The effect of sterilization and storage on the viscoelastic properties of human tendon allografts - Continued: Storage for 0 to 4 months.

The role of donor-derived tendons, also known as allografts, in anterior cruciate ligament replacement surgeries is steadily increasing. Before surgery, temporary storage and, in most cases, sterilization are essential. It is, thus, crucial to determine how these procedures alter the grafts' biomechanical properties. The purpose of this research was to analyze the effect of different sterilization methods (native, frozen, frozen + 21 kGy gamma irradiation, frozen + 21 kGy electron beam irradiation) and storage durations (0 to 4 months) on the deformation and creep of two tendon types (tibialis anterior, peroneus longus). 80 tibialis anterior and 83 peroneus longus tendons from 51 human cadavers were included. The samples were removed, placed in a radio-cryoprotectant solution, then slowly cooled, sterilized and stored at -80 °C. All groups were subject to 60 s static creep test with 250 N load. Deformation during the loading phase, creep during static loading, and the ratio of these two were evaluated. Deformation at the end of the loading phase and creep consistently exhibited significantly smaller values in the tibialis anterior compared to the peroneus longus type, as well as in electron beam-sterilized grafts as opposed to gamma beam-sterilized ones. Prolonged storage periods (within 0 to 4 months) resulted in a notable increase in these values, particularly in deformation. Based on the experimental data, the tibialis anterior tendon type and sterilization by gamma beam irradiation are better choices for anterior cruciate ligament reconstruction than the peroneus longus and sterilization by electron beam. Increased storage time affects negatively the evaluated mechanical properties.

Effects of SCCO2, Gamma Irradiation, and Sodium Dodecyl Sulfate Treatments on the Initial Properties of Tendon Allografts.

Sterile and decellularized allograft tendons are viable biomaterials used in reconstructive surgeries for dense connective tissue injuries. Established allograft processing techniques including gamma irradiation and sodium dodecyl sulfate (SDS) can affect tissue integrity. Supercritical carbon dioxide (SCCO2) represents a novel alternative that has the potential to decellularize and sterilize tendons with minimized exposure to denaturants, shortened treatment time, lack of toxic residues, and superior tissue penetration, and thus efficacy. This study attempted to develop a single-step hybrid decellularization and sterilization protocol for tendons that involved SCCO2 treatment with various chemical additives. The processed tendons were evaluated with mechanical testing, histology, scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy. Uniaxial mechanical testing showed that tendons treated with SCCO2 and additive NovaKillTM Gen2 and 0.1% SDS had significantly higher (p < 0.05) ultimate tensile stress (UTS) and Young's modulus compared to gamma-irradiated and standard-SDS-treated tendons. This was corroborated by the ultrastructural intactness of SCCO2-treated tendons as examined by SEM and FTIR spectroscopy, which was not preserved in gamma-irradiated and standard SDS-treated tendons. However, complete decellularization was not achieved by the experimented SCCO2-SDS protocols used in this study. The present study therefore serves as a concrete starting point for development of an SCCO2-based combined sterilization and decellularization protocol for allograft tendons, where additive choice is to be optimized.

Radioprotection and cross-linking of allograft bone in the presence of vitamin E.

Bone allografts are the preferred method for bone augmentation in over 500,000 orthopedic surgical procedures in the US. Sterilization by ionizing radiation is the most effective method of minimizing the bioburden of bone allografts; however, radiation causes chain scission of collagen, resulting in the reduction of the allografts' mechanical strength. In this study, we doped bone allografts with vitamin E as radioprotectant using a novel two-step process to protect the collagen architecture against radiation damage and to preserve the mechanical strength of the construct. In addition, combining the radioprotectant with a cross-linking agent further minimized collagen degradation and further preserved the mechanical strength of the allografts. Both vitamin E and combined vitamin E/genipin-treated allograft were less cytotoxic to both osteoblasts and osteoclasts when compared to irradiated-only allografts. Host bone-allograft unionization was faster in a rat calvaria defect model with vitamin E-treated and combined vitamin E and genipin-treated allograft when compare to irradiated-only allografts. This method can enable the efficient and uniform radioprotective treatment of bone allograft of desired shapes for sterilization with improved mechanical strength and biointegration.

