Electron Beam Sterilization Does Not Have a Detrimental Effect on the Ability of Extracellular Matrix Scaffolds to Support In Vivo Ligament Healing

Abstract

Purpose

Extra-cellular matrix (ECM) scaffolds have been used to enhance anterior cruciate ligament (ACL) repair in large animal models. To translate this technology to clinical care, identifying a method, which effectively sterilizes the material without significantly impairing in vivo function, is desirable.

Methods

16 Yorkshire pigs underwent ACL transection and were randomly assigned to bridge-enhanced ACL repair – primary suture repair of the ACL with addition of autologous blood soaked ECM scaffold - with either 1) an aseptically processed ECM scaffold, or 2) an electron beam irradiated ECM scaffold. Primary outcome measures included sterility of the scaffold and biomechanical properties of the scaffold itself and the repaired ligament at eight weeks after surgery.

Results

Scaffolds treated with 15kGy electron beam irradiation had no bacterial or fungal growth noted, while aseptically processed scaffolds had bacterial growth in all tested samples. The mean biomechanical properties of the scaffold and healing ligament were lower in the electron beam group; however, differences were not statistically significant.

Conclusions

Electron beam irradiation was able to effectively sterilize the scaffolds. In addition, this technique had only a minimal impact on the in vivo function of the scaffolds when used for ligament healing in the porcine model.

Introduction

Many tissue engineering technologies employ use of a scaffold of collagen or other extracellular matrix (ECM) proteins. These preclinical studies often utilize an ECM scaffold which had been processed using “aseptic” techniques, where all processing steps are conducted in a clean environment, using sterile instruments, glassware and specific techniques to avoid bacterial, fungal or viral contamination. The translation of these scaffolds to clinical trials requires an additional degree of sterility than that typically required for preclinical studies.

Terminal sterilization refers to the sterilization of a product within a sterile barrier system, usually at the end of production. The goal of terminal sterilization is to eliminate any microbial and viral contamination that may have been introduced during the manufacturing process.¹ In contrast to aseptic processing, terminal sterilization can provide more repeatable process control and a high assurance of sterility for tissue engineered scaffolds.² According to the Association of Advancement of Medical Instrumentation (AAMI) the current sterility assurance level (SAL) an implantable medical device has to fulfill is 10^-6, meaning that less than one device in a million has detectable contamination.³ Irradiation is routinely used to sterilize implantable medical devices;⁴ however, ECM scaffolds can undergo biochemical and structural alterations during the process of terminal sterilization by irradiation.^{5; 6} For example, Hoburg et al. reported that electron beam irradiation of an allograft tendon used for ACL reconstruction yielded a higher mechanical failure load than a graft irradiated with gamma irradiation.⁷ However, the influence of irradiation on the performance of biological scaffolds in tissue engineering functions has been less studied. To our best knowledge, this is the first report of terminal sterilization for an ECM -based scaffold and the effect of that sterilization on in vivo healing in a validated large animal model.

The aims of this study were to determine if an ECM scaffold, previously used successfully for preclinical studies of bridge-enhanced ACL repair,^8-10 could be terminally sterilized using low dose 15kGy electron beam irradiation, and whether the irradiation of the ECM would have a significant impact on the biomechanical performance of the scaffold itself as well as the repaired ACL after 8 weeks of healing.

Materials and Methods

Study Design

ECM scaffolds were manufactured using Good Laboratory Practice with Design Control and sent for sterility testing (Microtest/Charles River Laboratories) after aseptic processing or aseptic processing and terminal sterilization with 15kGy of electron beam irradiation (Synergy). Institutional Animal Care and Use Committee approvals were obtained prior to the study. Sixteen Yorkshire pigs with closed tibial and femoral physes [age (mean±SD): 91.3±4.9 days; weight: 35.0±2.0 kg] underwent ACL transection on the right hind-limb and were randomized into two experimental groups: 1) bridge-enhanced ACL repair using a non-terminally sterilized aseptically processed scaffold (ASEPTIC) and 2) bridge-enhanced ACL repair using the same scaffold treated with 15kGy electron beam irradiation (EBEAM). The left hind-limb was left intact as a control for both groups. All animals were allowed to heal for 8 weeks and then were assessed biomechanically and histologically.

