been disabling pain following loss of a meniscus in a skeletally mature individual. Overall, healing of the graft to the capsule occurs in up to 80% of all transplants. Revascularization and cell repopulation is found in all grafts but is highly variable. The risk for graft failure seems to be greater with irradiated grafts and in patients with grade III or IV osteoarthritic changes. In most series, patients experienced a decrease in pain and an increase in activity level postoperatively. In many series, concominant surgery (cruciate ligament reconstruction or osteotomy) had been performed. Meniscus replacement with frozen or cryopreserved allografts seems to give the most promising short-term results in patients with post-meniscectomy pain. Controlled, randomized prospective studies are needed to confirm a long-term benefit and better define transplantation indications. Viable meniscus allografts seem to survive transplantation, as donor cells were found in the graft after 2 years. Clinically, pain was reduced and activity increased following transplantation, but after 4 years some of these gains were lost. There was no correlation between postoperative findings on MRI and clinical outcome. Meniscal replacement with a quadriceps tendon autograft in humans resulted in pain reduction, but at second-look arthroscopy, only 2 of 9 tendon autografts looked like a meniscus. Six were in position but still looked like tendons. Total medial meniscus replacement by quadriceps tendon autrograft is still an experimental procedure. There is no proof at present that meniscal substitutes (meniscus allografts or tendon autografts) in humans can protect the hyaline cartilage of the knee from the degeneration, following loss of a meniscus. There is some evidence in animal experiments that under circumstances not yet exactly known, a meniscus substitute can have a protective effect on articular cartilage. Three factors have been identified that prevent proper meniscal function: poor fixation of the meniscal horns, no contact of the graft with the articulating surfaces under load and incorrect positioning of the horns. Meniscal allograft transplantation sensitizes humoral and cell mediated immune systems. Bone plugs attached to meniscal allograft tissue may increase cell surface antigenicity. Deep freezing and especially freeze drying of meniscal tissue decreases host immunogenicity. Cryopreservation maintains the content of donor HLA encoded antigens and is likely more sensitizing to the host. The clinical importance of immune responses to meniscal allografts is not known, but it has not been shown to result in graft failure or rejection. Prospective studies are needed.     

Osteochondral allograft transplantation

Experience with fresh osteochondral allografting for cartilage defects in the knee now extends two decades. Clinical outcomes and basic scientific investigations have supported the theoretic basis for this procedure. At the University of California, San Diego, our experience has encouraged us to continue to offer this procedure as a primary treatment for both large and small articular cartilage defects in the young knee. The success rate of fresh osteochondral allografting, particularly in isolated femoral condylar defects, compares favorably with other presently available cartilage repair and resurfacing techniques. In our second hundred cases, which we are currently evaluating, failure of monopolar allografts has been exceedingly rare in short-term follow-up. Fresh osteochondral allografting also appears to be effective in treating larger osteochondral lesions, where there are few other attractive alternatives. Fresh osteochondral allografts can thus be used to treat a wide spectrum of articular pathology. Technical refinements, and improvement in our understanding of graft-host interaction, as well as chondrocyte biology, should continue to improve clinical results. Disadvantages of fresh osteochondral allografting include the relative paucity of donor tissue, complexities in procurement and handling, and the possibility of disease transmission through the transplantation of fresh tissue. At present, only institutions that have overcome these obstacles seem capable of routinely performing this type of articular cartilage transplantation. In the future, as tissue banking and cartilage storage technology advance, fresh allograft tissue may become more available, allowing more widespread use of fresh osteochondral allografting in the treatment of articular cartilage lesions.     

Lyophilization does not inactivate infectious retrovirus in systemically infected bone and tendon allografts

BACKGROUND: A review of multiple transplantations of human immunodeficiency virus-infected musculoskeletal allografts found that recipients of lyophilized (freeze-dried) bone or tendon from an infected donor all tested negative for human immunodeficiency virus. The finding that 75% of the recipients of fresh-frozen bone from the same donor contracted human immunodeficiency virus has led to speculation that freeze-drying may render retroviral-infected musculoskeletal allografts noninfectious. HYPOTHESIS: Lyophilization does not inactivate retrovirus in systemically infected bone and tendon. STUDY DESIGN: Controlled laboratory study. METHODS: Tendons and cortical bone segments from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cultured in the presence of fresh-frozen or freeze-dried cortical bone or tendon segments. At each passage, feline leukemia virus p27 antigen was measured in media by enzyme-linked immunosorbent assay, and feline leukemia virus (pro)viral nucleic acids were quantified by real-time quantitative polymerase chain reaction in the DNA extracted from cells. RESULTS: Enzyme-linked immunosorbent assay results and quantitative polymerase chain reaction results demonstrated retroviral antigen and proviral DNA in all cultured cell replicates after exposure to fresh-frozen or freeze-dried bones or tendons. CONCLUSION: Freeze-drying (lyophilization) of retroviral-infected cortical bone and tendon does not inactivate retrovirus. Clinical Relevance: These results conclusively demonstrate that freeze-drying should not be relied on to inactivate infectious retrovirus in systemically infected musculoskeletal allografts.     