Lyophilized and gamma-sterilized allogeneic bone implant used as a spacer for advancement of a modified tibial tuberosity in the treatment of cranial cruciate ligament disease in dogs.

The objective of this study was to evaluate the use of a lyophilized and gamma-sterilized allogeneic freeze-dried bone wedge as a spacer for advancement of a modified tibial tuberosity (mTTA) in 16 knees that were clinically diagnosed with cranial cruciate ligament disease. Patients underwent radiography before the surgical procedure as well as immediately after surgery and at 30, 60, 90 and 120 days post-surgery, and their locomotion was evaluated at the same time points except for the immediate postoperative period. The surgical wounds were evaluated for signs of infection and rejection of the bone implant. Locomotion was graded on a scale of 0-5, with 0 indicating no limping and 5 indicating limb functional impotence. The "tibial-tibial bone-tibial implant" interfaces were evaluated radiographically, and each interface was assigned scores of 0-3, with 0 indicating no contact between the implant and adjacent bone and 3 indicating a bone bridge throughout the interface. The patients showed good clinical and radiographic recovery. The lyophilized bone spacer allowed for easy storage and transport and rapid and satisfactory execution of mTTA while showing resistance to drilling and fixation with screws in 87.5% of cases and a mean surgical time of 45.9 minutes. No immunogenic reactions were observed in 93.7% of the cases. One patient presented infection of the surgical focus, which showed remission after antimicrobial therapy. All patients showed functional recovery of the operated limb, with the number of clinically healthy patients being higher than those with claudication at 120 days (p ≤ 0.05). In all patients, it was possible to verify the incorporation of the bone implant into the tibia. Bone union occurred progressively, and the degrees of bone union observed on radiographs at postoperative days 60, 90, and 120 were significantly greater (p < 0.05) than those observed in the immediate postoperative period and at 30 days.

Ribose pre-treatment can protect the fatigue life of γ-irradiation sterilized bone.

Structural bone allografts are often sterilized with γ-irradiation to decrease infection risk, which unfortunately degrades the bone collagen connectivity, making the bone weak and brittle. In previous studies, we successfully protected the quasi-static mechanical properties of human cortical bone by pre-treating with ribose, prior to irradiation. This study focused on the quasi-static and fatigue tensile properties of ribose treated irradiated sterilized bone allografts. Seventy-five samples were cut from the mid-shaft diaphysis of human femurs into standardized dog-bone shape geometries for quasi-static and fatigue tensile testing. Specimens were prepared in sets of three adjacent specimens. Each set was made of a normal (N), irradiated (I) and ribose pre-treated + irradiation (R) group. The R group was incubated in a 1.2 M ribose solution before γ-irradiation. The quasi-static tensile and decalcified tests were conducted to failure under displacement control. The fatigue samples were tested under cyclic loading (10 Hz, peak stress of 45MP, minimum-to-maximum stress ratio of 0.1) until failure or reaching 10 million cycles. Ribose pre-treatment significantly improved significantly the mechanical properties of irradiation sterilized human bone in the quasi-static tensile and decalcified tests. The fatigue life of the irradiated group was impaired by 99% in comparison to the normal control. Surprisingly, the R-group has significantly superior properties over the I-group and N-group (p < 0.01, p < 0.05) (> 100%). This study shows that incubating human cortical bone in a ribose solution prior to irradiation can indeed improve the fatigue life of irradiation-sterilized cortical bone allografts.

Pre-clinical evaluation of bone allograft toughened with a novel sterilization method: An in vivo rabbit study.