Preparation of the extra-cellular matrix scaffold

The ECM scaffolds (MIACH, Boston Children's Hospital, Boston, MA) for the ASEPTIC and EBEAM group stem from a single production lot and were manufactured as previously described.⁹ Briefly, bovine connective tissue was solubilized to produce a slurry of ECM proteins with a collagen concentration of greater than 10 mg/ml. The slurry was lyophilized to form a porous cylinder 22 mm in diameter and 30 mm in length. For all scaffolds, aseptic processing techniques were used and the scaffolds were packaged in sterile protective pouches at room temperature and shielded from light.

Electron Beam Sterilization

Scaffolds for the EBEAM group underwent 15kGy of electron beam irradiation (Synergy Health, Saxonburg, PA) in accordance with the industrial standard ISO 11137-2. A dose map was performed before irradiation of the scaffolds to assure similar radiation doses for each scaffold [dose (minimum, maximum, mean±SD): 14.7 kGy, 15.3 kGy, 15±0.2 kGy]. After terminal sterilization, the scaffolds were stored in the sterile protective pouches at room temperature and shielded from light.

Sterility Testing

ASEPTIC and EBEAM scaffolds were tested for sterility in accordance to USP (United States Pharmacopeia) <71> (Microtest Laboratories, Agawam, MA, USA).¹¹ As described by the contractor, randomly chosen scaffolds from each lot (ASEPTIC, EBEAM) were incubated in either trypticase soy broth – for detection of common aerobic and facultatively anaerobic bacteria – or fluid thioglycollate medium – a differential medium to determine the oxygen requirements of microorganisms. For each media type, 10% of the scaffolds from each lot were incubated in the media at 32.5 ± 2.5 °C for 7 days. Samples were evaluated visually for cloudiness or pellicle formation as an indicator of bacterial and fungal growth. Additionally, a bacteriostasis/fungostasis test was performed on the scaffolds, during which scaffolds were transferred to growth media, inoculated with microorganisms and monitored for growth, to validate the results of the above mentioned sterility test. EBEAM Scaffolds were tested for presence of Bovine Rhinotracheitis Virus, Parainfluenza Virus Type 3, Bovine Adenovirus-3, Bovine Parvovirus, Bovine Respiratory Syncytial Virus, Bovine Viral Diarrhea Virus-NY1, Bluetongue Virus-10, Rabies Virus ERA Strain, and Reovirus-3 in accordance to Title 9 of the Code of Federal Regulations (Charles River Laboratories, Malvern, PA).^12-14

Scaffold Mechanical Testing

10 aseptically processed and 10 scaffolds sterilized with 15 kGy electron beam irradiation were tested in uniaxial compression on an Instron 5542 test frame (Norwood, MA). Samples were to cut to approximately 5 mm in length and 8 mm in diameter using a biopsy punch and allowed to equilibrate to room temperature and humidity for 24 hours. A pre-load of 1 N was applied to account for non-uniform surfaces and to avoid data capture while samples were not in full contact with compression platens. The upper cross-head was depressed downward at a rate of 0.1% strain/second. The linear elastic modulus, yield stress, and collapse modulus were calculated for both groups. The yield stress was calculated based on the intersection of the linear regression of the linear elastic modulus and the collapse modulus. All data analysis was performed with Bluehill Materials Testing Software Version 2.

Surgical Technique: Bridge-enhanced ACL repair

Bridge-enhanced ACL repair was performed as previously described.⁹ Briefly, the ACL was transected at the junction of the proximal and middle thirds, and a variable depth suture of #1 Vicryl (Ethicon, Somerville, NJ) was placed in the tibial stump. The knee was stabilized with a suture stent anchored proximally and distally with a cortical button (Endobutton™, Smith & Nephew, Andover, MA). An ECM scaffold was secured between the two ends of the transected ACL and five cc of autologous whole blood was used to saturate the scaffold in situ (Figure 1).

Figure 1. Surgical technique.

