Characterization of the mechanical properties of bovine cortical bone treated with a novel tissue sterilization process

Over the past decade chemical processing and engineering of musculoskeletal tissue (tendon and bone) has improved dramatically. The use of bone allograft and xenograft in reconstructive orthopedic and maxillofacial surgeries is increasing, yet severe complications can occur if the material is contaminated in any way. A novel tissue sterilization process, BioCleanse^®, has been developed to clean and sterilize musculoskeletal tissue for implantation. The present study was designed to determine the effect of this novel cleaning process on the biomechanical properties of bovine cortical bone prior to implantation. The mechanical properties of treated bovine bone material were compared to human samples with respect to failure under compression, shear and three-point bending. The data demonstrate that bovine bone treated with the novel sterilization procedure has favorable biomechanical properties compared to that of human bone treated in a similar fashion.     

Inactivation of enveloped and non-enveloped viruses on seeded human tissues by gamma irradiation

Human tissue allografts are widely used in a variety of clinical applications with over 1.5 million implants annually in the US alone. Since the 1990s, most clinically available allografts have been disinfected to minimize risk of disease transmission. Additional safety assurance can be provided by terminal sterilization using low dose gamma irradiation. The impact of such irradiation processing at low temperatures on viruses was the subject of this study. In particular, both human tendon and cortical bone samples were seeded with a designed array of viruses and the ability of gamma irradiation to inactivate those viruses was tested. The irradiation exposures for the samples packed in dry ice were 11.6-12.9 kGy for tendon and 11.6-12.3 kGy for bone, respectively. The viruses, virus types, and log reductions on seeded tendon and bone tissue, respectively, were as follows: Human Immunodeficiency Virus (RNA, enveloped), >2.90 and >3.20; Porcine Parvovirus (DNA, non-enveloped), 1.90 and 1.58; Pseudorabies Virus (DNA, enveloped), 3.80 and 3.79; Bovine Viral Diarrhea Virus (RNA, enveloped), 2.57 and 4.56; and Hepatitis A Virus (RNA, non-enveloped), 2.54 and 2.49, respectively. While proper donor screening, aseptic technique, and current disinfection practices all help reduce the risk of viral transmission from human allograft tissues, data presented here indicate that terminal sterilization using a low temperature, low dose gamma irradiation process inactivates both enveloped and non-enveloped viruses containing either DNA or RNA, thus providing additional assurance of safety from viral transmission.     

Sterilization of Allograft Bone: is 25 kGy the Gold Standard for Gamma Irradiation?

For several decades, a dose of 25 kGy of gamma irradiation has been recommended for terminal sterilization of medical products, including bone allografts. Practically, the application of a given gamma dose varies from tissue bank to tissue bank. While many banks use 25 kGy, some have adopted a higher dose, while some choose lower doses, and others do not use irradiation for terminal sterilization. A revolution in quality control in the tissue banking industry has occurred in line with development of quality assurance standards. These have resulted in significant reductions in the risk of contamination by microorganisms of final graft products. In light of these developments, there is sufficient rationale to re-establish a new standard dose, sufficient enough to sterilize allograft bone, while minimizing the adverse effects of gamma radiation on tissue properties. Using valid modifications, several authors have applied ISO standards to establish a radiation dose for bone allografts that is specific to systems employed in bone banking. These standards, and their verification, suggest that the actual dose could be significantly reduced from 25 kGy, while maintaining a valid sterility assurance level (SAL) of 10⁻⁶. The current paper reviews the methods that have been used to develop radiation doses for terminal sterilization of medical products, and the current trend for selection of a specific dose for tissue banks.     

Biomechanical properties of sterilized human auditory ossicles.