Bone allografts often undergo γ-irradiation sterilization to decrease infection risk. However this consequently degrades bone collagen and makes the allograft brittle. Our laboratory has previously found that pre-treatment with ribose ex vivo protects the bone. However, it remains unclear whether or not ribose-treated γ-irradiated allografts are able to unite and remodel in vivo. Using New Zealand White rabbits (NZWr), we aimed to evaluate if ribose-treated allografts can unite with host bone (compared to untreated (fresh-frozen) and conventionally-irradiated allografts). A critically-sized defect was created in the radii of NZWr and reconstructed with allografts fixed with an intramedullary Kirschner wire. Healing and union were assessed at 2, 6, and 12 weeks post operation, with radiographs, µCT, static and dynamic histomorphometry, backscatter electron microscopy, and torsion testing. Intramedullary fixation achieved stable reconstructions and bony union in all groups and no differences were found in the radiographic and biomechanical parameters tested. Interestingly, γ-irradiated allografts had significantly less bone volume due to evident resorption of the grafts. In contrast, ribose pre-treatment protected γ-irradiated allografts from this bone loss, with results similar to the fresh frozen controls. In conclusion, ribose-pretreated γ-irradiated allografts were able to unite in vivo. In addition to achieving bony union with host bone, ribose pre-treatment may protect against allograft resorption. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Autograft or Allograft? Irradiated or Not? A Contrast Between Autograft and Allograft in Anterior Cruciate Ligament Reconstruction: A Meta-analysis.

PURPOSE: To compare the clinical outcomes and adverse events associated with irradiated and nonirradiated allografts in anterior cruciate ligament (ACL) reconstruction. METHODS: PubMed, Web of Science, and EMBASE were searched for randomized controlled trials from January 1990 to March 2018 to compare autograft with allograft in ACL reconstruction. Both objective and subjective outcomes of the function and adverse events were meta-analyzed. Two comparisons were summarized: (1) autograft and nonirradiated allograft and (2) autograft and irradiated allograft. The bias risk was based on the Cochrane Handbook for Systematic Reviews of Interventions. The overall risk ratio or weighted mean difference was calculated using a fixed- or random-effects model. Heterogeneity between studies was evaluated by the Q and the I2 statistics. RESULTS: Eleven trials were included in this review for meta-analysis. A total of 1,172 patients were involved (465 autograft and 461 nonirradiated allograft; 141 autograft and 138 irradiated allograft patients). The average follow-up varied from 2 to >10 years. The mean patient age varied from 22 to 32.8 years. The total failure rate was 2.5%. Our analyses demonstrated better clinical outcomes in autograft than irradiated allograft, which could be observed clearly through the International Knee Documentation Committee score (3.84; 95% confidence interval [CI], 1.93-5.76; P < .0001; I2 = 0%), Lysholm score (2.94; 95% CI, 0.66-5.22; P = .01; I2 = 0%), and Tegner score (0.14; 95% CI, -0.08 to 0.36; P = .22; I2 = 0%) with fewer adverse events 0.20 (95% CI, 0.11-0.39; P < .00001; I2 = 0%). There were no significant differences in autograft and nonirradiated allograft groups (P = .47, P = .27, P = .24, and P = .24, respectively). CONCLUSIONS: Autograft offered greater advantages in functional outcomes and adverse events than irradiated allograft in ACL reconstruction; however, there were no significant differences between autograft and nonirradiated allograft in ACL reconstruction. LEVEL OF EVIDENCE: Level II, meta-analysis of Level I and Level II studies.

Long-term results of acetabular reconstruction using irradiated allograft bone.

AIMS: The management of acetabular defects at the time of revision hip arthroplasty surgery is a challenge. This study presents the results of a long-term follow-up study of the use of irradiated allograft bone in acetabular reconstruction. PATIENTS AND METHODS: Between 1990 and 2000, 123 hips in 110 patients underwent acetabular reconstruction for aseptic loosening, using impaction bone grafting with frozen, irradiated, and morsellized femoral heads and a cemented acetabular component. A total of 55 men and 55 women with a mean age of 64.3 years (26 to 97) at the time of revision surgery are included in this study. RESULTS: At a mean follow-up of 16.9 years, there had been 23 revisions (18.7%), including ten for infection, eight for aseptic loosening, and three for dislocation. Of the 66 surviving hips (58 patients) that could be reassessed, 50 hips (42 patients; 75.6%) were still functioning satisfactorily. Union of the graft had occurred in all hips with a surviving implant. Survival analysis for all indications was 80.6% at 15 years (55 patients at risk, 95% confidence interval (CI) 71.1 to 87.2) and 73.7% at 20 years (eight patients at risk, 95% CI 61.6 to 82.5). CONCLUSION: Acetabular reconstruction using frozen, irradiated, and morsellized allograft bone and a cemented acetabular component is an effective method of treatment. It gives satisfactory long-term results and is comparable to other types of reconstruction. Cite this article: Bone Joint J 2018;100-B:1449-54.