A – Knee capsule is opened through a lateral arthrotomy, part of the retropatellar fat pad is resected and the ACL is transected in the mid section. B - Bone tunnels for the suture stent are drilled through the tibial and femoral ACL attachment and an endobutton loaded with three suture loops is passed through the femoral drill hole. C – A separate suture loop is stitched to the tibial ACL stump and the ECM scaffold is threaded on two of the suture loops loaded on the femoral endobutton. D – The same two suture loops are passed through the tibial drill hole and knotted over another endobutton. Care is taken to keep the suture stent under tension with the knee in extension. The scaffold is soaked with autologous whole blood. One end of the suture loop sutured to the tibial ACL stump is tied to one end of the suture loop from the femoral endobutton that has not been passed through the scaffold or the tibial drill hole. The suture connection is tensioned by pulling the lose end of the femoral suture end. E – The free suture ends from tibial stump and femoral endobutton are tied together and the knee is closed in layers. (Published with permission from Vavken et al. 2012).

ACL – Anterior cruciate ligament

ECM –extracellular matrix

Following surgery, the animals were housed in individualized pens, without restrictions. The weight bearing status was evaluated by one observer during the mandatory 7-day post-surgical evaluation twice a day qualitatively on a scale from 0-2 (0 = no weight bearing, 1 = touching ground with toe tips, 2 = full weight bearing). After eight weeks, the animals were euthanized and the hind-limbs were harvested. The limbs were wrapped in a cloth soaked with saline and immediately frozen at -20°C until biomechanical testing.

Biomechanical testing

Biomechanical testing was performed as previously described.⁸ Briefly, the femur and tibia were cleaned of any soft tissue while leaving the joint capsule intact and were mounted in PVC pipes using quick-set epoxy (Smooth-On Inc., Easton, PA). Biomechanical test procedures to obtain the linear stiffness, yield tensile load, maximum tensile load, and anteroposterior (AP) knee laxity were performed using a servohydraulic load frame and custom test fixtures (MTS 810; MTS Systems Corporation, Eden Prairie, MN).⁸ All investigators were blinded to the treatment group during specimen preparation and biomechanical testing. AP knee laxity was measured at 30, 60, and 90 of knee flexion by applying fully-reversed, sinusoidal anterior-posterior directed shear loads of ±40 N at 0.0833 (1/12) Hz for 12 cycles.¹⁰ After laxity testing, all remaining tissues with exception of the ACL were removed and the ACL dimensions were measured using a caliper with an accuracy of 0.1mm. Length was measured from the center of tibial to femoral insertion sites, width and depth at half of the total length of the ACL. The samples were tested at a displacement rate of 20 mm/min and the load-displacement data were recorded at 100 Hz.¹⁵ The yield load, maximum load, and linear stiffness were determined from the load-displacement data. The data were all reported after normalization to the contralateral intact knee (surgical/contralateral control).

Qualitative histological assessment of ligaments, synovium and popliteal lymph nodes

Qualitative histological assessments were conducted for the anterior cruciate ligaments, synovium, and popliteal lymph nodes from both treatment groups. Tissues were removed at necropsy, fixed, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E). Slides were evaluated by a board certified veterinary pathologists (JK, MLH). Criteria for histopathologic analysis of the ACL were adapted from appendix E of ISO 10993-6 ¹⁶ and criteria for synovium from Cake et al.¹⁷ (Table 1)

Table 1.

Histologic criteria used to score the anterior cruciate ligament, synovium, and popliteal lymph nodes from the knees of both groups of animals.