Bone allograft material is treated with sterilization methods to prevent the transmission of diseases from the donor to the recipient. The effect of some of these treatments on the integrity of the bone is unknown. This study was performed to evaluate the effect of several sterilization methods on the mechanical behaviour of human middle ear bones. Due to the size and composition of the bones (approximately 1.5 mm diameter by 4 mm long), mechanical testing options were limited to the traditional platens compression test. Experiments were first performed with synthetic bone to evaluate the precision of this test applied to small specimens. Following this, fresh frozen human ossicles were thawed and sterilized with (i) 1 N NaOH (n = 12); (ii) 0.9% LpH, a phenolic solution (n = 12); or (iii) steam at 134 degrees C (n = 18). A group of 26 control specimens did not receive any sterilization treatment. Material and structural properties were determined from axial compression testing. Results from the synthetic bone showed that the test was reproducible, with standard deviations less than 20% of the means. Significant differences occurred in stiffness and ultimate force values between NaOH-treated and autoclaved bones when compared to normals (p<0.05), but not for LpH-treated bones. LpH is not approved for medical use, so NaOH is the most appropriate of the treatments studied for the sterilization of ossicle allografts.     

Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts

The increased use of allograft tissue for musculoskeletal repair has brought more focus to the safety of allogenic tissue and the efficacy of various sterilization techniques. Gamma irradiation is an effective method for providing terminal sterilization to biological tissue, but it is also reported to have deleterious effects on tissue mechanics in a dose-dependent manner. At irradiation ranges up to 25 kGy, a clear relationship between mechanical strength and dose has yet to be established. The aim of this study was to investigate the mechanical properties of bone and soft tissue allografts, irradiated on dry ice at a low absorbed dose (18.3–21.8 kGy) and a moderate absorbed dose (24.0–28.5 kGy), using conventional compressive and tensile testing, respectively. Bone grafts consisted of Cloward dowels and iliac crest wedges, while soft tissue grafts consisted of patellar tendons, anterior tibialis tendons, semitendinosus tendons, and fascia lata. There were no statistical differences in mechanical strength or modulus of elasticity for any graft irradiated at a low absorbed dose, compared to control groups. Also, bone allografts and two soft tissue allografts (anterior tibialis and semitendinosus tendon) that were irradiated at a moderate dose demonstrated similar strength and modulus of elasticity values to control groups. The results of this study support the use of low dose and moderate dose gamma irradiation of bone grafts. For soft tissue grafts, the results support the use of low dose irradiation.     

Alterations in damage processes in dense cancellous bone following gamma-radiation sterilization

Structurally intact cancellous bone allograft is an attractive tissue form because its high porosity can provide space for delivery of osteogenic factors and also allows for more rapid and complete in-growth of host tissues. Gamma radiation sterilization is commonly used in cancellous bone allograft to prevent disease transmission. Commonly used doses of gamma radiation sterilization (25–35 kGy) have been shown to modify cortical bone post-yield properties and crack propagation but have not been associated with changes in cancellous bone material properties. The purpose of this study was to determine the effects of irradiation on the elastic and yield properties and microscopic tissue damage processes in dense cancellous bone. Cancellous bone specimens (13 control, 14 irradiated to 30 kGy) from bovine proximal tibiae were tested in compression to 1.3% apparent strain and examined for microscopic tissue damage. The yield strain in irradiated specimens (0.93±0.11%, mean±SD) did not differ from that in control specimens (0.90±0.11%, p=0.44). No differences in elastic modulus were observed between groups after accounting for differences in bone volume fraction. However, irradiated specimens showed greater residual strain (p=0.01), increased number of microfractures (p=0.02), and reduced amounts of cross-hatching type damage (p<0.01). Although gamma radiation sterilization at commonly used dosing (30 kGy) does not modify elastic or yield properties of dense cancellous bone, it does cause modifications in damage processes, resulting in increased permanent deformation following isolated overloading.     