Effects of supercritical fluid CO2 and 25 kGy gamma irradiation on the initial mechanical properties and histological appearance of tendon allograft.

Tendon allografts, when autograft options are limited or when obtaining an autograft is not aligned with the patients' best interest, play an important role in tendon and ligament reconstruction. To minimize the risk of infectious disease transmission tissue banks perform screening tests and the allografts cleaned are sterilized. The current study examines and compares the initial mechanical properties and histological appearance of supercritical CO2 (SCCO2)-treated and gamma-irradiated porcine extensor tendons. Thirty intact porcine forelimb extensor tendons randomized equally into three groups: control group, gamma-irradiation group, and SCCO2-treated group. Once treated, histological assessment and histomorphologic measurements were made on the histological sections obtained from each tendon while stiffness and ultimate failure loads were evaluated from tensile testing. Histological evaluation of gamma-irradiated tendons showed significant disruption to the hierarchical morphology of the fascicle bundles, which was not evident in SCCO2-treated specimens. Histomorphologic measurements showed a significant increase for measured dead space (void) between tendon fibrils of the gamma-irradiated group comparing to both control and SCCO2 treated groups (p < 0.01). There was a significant reduction in the ultimate failure load for tendons treated by gamma-irradiation compared to the control group (p < 0.05). No statistically significant difference was detected between control and SCCO2-treated tendons in the ultimate failure load. Stiffness values were not significantly different between three-study groups. This study suggests that while gamma-irradiation has a deleterious effect on mechanical properties of tendon tissue, SCCO2 does not alter the biomechanical properties and the histological structure of porcine extensor tendons.

How Variable Are Achilles Allografts Used for Anterior Cruciate Ligament Reconstruction? A Biomechanical Study.

BACKGROUND: Allograft tendon used in anterior cruciate ligament reconstruction (ACLR) requires sterilization before implantation. Low-dose gamma irradiation is a means of sterilization that may minimize tissue damage. PURPOSE: To quantify the variability in mechanical properties between low-dose irradiated Achilles tendon allografts used for ACLR. STUDY DESIGN: Descriptive laboratory study. METHODS: A total of 15 intact outer-third Achilles tendon allograft specimens were collected from the remains of full Achilles allografts used for intraoperative ACLR at a single hospital. All grafts were obtained from a single tissue bank and underwent proprietary disinfection and low-dose gamma irradiation (1.5-2.5 Mrad). Biomechanical testing was carried out to measure tendon elongation, failure location during tensile testing, maximum stress, maximum strain, and modulus of elasticity. The mean and standard deviation were calculated for each outcome measure, and the variability between specimens was calculated by the coefficient of variation (CV). The effect of donor age on graft material properties was examined by use of linear regression. One-way analysis of variance was performed to compare differences in the mechanical properties across failure locations. RESULTS: During cyclic testing, tendon elongation averaged 1.4% ± 1.6% with a CV of 118%. During failure testing, the maximum stress averaged 12.2 ± 4.1 MPa, maximum strain averaged 21.0% ± 6.3%, and modulus of elasticity averaged 95.5 ± 30.8 MPa. The CVs for maximum stress, maximum strain, and modulus of elasticity were 34%, 30%, and 32%, respectively. Ten tendons failed in the midsubstance and 5 failed at the tendon-bone enthesis. No differences were noted in mechanical properties between grafts that failed in the midsubstance versus those that failed at the enthesis. Donor age did not correlate with allograft elongation during cyclic load or any of the material property measures during failure testing. CONCLUSION: The variabilities in the material properties and graft elongation during cyclic loading of Achilles tendon allografts used in ACLR fall within the range of properties reported in the literature for other ACLR allografts. Material properties do not differ by donor age or graft failure location observed during failure testing. CLINICAL RELEVANCE: Surgeons should be aware that there exists considerable variation in the mechanical properties of Achilles allograft tendons used for ACLR. This variability is difficult to detect by tissue bank screening or the treating surgeon's inspection and may contribute to the heterogeneity in outcomes of allograft ACLR.