ANTERIOR CRUCIATE LIGAMENT		SYNOVIUM		POPLITEAL LYMPH NODE
CRITERIA	pts	CRITERIA	pts	CRITERIA	pts
INFLAMMATION (Macrophages, polymorphonuclear, plasma, giant cells, scored separately)	0-20	INFLAMMATION (Macrophages, polymorphonuclear, plasma, giant cells, scored together)	0-4	SINUS HISTIOCYTOSIS	0-4
				None	0
				Minimal	1
None observed	0	None	0	Mild	2
Rare, 1-5/40× field	1	Minimal – Scattered small aggregates	1	Moderate	3
5-10/40× field	2	Mild – More numerous small aggregates or diffuse infiltration by small numbers of cells	2	Severe	4
Heavy Infiltrate	3
Packed	4			FOLLICULAR HYPERPLASIA	0-4
		Moderate – Numerous large aggregates or diffuse infiltration by a large number of cells	3	None	0
NEOVASCULARIZATION	0-4			Minimal	1
None	0			Mild	2
Minimal capillary proliferation, focal, 1-3 buds	1	Marked – Diffuse infiltration by large numbers of cells	4	Moderate	3
				Severe	4
Groups of 4-7 capillaries with supporting fibroelastic structures	2	NEOVASCULARIZATION	0-4
Broad band of capillaries with supporting structures	3	0-10 vascular formations/60× field	0
		11-15 vascular formations/60× field	1
Extensive band of capillaries with fibroelastic structures	4	16-20 vascular formations/60× field	2
		21-25 vascular formations/60× field	3
		26-30 vascular formations/60× field	4
FIBROSIS	0-4
None	0	FIBROSIS	0-4
Narrow band	1	None	0
Moderately thick band	2	Minimal subintimal fibrosis – loose wavy fibers separated by edema	1
Thick band	3
Extensive band	4	Mild subintimal fibrosis – thicker and more organized fibers extending slightly further from the synoviocyte lining layer	2
FATTY INFILTRATE	0-4
None	0	Moderate subintimal fibrosis – thick bands of fibrosis extending subintimally	3
Minimal amount of fat associated with fibrosis	1	Marked subintimal fibrosis – diffuse marked fibrosis	4
Several layers of fat and fibrosis	2	SYNOVIAL HYPERPLASIA	0-4
Elongated and broad accumulation of fat cells at the implant site	3
Extensive fat completely surrounding the implant	4	Normal synoviocyte lining layer, 1-2 cell layers thick	0
		Minimal hyperplasia – Focal areas of synoviocyte hyperplasia that are 3-4 cell layers thick	1
		Mild hyperplasia – Diffusely 3-4 cell layers thick +/- focal areas up to 5 cell layers thick	2
		Moderate hyperplasia – Diffusely 5-7 cell layers thick +/- focal areas up to 10 layers thick	3
		Marked hyperplasia - Diffusely 8+ cell layers thick	4

Statistical Analysis

Generalized estimating equations were used to model the surgical and intact limb values as a function of treatment group for all biomechanical outcome measures. Shapiro-Wilk test was performed to test for normality of the data. All models displayed a normal distribution. Intact limbs were compared between groups to determine any treatment effects on those contralateral limbs. No differences in the biomechanical data were determined between the intact limbs of the treatment groups, therefore results from surgical limbs were compared between treatment groups after normalizing them to their intact limb results. The difference between the surgical and intact limbs was used to evaluate AP laxity. All comparisons were performed using the student t-test with an alpha at 0.05 considered statistically significant. We report Cohen's d as measure of effect size (calculated as mean difference divided by the pooled standard deviation). The number of animals per group was determined by a priori power calculation to provide greater than 90 percent power to detect a 20% difference in linear stiffness, yield load, and maximum load between the groups with a standard deviation of 10% based on previous studies.^{8; 10; 18; 19} An effect size of > 0.8 was considered substantial. Geometric means and confidence intervals were reported. All statistical analysis was performed with SAS® Analytics Software (SAS Institute Inc., Cary, NC, USA).

Results

Terminal Sterilization

For the aseptically processed scaffolds, five out of five tested specimens showed turbidity and pellicle formation on incubation in either the trypicase soy broth or thioglycollate media after 7 days in culture. In contrast, none of the five tested terminally sterilized scaffolds developed turbidity in either media after 7 days in culture. The bacteriostasis/fungostasis validation test showed no growth-inhibiting properties of the aseptically processed non-terminally sterilized or electron beam irradiated ECM scaffolds. In vitro tests for bovine adventitious viral agents did not detect any contamination of the EBEAM samples with bovine adventitious viruses.

Scaffold Mechanical Testing

While the average linear elastic modulus and yield stress increased and collapse modulus all decreased approximately 30 % in the EBEAM scaffolds, none of the differences were statistically significant (Table 2, P≥ 0.096 for all comparisons).

Table 2.

Linear Elastic Modulus (LEM), Yield Stress, and Collapse Modulus (CM) of ASEPTIC and EBEAM scaffolds, n = 10 (P-values > 0.096 for all comparisons).