Quantitation of lipid in biological tissue by chemical shift magnetic resonance imaging

A method combining several previously used approaches is described for the rapid, accurate quantitation of the fat content of biological tissue based on chemical shift images (CSI) corrected for magnetic field inhomogeneity, and compensated for T₁, and T₂, effects. The gravimetrically determined lipid content of fatty tissues (pork fat, rabbit and human liver) that had been differentially depleted of lipid by chloroform extraction correlated well (r = 0.99) with the lipid image intensities of the respective tissues. This multi-point CSI method was used to quantitate lipid in fresh fatty human liver tissue (wet and dry) containing varying amounts of lipid. Plots of integrated lipid intensity versus tissue lipid content gave straight parallel lines for hydrated (r = 0.94) and dehydrated (r = 0.98) tissues, permitting determination of a proportionality constant for measuring absolute amounts of lipid present in a specific biological tissue. These results suggest the feasibility of using the method in vivo for absolute quantitation of lipid in tissues of agricultural (e.g. pork, beef) and medical (e.g. human liver) interest.     

Osteochondral allografts in the treatment of articular cartilage injuries of the knee

Osteochondral allografts have demonstrated encouraging clinical and scientific success in the treatment of full-thickness articular cartilage defects in multiple anatomic locations including the knee. The use of fresh grafts has shown the greatest potential for clinical success. There has been growing interest in cryopreservation techniques and the use of cryopreserved grafts owing to the delay in obtaining grafts secondary to regulatory testing, encouraging laboratory data surrounding their use, and the potential for more effective tissue banking. This article reviews the indications, operative technique, and clinical outcomes using osteochondral allografts for full-thickness articular cartilage defects in the knee.     

颌面部破片复合冲击伤后软组织损伤特点及其临床意义

目的　探讨颌面部破片复合冲击伤 (破 -冲伤 )后软组织损伤特点及其临床意义。方法　 15只犬按伤后 0 ,6,2 4 ,72h、7d随机分为 5组 ,每组 3只。采用雷管、滑膛枪以及同步测速及测压系统 ,建立颌面部爆炸伤模型 ,造成犬颌面部破 -冲伤。记录致伤参数 ,观察动物伤情及局部距创缘不同距离的软组织伤后各时相点的病理变化。　结果　 15只犬均呈左咬肌区软组织切线伤 ,创面污染严重 ,距伤区创缘 0～ 0 .8cm处肌组织完全坏死 ,0 .9～ 1.6cm处肌组织呈灶性坏死 ,正常组织与坏死组织相嵌存在 ;皮肤坏死范围距创缘 0 .2cm。　结论　颌面部破 -冲伤后软组织损伤范围广 ,污染严重 ,肌组织清创范围应距创缘 0 .9～ 1.6cm ,皮肤切除不小于 0 .2cm。     

Effects of a novel sterilization process on soft tissue mechanical properties for anterior cruciate ligament allografts

BACKGROUND: Allograft anterior cruciate ligament reconstruction provides benefits such as earlier return to activities and less pain, but concerns remain regarding potential infection and biomechanical stability. HYPOTHESIS: There is no difference in biomechanical properties of soft tissue allografts treated with the Biocleanse tissue sterilization process compared with irradiated and fresh-frozen allografts. STUDY DESIGN: Controlled laboratory study. METHODS: Thirty-six tibialis anterior allografts were equally divided between Biocleanse, irradiated, and fresh-frozen groups. Grafts were measured for cross-sectional area and looped over a smooth rod with the free sutured ends of the graft fixed in custom clamps. Specimens were tensioned to 10 N for 2 minutes and then loaded between 50 and 300 N for 1000 cycles followed by a failure test. Data for creep (mm); stiffness (N/mm) at cycles 1, 10, 100, and 1000; failure load (N); and failure stress (MPa) were compared with a one-way analysis of variance (P < .05). RESULTS: There were no statistically significant differences in creep between groups. Sterilized groups (irradiated = 144.7 +/- 17.7 N/mm and Biocleanse = 146.5 +/- 28.2N/mm) were significantly stiffer during the first cycle than the fresh-frozen group (117.8 +/- 15.7 N/mm, P < .005) without statistically significant differences for subsequent cycles. There were no differences between groups for either failure load (fresh-frozen = 1665 +/- 291.3 N, irradiated = 1671.9 +/- 290.2 N, Biocleanse = 1651.6 +/- 377.4 N) or failure stress. CONCLUSION: Data for "time-zero" physiologic stiffness and failure loads indicate that the Biocleanse process does not adversely affect the biomechanical properties of the allograft material. CLINICAL RELEVANCE: This novel sterilization technique may provide surgeons with potential allograft material with similar biomechanical properties to native tissue.     

Overview of procurement, processing, and sterilization of soft tissue allografts for sports medicine

Musculoskeletal allografts are commonly used in orthopedic surgery and have become increasingly popular. Their indications have widened as an alternatives to autografts. A tissue processing industry has secondarily evolved. An increasing number of accredited tissue banks are providing donor screening, procurement, processing, storage, and distribution of tissue. Multiple factors play a role for a graft to be successfully implanted: sterility, reduction of antigenicity, and preservation of its biologic and biomechanical properties. A rare but catastrophic complication that has raised concern is disease transmission. Controversies exist on which is the best way to produce a strong, disease-free graft. There is no current standard, but as allograft technology evolves, surgeons need to be aware of the regulations and policies surrounding allograft tissue procurement and processing to provide the best outcomes in transplanted patients.