Irradiated allograft versus autograft for anterior cruciate ligament reconstruction: A meta-analysis and systematic review of prospective studies.

BACKGROUND: Irradiated allografts and autografts are commonly used for anterior cruciate ligament (ACL) reconstruction. The outcomes between these two grafts are controversial. This meta-analysis and systematic review of prospective comparative studies was performed to compare the clinical outcomes, including knee functionality, stability, subjective evaluation, complications, and failure, of irradiated allografts and autografts in primary ACL reconstruction. MATERIALS AND METHODS: PubMed, Embase, and the Cochrane Library were searched from database inception to 12 August 2017 to identify prospective studies that compared irradiated allografts with autografts for primary ACL reconstruction. Randomized controlled trials were included in the meta-analysis. Prospective cohort studies were included in the systematic reviews. Two reviewers independently assessed the study quality and extracted relevant data. Statistical heterogeneity among the trials was evaluated by the chi-square and I-square tests. RESULTS: Four randomized controlled trials and two prospective cohort studies involving 18,835 patients met the inclusion criteria. In the meta-analysis, significant differences were observed in knee stability and subjective evaluation with respect to the KT-2000 score (p < .0001), pivot shift test (p = .001), anterior drawer test (p = .0001), Lachman test (p = .0002), subjective International Knee Documentation Committee (IKDC) score (p < .0001), Cincinnati knee score (p = .04), Lysholm score (p = .01), and Tegner score (p = .03). However, the differences in functional assessment in terms of the overall IKDC score (p = .21), range of motion (p = .94), Harner's vertical jump test (p = .09), Daniel's one-leg hop test (p = .50), and complication rate (p = .34) were not significant between the two groups. Failure was reported in two prospective cohort studies in 302 of 14,829 (2%) patients in the autograft group and 157 of 3941 (4%) patients in the irradiated allograft group. CONCLUSION: Irradiated allografts are inferior to autografts for patients undergoing primary ACL reconstruction with respect to knee stability and subjective evaluation. However, no significant differences were found between the two groups in terms of function and complication. The robustness of the findings might need to be further validated because of the limited number of randomized controlled trials. More randomized controlled trials with longer follow-ups are required to further evaluate the failure rate in the two groups.

The effect of ribose pre-treatment of cortical bone on γ-irradiation sterilization effectiveness.

Reconstruction of large skeletal defects is a significant and challenging issue. Tissue banks often use γ-irradiation (15-35 kGy) to sterilize bone allografts, which, however, drastically impairs the post-yield mechanical properties. In previous studies, we reported the development of a method that protects human bone collagen connectivity through ribose crosslinking while still undergoing γ-irradiation. Given these promising results, the next step was to determine if the presence of ribose within the bone tissue would interfere with the effectiveness of the γ-irradiation sterilization against bacteria. This study had two stages. The aim of the first stage was to assess the protective effect of ribose in solution using a Bacillus pumilus spore strip model. The aim of the second stage was to assess the protective effect of ribose (R) on spores within a human cortical bone model in comparison to conventionally irradiated bone (I). Treatment of B. pumilus spore strips with ribose in solution led to temperature-dependent effects on spore viability versus spore strips treated with PBS alone. Ribose solution at 60 °C led to a notable two logs decrease in spore count relative to PBS at 60 °C. In the human bone model, the number of spores in the I and R groups were greatly decreased in comparison to the non-irradiated N group. No spore colonies were detected in the R group (n = 4) whereas two of the four plates of group I formed colonies. This study provides evidence that the method of pre-treating bone with ribose crosslinking prior to irradiation sterilization, while improving irradiation sterilized bone allograft quality, also may improve the effectiveness of the sterilization process.

Development of a novel method for the strengthening and toughening of irradiation-sterilized bone allografts.