Solvent-dehydrated calvarial allografts in craniofacial surgery

Craniofacial surgery almost always requires the use of bone grafting. Although autografts are the standard procedure for bone grafting, it is sometimes not possible to harvest bone, and autografts have particular risks. The use of allograft bone provides a reasonable alternative to meet the need for graft material. Solvent dehydration is a multistage procedure in which human cadaveric bone is processed by osmotic exchange baths and gamma sterilization. This processing avoids the risk of infection transmission, decreases antigenicity, and does not weaken the mechanical properties of the bone. Solvent-dehydrated, gamma-irradiated human calvarial bone allografts were used for reconstruction of craniofacial deformities in 24 patients between 1988 and 2002. Resorption of the allografts and results of the surgical intervention were evaluated with plain radiographs and three-dimensional computed tomography 12 months after surgery, in 21 patients. Serologic tests for human immunodeficiency virus-1 antibody, hepatitis B surface antigen, and hepatitis C antigen were also performed. Biopsy specimens were taken from the allografts. Average follow-up in this group was 30 months (range, 8 to 60 months), and results of serologic tests were negative in all patients. Seventy-one percent of the patients (15 of 21) showed no resorption, with partial and complete allograft fusion. One patient had nearly total graft loss and the remaining five patients had 10 to 25 percent graft resorption. Rigid fixation of the allograft, contact with the dura and periosteum, and prevention of dead spaces around the allograft are the most important factors in achieving a satisfactory result. In solvent-dehydrated bone allografts, sterilization and antigenic tissue cleaning are achieved after several steps with a minimal dose of radiation. The result is a nonantigenic, sterile mechanical scaffold that can tolerate external forces. Although autografts are the standard in craniofacial surgery, solvent-dehydrated calvarial bone allografts produced successful results in selected cases.     

Effects of gamma irradiation on mechanical properties of defatted trabecular bone allografts assessed by speed-of-sound measurement

New sterilization methods for human bone allografts may lead to alterations in bone mechanical properties, which strongly influence short- and medium-term outcomes. In many sterilization procedures, bone allografts are subjected to gamma irradiation, usually with 25 KGy, after treatment and packaging. We used speed-of-sound (SOS) measurements to evaluate the effects of gamma irradiation on bone. All bone specimens were subjected to the same microbial inactivation procedure. They were then separated into three groups, of which one was treated and not irradiated and two were exposed to 10 and 25 KGy of gamma radiation, respectively. SOS was measured using high- and low-frequency ultrasound beams in each orthogonal direction. SOS and Young modulus were altered significantly in the three groups, compared to native untreated bone. Exposure to 10 or 25 KGy had no noticeable effect on the study variables. The impact of irradiation was small compared to the effects of physical or chemical defatting. Reducing the radiation dose used in everyday practice failed to improve graft mechanical properties in this study.     

Disinfection of human musculoskeletal allografts in tissue banking: a systematic review

Musculoskeletal allografts are typically disinfected using antibiotics, irradiation or chemical methods but protocols vary significantly between tissue banks. It is likely that different disinfection protocols will not have the same level of microorganism kill; they may also have varying effects on the structural integrity of the tissue, which could lead to significant differences in terms of clinical outcome in recipients. Ideally, a disinfection protocol should achieve the greatest bioburden reduction with the lowest possible impact on tissue integrity. A systematic review of three databases found 68 laboratory and clinical studies that analyzed the microbial bioburden or contamination rates of musculoskeletal allografts. The use of peracetic acid–ethanol or ionizing radiation was found to be most effective for disinfection of tissues. The use of irradiation is the most frequently published method for the terminal sterilization of musculoskeletal allografts; it is widely used and its efficacy is well documented in the literature. However, effective disinfection results were still observed using the BioCleanse? Tissue Sterilization process, pulsatile lavage with antibiotics, ethylene oxide, and chlorhexidine. The variety of effective methods to reduce contamination rate or bioburden, in conjunction with limited high quality evidence provides little support for the recommendation of a single bioburden reduction method.     

Effect of gamma sterilization on microhardness of the cortical bone tissue of bovine femur in presence of N-Acetyl-L-Cysteine free radical scavenger

Gamma sterilization is usually used to minimize the risk of infection transmission through bone allografts. However, it is believed that gamma irradiation affects the mechanical properties of allografts and free radical scavengers can be used to alleviate the radiation-induced degradation of these properties. The aim of this study was to investigate the radioprotective effects of N-Acetyl-L-Cysteine (NAC) free radical scavenger on the material properties of sterilized bovine cortical bone at microstructure level. Forty-two cortical tissue specimens were excised from three bovine femurs and irradiated to 35 and 70 kGy gamma rays in the presence of 5, 50, and 100 mM concentrations of NAC. The localized variations in microhardness were evaluated via indentation in the radial and longitudinal directions to examine different regions of the microstructures of the specimens, including the osteonal and interstitial tissues. A significant increase was observed in the hardness of osteonal, interstitial, and longitudinal combined microstructures exposed to 35 and 70 kGy radiations (P < 0.05), whereas a relative reduction of the hardness was observed in the radial direction. Furthermore, it was found that the application of 50 and 100 mM NAC during gamma irradiation significantly subsided the hardening in longitudinal combined microstructure. Moreover, the reduction of hardness in radial direction was suppressed in the presence of 100 mM of NAC. In conclusion, the results indicated that NAC free radical scavenger can protect the cortical bone against deteriorative effects of ionizing radiation and can be used to improve the material properties of sterilized allografts.     