Scaffold Group	LEM [kPa]	95% CI	Yield Stress [kPa]	95% CI	CM [kPa]	95% CI
ASEPTIC	5317	8299 2335	304.9	435.2 174.5	331.1	467.9 194.3
EBEAM	6952	8998 4906	390.2	471.5 308.8	239.3	363.5 115.0
P-value	0.170		0.096		0.133

95% CI – 95% confidence interval

LEM – Linear Elastic Modulus

CM – Collapse Modulus

Animal welfare

All animals recovered well from surgery. Full weight bearing status was achieved within 48-72 hours for all groups. No infections or other adverse events were observed in any of the animals.

ACL dimensions

Comparisons of ACL length, width, depth and volume between the two groups did not yield any significant differences or substantial effect size (Table 2, P≥ 0.48, d≤ 0.63 for all comparisons).

Biomechanics – Structural Properties and AP laxity

Although the mean values for all of the mechanical properties were lower in the group treated with electron beam irradiation, these differences were not statistically significant in comparing the linear stiffness, yield load, maximum load ratios or AP laxity values of the two groups (Table 3, P≥ 0.391, d≤ 0.43 for all comparisons).

Table 3.

Comparison of ACL dimensions at two months after surgery at harvest (ACL length measured from center of tibial to femoral insertion site [mm], width and depth measured in the middle of the ACL [mm], volume is product of length, width and depth [mm³]) between ASEPTIC and EBEAM treatment groups. n = 8. No comparisons are considered significant with P-values >0.48 or substantially different with Cohen's d< 0.63 for all comparisons.

Surgical Group	ACL Length	95% CI	ACL Width	95% CI	ACL Depth	95% CI	ACL Volume	95% CI
ASEPTIC	31.8	34.2 29.4	5.0	7.0 3.0	11.8	15.0 8.7	1931	2903 985
EBEAM	32.8	34.8 30.7	4.5	6.1 2.8	11.0	15.1 6.9	1730	2789 671
P-value	0.48		0.62		0.72		0.75
d-value	0.63		0.004		0.07		0.18

95% CI – 95% confidence interval

ACL – Anterior cruciate ligament

Qualitative histological assessment

Both the ASEPTIC and EBEAM scaffolds were completely resorbed by 8 weeks after surgery. Inflammation in the repaired ACLs was classified as minimal to mild at eight weeks in both groups (Figures 2&3). Mononuclear cells were observed to infiltrate the wound site. Inflammation was more prominent and severe around residual suture material (Figure 2B). At eight weeks after surgical repair, remodeling of the fibrotic tissue to mature collagen was beginning to occur in both groups equally, but areas of disorganized fibrosis were still visible (Figure 2A). Neovascularization, identified as small groups of new capillaries was noted; however, fatty infiltration of the tissue was not seen (Figure 2AB). At eight weeks, signs of chronic inflammation were seen in the deep synovial tissue of both groups, with a thin layer of synovial cells lining the joint surface (Figure 2C&3). In the popliteal lymph nodes, mild sinus histiocytosis and follicular hyperplasia were seen in the lymph nodes of both treatment groups (Figure 2D&3). Overall, there were no significant differences in the histologic appearance of the ligament, synovium or popliteal lymph nodes for the two treatment groups.

Figure 2. Representative histological sections from ACL, synovium, and popliteal lymph node 8 weeks after ACL injury and repair utilizing aseptically processed (ASEPTIC) and electron beam irradiated (EBEAM) ECM scaffolds.

A – H&E stained ACL sections at 20× magnification with smaller pictures showing sections recorded using polarized light to visualize collagen crimp (black bar = 800 μm). Moderate to marked fibrosis with large bands of fibrosis in both groups (black arrows). Additionally, under polarized light, mature collagen appears well organized (white arrows); B - H&E stained ACL sections at 400× magnification to visualize cells (Black bar = 40μm). Little areas of inflammation may represent foci in which suture material was recently cleared or in which suture material was present just out of the plane of section in the aseptically processed group (black arrows). Areas of inflammation surrounding small pieces of suture characteristic of a foreign body reaction in the EBEAM group (black arrows). C - H&E stained synovium sections at 40× magnification for a general view (Black bar = 400 μm). Intimal hyperplasia was minimal and most of the synovium was covered by a normal, thin layer of synovial cells (black arrows) and blood vessels (green arrows) consistent with neovascularization were visible in some sections in both groups; D - H&E stained popliteal lymph node sections at 40× magnification for a general view (Black bar = 400 μm). Prominent sinus histiocytosis consistent with mild lymphadenopathy as a result of draining inflammation from the implant site and is not considered an adverse reaction in both groups.