Reconstruction of large skeletal defects is a significant and challenging issue. Bone allografts are often used for such reconstructions. However, sterilizing bone allografts by using γ-irradiation, damages collagen and causes the bone to become weak, brittle and less fatigue resistant. In a previous study, we successfully protected the mechanical properties of human cortical bone by conducting a pre-treatment with ribose, a natural and biocompatible agent. This study focuses on examining possible mechanisms by which ribose might protect the bone. We examined the mechanical properties, crosslinking, connectivity and free radical scavenging potentials of the ribose treatment. Human cortical bone beams were treated with varying concentration of ribose (0.06-1.2 M) and γ-irradiation before testing them in 3-point bending. The connectivity and amounts of crosslinking were determined with Hydrothermal-Isometric-Tension testing and High-Performance-Liquid-Chromatography, respectively. The free radical content was measured using Electron Paramagnetic Resonance. Ribose pre-treatment improved the mechanical properties of irradiation sterilized human bone in a pre-treatment concentration-dependent manner. The 1.2 M pre-treatment provided >100% of ultimate strength of normal controls and protected 76% of the work-to-fracture (toughness) lost in the irradiated controls. Similarly, the ribose pre-treatment improved the thermo-mechanical properties of irradiation-sterilized human bone collagen in a concentration-dependent manner. Greater free radical content and pentosidine content were modified in the ribose treated bone. This study shows that the mechanical properties of irradiation-sterilized cortical bone allografts can be protected by incubating the bone in a ribose solution prior to irradiation.

Does a different dose of gamma irradiation have the same effect on five different types of tendon allografts? - a biomechanical study.

INTRODUCTION: The goals of our study were to evaluate the biomechanical differences between five tendons and the changes in biomechanical properties caused by irradiation. METHODS: Achilles, quadriceps, semitendinosus + gracilis (STG), tibialis anterior (TA) and the peroneus longus (PL) were harvested from 30 donors. Group A contained 50 tendons without gamma irradiation. The groups were irradiated with a dose of 21 kGy (group B 50 tendons) and with a dose of 42 kGy (group C 50 tendons). The grafts were soaked in a radio-protectant solution and frozen at -80 °C. Cyclic loading tests were performed followed by load to failure tests. Young modulus of elasticity, maximum force, strain at tensile strength and strain at rupture were calculated. RESULTS: The Achilles tendons had significantly lower Young modulus than the TA (p = 0.0036) in group A. The Achilles showed significantly lower than PL (p = 0.000042) and TA (p = 0.00142) in group B and C. The quadriceps and the ST (p = 0.0037) provided poorer values than the TA (p = 0.0432) in group C. We found no difference in maximum loads among the tendons in group A. The maximum load of the Achilles and quadriceps showed better results than the PL (p = 0.0016), (p = 0.0018) and the STG (p = 0.0066), (p = 0.0019) in group C. The TA had similar results like the Achilles and quadriceps. DISCUSSION AND CONCLUSIONS: The vulnerability of gamma irradiation of TA was less than Achilles and quadriceps tendons.

The effects of low-dose radiotherapy on fresh osteochondral allografts: An experimental study in rabbits.

OBJECTIVE: The aim of this study was to investigate the effects of low-dose fractionated radiotherapy on cartilage degeneration after distal femoral fresh massive osteochondral allograft transplantation. METHODS: Twenty-four New Zealand White rabbits were divided into three groups of 8 rabbits each. All rabbits underwent distal femoral medial condyle fresh massive osteochondral allograft transplantation from California rabbits. The group 1 underwent transplantation without any preliminary process. The group 2 underwent fractionated local radiotherapy of 100 cGy for five days starting on the transplantation day. The group 3 included the rabbits to which the grafts transplanted after radiating in vitro by a single dose radiation of 1500 cGy. The hosts were sacrificed twelve weeks later. Anteroposterior and lateral radiographs were taken. Synovial tissue, cartilaginous tissue, and subchondral bone were assessed histopathologically. RESULTS: Nonunion was present in three cases of group 2 and one of group 3 in which cartilage degeneration was more severe. Synovial hypertrophy and pannus formation were more obvious in non-radiated rabbits. Hypocellularity and necrosis of the subchondral bone were rare in group 2. More cartilage tissue impairment was present in group 3 compared to group 1. CONCLUSION: In osteochondral massive allograft transplantations, the immune reaction of the host could be precluded with radiotherapy, and the side-effects can be prevented by low-dose fractionated regimen. The total dose of fractionated radiotherapy for an immune suppression should be adjusted not to damage the cartilage tissue, but to avoid articular degeneration in the long term.