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.     

Radioprotection provides functional mechanics but delays healing of irradiated tendon allografts after ACL reconstruction in sheep

Successful protection of tissue properties against ionizing radiation effects could allow its use for terminal sterilization of musculoskeletal allografts. In this study we functionally evaluate Achilles tendon allografts processed with a previously developed radioprotective treatment based on (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) crosslinking and free radical scavenging using ascorbate and riboflavin, for ovine anterior cruciate ligament reconstruction. Arthroscopic anterior cruciate ligament (ACL) reconstruction was performed using double looped allografts, while comparing radioprotected irradiated and fresh frozen allografts after 12 and 24 weeks post-implantation, and to control irradiated grafts after 12 weeks. Radioprotection was successful at preserving early subfailure mechanical properties comparable to fresh frozen allografts. Twelve week graft stiffness and anterior-tibial (A-T) translation for radioprotected and fresh frozen allografts were comparable at 30 % of native stiffness, and 4.6 and 5 times native A-T translation, respectively. Fresh frozen allograft possessed the greatest 24 week peak load at 840 N and stiffness at 177 N/mm. Histological evidence suggested a delay in tendon to bone healing for radioprotected allografts, which was reflected in mechanical properties. There was no evidence that radioprotective treatment inhibited intra-articular graft healing. This specific radioprotective method cannot be recommended for ACL reconstruction allografts, and data suggest that future efforts to improve allograft sterilization procedures should focus on modifying or eliminating the pre-crosslinking procedure.     

Sterilization of sealed PVDF pouches containing decellularized scaffold by electrical stimulation

A terminal sterilization process for tissue engineering products, such as allografts and biomaterials is necessary to ensure complete removal of pathogenic microorganisms such as the bacteria, fungi, and viruses. However, it can be difficult to sterilize allografts and artificial tissue models packaged in wet conditions without deformation. In this study, we investigated the sterilization effects of electrical stimulation (ES) and assessed its suitability by evaluating sterility assurance levels in pouches at a constant current. Stability of polyvinylidene fluoride pouches was determined by a sterility test performed after exposure to five microorganisms (Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans) for 5 days; the sterility test was also performed with decellularized human dermal tissues inoculated with the five microorganisms. Sterilization using ES inactivated microorganisms both inside and outside of sealed pouches and caused no damage to the packaged tissue. Our results support the development of a novel system that involves ES sterilization for packaging of implantable biomaterials and human derived materials.     

Interfacial Strength of Cement Lines in Human Cortical Bone

In human cortical bone, cement lines (or reversal lines) separate osteons from the interstitial bone tissue, which consists of remnants of primary lamellar bone or fragments of remodeled osteons. There have been experimental evidences of the cement line involvement in the failure process of bone such as fatigue and damage. However, there are almost no experimental data on interfacial properties of cement lines in human cortical bone. The objective of this study is to design and assemble a precision and computer controlled osteon pushout microtesting system, and to experimentally determine the interfacial strength of cement lines in human cortical bone by performing osteon pushout tests. Thirty specimens were prepared from humeral diaphyses of four human subjects. Twenty specimens were tested under the condition of a small hole in the supporting plate, in which the cement line debonding occurred. The cement line interfacial strength ranged from 5.38 MPa to 10.85 MPa with an average of 7.31±1.73 MPa. On the other hand, ten specimens were tested under the condition of a large hole in the supporting plate, in which the shear failure inside osteons was observed. The specimens tested under the condition of the large hole resulted in an average shear strength of 73.71±15.06 MPa, ranging from 45.97 MPa to 93.74 MPa. Therefore, our results suggest that the cement line interface between osteon and interstitial bone tissue is weaker than that between bone tissue lamellae.     

Quality assessment for processed and sterilized bone using Raman spectroscopy

To eliminate the potential for infection, many tissue banks routinely process and terminally sterilize allografts prior to transplantation. A number of techniques, including the use of scanning electron microscopy, bone graft models, and mechanical property tests, are used to evaluate the properties of allograft bone. However, as these methods are time consuming and often destroy the bone sample, the quality assessment of allograft bones are not routinely performed after processing and sterilization procedures. Raman spectroscopy is a non-destructive, rapid analysis technique that requires only small sample volumes and has recently been used to evaluate the mineral content, mineral crystallinity, acid phosphate and carbonate contents, and collagen maturity in human and animal bones. Here, to establish a quality assessment method of allograft bones using Raman spectroscopy, the effect of several common sterilization and preservation procedures on rat femoral bones were investigated. We found that freeze-thawing had no detectable effects on the composition of bone minerals or matrix, although heat treatment and gamma irradiation resulted in altered Raman spectra. Our findings suggest Raman spectroscopy may facilitate the quality control of allograft bone after processing and sterilization procedures.     

The effects of prolonged deep freezing on the biomechanical properties of osteochondral allografts

Musculo-skeletal allografts sterilized and deep frozen are among the most common human tissue to be preserved and utilized in modern medicine. The effects of a long deep freezing period on cortical bone has already been evaluated and found to be insignificant. However, there are no reports about the influences of a protracted deep freezing period on osteochondral allografts. One hundred osteochondral cylinders were taken from a fresh specimen and humeral heads of 1 year, 2 years, 3 years and 4 year old bones. Twenty chips from each period, with a minimum of 3 chips per humeral head. Each was mechanically tested by 3 point compression. The fresh osteochondral allografts were significantly mechanically better than the deep frozen osteochondral allografts. There was no statistical significant time dependent difference between the deep frozen groups in relation to the freezing period. Therefore, we conclude that, from the mechanical point of view deep freezing of osteochondral allografts over a period of 4 years, is safe without further deterioration of the biomechanical properties of the osteochondral allografts.     

Platelet concentrate as an additive to bone allografts: a laboratory study using an uniaxial compression test

Chemical cleaning procedures of allografts are destroying viable bone cells and denaturing osteoconductive and osteoinductive proteins present in the graft. The aim of the study was to investigate the mechanical differences of chemical cleaned allografts by adding blood, clotted blood; platelet concentrate and platelet gel using a uniaxial compression test. The allografts were chemically cleaned, dried and standardized according to their grain size distribution. Uniaxial compression test was carried out for the four groups before and after compacting the allografts. No statistically significant difference was found between native allografts, allografts mixed with blood, clotted blood, platelet concentrate and platelet concentrate gel regarding their yield limit after compaction. The authors recommend to chemical clean allografts for large defects, optimize their grain size distribution and add platelet concentrate or platelet rich plasma for enhancing as well primary stability as well bone ingrowth.     

Sporicidal efficacy of genipin: a potential theoretical alternative for biomaterial and tissue graft sterilization

Terminal sterilization of musculoskeletal allografts by gamma radiation minimizes the risk of disease transmission but impairs allograft mechanical properties. Commonly employed crosslinking agents can sterilize tissues without affecting mechanical properties adversely; however, these agents are toxic. Genipin is reported to be a benign crosslinking agent that strengthens mechanical properties of tissues; however, the antimicrobial capacity of genipin is largely unknown. The present study’s aims were: (1) to assess the sporicidal potential of genipin, (2) to improve antimicrobial capacity by changing chemical and physical treatment conditions. To establish genipin’s sterilization potential Bacillus subtilis var. niger spore strips were treated with 0–10 % genipin in PBS or in 1:1 DMSO:PBS up to 72 h at room temperature (RT). Sterilizing doses and concentrations of genipin were used to treat B. pumilus and Geobacillus stearothermophilus spores to assess broader spectrum sporicidal activity of genipin. Scanning electron microscopy (SEM) was performed to evaluate gross morphological changes after genipin treatment. Optimal sterilization conditions were determined by evaluating the effects of temperature (RT-50 °C), DMSO:PBS ratio (0:100–100:0), and treatment duration (24–72 h) on B. subtilis. Genipin penetration of full thickness bovine patellar tendon and cortical bone specimens was observed to assess the feasibility of the agent for treating grafts. Initial studies showed that after 72 h of treatment at RT with 0.63–10 % genipin/DMSO:PBS B. subtilis spore strips were sterilized; 0.63 % genipin/PBS did not sterilize spore strips at 72 h at RT. Genipin doses and concentrations that sterilized B. subtilis spore strips sterilized B. pumilus and G. stearothermophilus spore strips. SEM revealed no gross morphological differences between untreated and treated spores. Treatment optimization resulted in sterilization within 24 h with 100 % PBS, and DMSO facilitated sporicidal activity. Genipin penetrated full thickness patellar tendon specimens and 3.72 ± 0.58 mm in cortical bone specimens. Genipin sterilizes B. subtilis, B. pumilus, and G. stearothermophilus spore strips. It penetrates soft and hard tissues at doses previously shown to be non-toxic and to improve mechanical strength in collagen-rich soft tissues. Further studies are indicated to assess genipin’s effects on the mechanical properties of genipin-sterilized grafts, the ability of genipin to eradicate infectious species other than spores, and to assess whether sterilant activity persists after penetrating tissues and biomaterials.     