Effect of a novel sterilization method on biomechanical properties of soft tissue allografts.

PURPOSE: Evaluate allograft tissue commonly used in soft tissue reconstruction to determine whether stiffness and strength were significantly altered after grafts were treated with different sterilization methods. Unprocessed, irradiated, and grafts treated with supercritical CO2 were compared. METHODS: Thirty-eight anterior or posterior tibialis tendons were obtained from a tissue bank (Allograft Innovations, Gainesville, FL). Group I was unprocessed, group II was sterilized with gamma irradiation (20-28 kGy), and group III was sterilized with supercritical CO2. The grafts were pretensioned to 89 N for 300 s. Specimens were then loaded from 50 to 300 N at 0.5 Hz for 250 cycles before being loaded to failure at 50 mm/min. Dependent variables were compared between sterilization groups with one-way ANOVA (P < 0.05) and equivalence trial. RESULTS: There was no significant difference in load to failure or failure stress among groups I, II, and III. Group III resulted in 27-36 % lower stiffness than group I and II. This difference was significant at 1, 10, 50, 100, and 250 cycles. There was no significant difference in stiffness between group I and group II. CONCLUSION: The two sterilization methods tested in this study do not affect allograft strength. The supercritical CO2 sterilization method resulted in significantly lower stiffness than unprocessed and irradiated allografts. However, the stiffness and strength of all groups tested were greater than that of published values of the native intact anterior cruciate ligament (ACL). This study provides previously unpublished mechanical test data on a new sterilization technique that will assist surgeons to decide which allograft to use in ACL reconstruction surgery. LEVEL OF EVIDENCE: III.

A Prospective Randomized Study of Anterior Cruciate Ligament Reconstruction With Autograft, γ-Irradiated Allograft, and Hybrid Graft.

PURPOSE: To compare the results of patients who underwent anterior cruciate ligament (ACL) reconstruction with autograft, γ-irradiated allograft, or hybrid graft in a prospective randomized study with a minimum clinical follow-up period of 5 years. METHODS: In this prospective, randomized, comparative study, 102 patients with ACL tears underwent ACL reconstruction with autograft (gracilis and semitendinosus tendons), γ-irradiated allograft (tibialis anterior tendons), or hybrid graft (γ-irradiated tibialis anterior tendon allograft and semitendinosus tendon autograft). Laboratory testing of the erythrocyte sedimentation rate and C-reaction protein level were performed; clinical results were evaluated with the KT-1000 arthrometer (MEDmetric, San Diego, CA), Lachman test, Lysholm score, Tegner activity score, and International Knee Documentation Committee evaluation (both objective and subjective). RESULTS: There were 32 patients in the autograft group, 31 in the hybrid graft group, and 32 in the γ-irradiated allograft group at last follow-up. No differences were found among the 3 groups regarding patient demographic data and the duration from injury to operation (P > .05). The C-reaction protein and erythrocyte sedimentation rate values were statistically higher in the γ-irradiated allograft group than in the other 2 groups on the third, seventh, and fourteenth days (P < .05). No significant differences were found between the autograft and hybrid graft groups (P > .05). The KT-1000 examination showed more anterior laxity in the γ-irradiated allograft group than in the other 2 groups (P < .05). No significant differences in the Lachman test and pivot-shift test findings were found among the 3 groups (P > .05). The Lysholm score, Tegner activity score, and International Knee Documentation Committee evaluation (subjective and objective) showed no differences among the 3 groups (P > .05). CONCLUSIONS: Patients undergoing primary ACL reconstruction with hybrid graft or autograft had satisfactory and similar objective and subjective clinical results. On KT-1000 measurement of anteroposterior knee laxity, both the autograft and hybrid graft groups showed statistically significant differences compared with the γ-irradiated allograft group. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Porphyrin-adsorbed Allograft Bone: A Photoactive, Antibiofilm Surface.

BACKGROUND: Allograft bone is commonly used to augment bone stock. Unfortunately, allograft is prone to bacterial contamination and current antimicrobial therapies are inadequate. Photoactivated porphyrins combat bacterial growth by production of reactive oxygen species (ROS); however, to our knowledge, they have not been tested in the setting of allograft bone. QUESTIONS/PURPOSES: We asked: (1) Does 5,10,15,20-tetrakis-(4-aminophenyl)-porphyrin (TAPP) stably adsorb to morselized, mineralized allograft? (2) Does Staphylococcus aureus acquire TAPP from TAPP-allograft? (3) Is TAPP-allograft antibacterial to S. aureus? (4) Is ROS production critical for antimicrobial activity? (5) Does illuminated TAPP-allograft dislodge biofilm? (6) Could other photoactive dyes (TAPP, TMPyP, TSP, THP, and methylene blue) confer antimicrobial properties to allograft? METHODS: TAPP adsorption to allograft (TAPP-allograft), its localization in S. aureus, and TAPP-allograft long-term stability were determined spectrophotometrically. Antimicrobial activity was measured while activated with light or in the dark during incubation with S. aureus or after allograft biofilm formation. Glutathione was added to illuminated TAPP-allograft to quench ROS and antimicrobial activity was determined. Light-dependent antimicrobial activity of other photoactive dyes (TMPyP, TSP, THP, and methylene blue) adsorbed to allograft was also tested. RESULTS: We found (1) porphyrins strongly adhere to bone allograft; and (2) the bacteria are not able to sequester TAPP from the TAPP-allograft; (3) when illuminated, TAPP-allograft is resistant to bacterial adherence; (4) the effects of TAPP are inhibited by the radical scavenger glutathione, indicating ROS-dependent antimicrobial activity; (5) illumination of TAPP-allograft disrupts biofilms; and, (6) other photoactive dyes impede biofilm formation on allograft bone in the presence of light. CONCLUSIONS: Porphyrins stably associate with allograft and are inactive until illuminated. Illuminated TAPP-allograft markedly reduces bacterial colonization, which is restored in the presence of radical scavengers. Finally, illuminated TAPP-allograft disrupts biofilms. CLINICAL RELEVANCE: The findings of this in vitro study suggest that loading bone allograft with biocompatible porphyrins before surgery might allow increased sterility of the allograft during implantation. Future testing in an animal model will determine if these in vitro activities can be used to prevent allograft-based infection in an establishing osteomyelitis.

Meniscal allograft sterilisation: effect on biomechanical and histological properties.

Sterilisation of allografts are a crucial step in ensuring safety and viability. Current sterilisation standards such as 25 kGy gamma irradiation (γ) can have adverse effects on the ultrastructure and biomechanical properties of allograft tissue. Supercritical CO2 (SCCO2) technology, represents an improved sterilisation process that potentially preserves tissue properties. This study aimed to test the effect of SCCO2 sterilisation on the biomechanical and histological properties of the meniscus and compare this to the current standard of γ. Thirty-two 18-month old ovine menisci were randomly assigned into three groups for sterilisation (SCCO2, γ and control). After treatment, biomechanical indentation testing (stiffness and stress relaxation) or histological analysis [percentage of void, cells and extracellular matrix (ECM) per slide] was undertaken. Both SCCO2 and gamma groups displayed an increase in stiffness and stress relaxation as compared to control, however, this difference was lesser in samples treated with SCCO2. No significant histological quantitative differences were detected between SCCO2 and control specimens. Gamma-treated samples demonstrated a significant increase in void and decrease in ECM. Interestingly, both treatment groups demonstrated a decreasing mean void and increasing ECM percentage when analysed from outer to inner zones. No significant differences were detected in all-endpoints when analysed by section. SCCO2 sterilisation represents a potential feasible alternative to existing sterilization techniques such as γ.