Effects of Hydrogen Peroxide Cleaning Procedures on Bone Graft Osteoinductivity and Mechanical Properties

Bone allografts are frequently used during orthopaedic trauma cases or other reconstructive procedures. Most allografts are processed and cleaned before use. Our goals were to determine if an improved cleaning procedure compromises the strength or osteoinductivity of a graft. We compared our improved cleaning procedure to our standard cleaning procedure on cortical bone allograft. The cleaning procedures are generally composed of a series of chemical steps with nonionic detergents, hydrogen peroxide, and alcohol under time and temperature control, subjected to ultrasonic agitation. We tested the compressive strength, impact strength, and shear strength following the standard and improved cleaning procedures. Osteoinductivity was tested in 4 groups, using the improved cleaning procedure with four different hydrogen peroxide cleaning times: 0, 1, 3, and 5 h. Osteoinductivity was evaluated in vivo, using a 28-day implant in the hamstring muscle of an athymic, nude mouse. Results demonstrated that osteoinductivity is maintained with cleaning in hydrogen peroxide for up to 1 h, and that compressive strength, impact strength, and shear strength were all unaffected by the improved cleaning procedure. The improved cleaning procedure therefore did not compromise the strength or osteoinductivity of cortical bone allografts in comparison to the standard procedure.     

Sterilization with electron beam irradiation influences the biomechanical properties and the early remodeling of tendon allografts for reconstruction of the anterior cruciate ligament (ACL)

Although allografts for anterior cruciate ligament (ACL) replacement have shown advantages compared to autografts, their use is limited due to the risk of disease transmission and the limitations of available sterilization methods. Gamma sterilization has shown detrimental effects on graft properties at the high doses required for sufficient pathogen inactivation. In our previous in vitro study on human patellar tendon allografts, Electron beam (Ebeam) irradiation showed less detrimental effects compared to gamma sterilization (Hoburg et al. in Am J Sports Med 38(6):1134-1140, 2010). To investigate the biological healing and restoration of the mechanical properties of a 34?kGy Ebeam treated tendon allograft twenty-four sheep underwent ACL replacement with either a 34?kGy Ebeam treated allograft or a non-sterilized fresh frozen allograft. Biomechanical testing of stiffness, ultimate failure load and AP-laxity as well as histological analysis to investigate cell, vessel and myofibroblast-density were performed after 6 and 12?weeks. Native sheep ACL and hamstring tendons (HAT, each n?=?9) served as controls. The results of a previous study analyzing the remodeling of fresh frozen allografts (n?=?12) and autografts (Auto, n?=?18) with the same study design were also included in the analysis. Statistics were performed using Mann-Whitney U test followed by Bonferroni-Holm correction. Results showed significantly decreased biomechanical properties during the early remodeling period in Ebeam treated grafts and this was accompanied with an increased remodeling activity. There was no recovery of biomechanical function from 6 to 12?weeks in this group in contrast to the results observed in fresh frozen allografts and autografts. Therefore, high dose Ebeam irradiation investigated in this paper cannot be recommended for soft tissue allograft sterilization.