ACL – Anterior cruciate ligament

ECM – Extra cellular matrix

H&E – Hematoxylin and Eosin

Figure 3. Histological Scoring of ACL, Synovium, and Popliteal Lymph Node.

Scatterplots (empty circles, n=8 per group) with mean (diamond) and 95% CI (range plots with caps) of ACL inflammation, neovascularization, fibrosis, and fatty infiltration, as well as synovial inflammation, neovascularization, fibrosis, and hypertrophy, and also popliteal lymph node sinus histiocytosis and follicular hyperplasia

ACL – Anterior cruciate ligament

PLN – Popliteal lymph node

Pts – Points

Discussion

Treatment of the ECM scaffold using 15 kGy electron beam irradiation resulted in effective sterilization of the scaffold. However, no significant reduction in biomechanical and histological performance of the ACL repair utilizing electron beam irradiated scaffolds was noted.

The industrial standard (ISO 11137-2) suggests 15 kGy irradiation dose will lead to a log reduction of 10^-6 colony forming units of bacteria and fungi when used on a material with a low initial bioburden.²⁰ Harvest of source material and the use of aseptic processing techniques can result in a scaffold with a relatively low bioburden, which can make this low dose irradiation effective for terminal sterilization of tissue engineering scaffolds. A recent study by Pruss et al. showed that a gamma irradiation dose of >30kGy was necessary to reduce a panel of human adventitious viral agents on frozen patellar tendon grafts by 4 log(10).²¹ In this current study, we were able to show that by combining selective material sourcing, aseptic technique and low dose electron beam sterilization, a final sterile product can be achieved. In the past, irradiation, particularly doses over 20kGy, have been shown to harm the collagen structure in vitro by direct splitting of the collagen chains,^{22; 23} whereas a 15 kGy dose seems resulted in less collagen degradation as well as a higher rate of cell adhesion of allograft materials in vitro.²⁴ While these prior results suggest there is a dose-dependent effect of irradiation for ECM-based medical devices in vitro, the results of the current study indicate that the use of 15 kGy electron beam irradiation did not have significant negative effects on the biomechanical or histological measures of ligament healing after eight weeks in vivo. This lack of difference is consistent with prior reports of irradiated allograft tendon when used for ACL reconstruction where the use of 25 kGy electron beam irradiated allografts in humans did not result in a significant difference in age adjusted KT-1000, IKDC knee score, or return to sport.²⁵

The histological analysis of the ligaments in the current study that received bridge-enhanced ACL repair with the terminally sterilized scaffold was consistent with an implant that was completely resorbed by eight weeks, resulting in a fibrovascular scar populated with only minimal inflammatory cells and a nascent vasculature. The results of this study demonstrated that EBEAM treatment did not change the healing and functional response of the repaired ligament versus the same outcomes achieved and reported previously using the existing ASEPTIC technique.^{9; 26; 27} No evidence of rejection or adverse reaction to the scaffold material was seen in the synovium or lymph nodes of either treatment group, as demonstrated by a thin layer of synovial cells lining the joint surface and only mild sinus histiocytosis and follicular hyperplasia in the lymph nodes, which is consistent with the inflammation associated with surgery.

The pig model in general has limitations common to all animal models of ACL injury treatment. The pig is a quadruped, and post-operative rehabilitation is difficult to control. However, porcine and human knees share multiple anatomic and biomechanical characteristics.^{28; 29} In addition, the Yorkshire pig has a similar hematologic system to humans, unlike other commonly used models for ACL reconstruction (for example, sheep and goat).³⁰ This is an important consideration for studies, such as this one, which utilize blood or its derivatives for bio-enhancement. Another limitation of the pig model is that the knee is smaller than an adult human knee; it is roughly the size of a young adolescent knee. Scale up of the technology for a fully grown adult knee will require use of a longer scaffold, which could influence the sterilization effectiveness for sterilization methods which are volume or thickness dependent. However, for this implant, the electron beam sterilization is carried out through the diameter of the scaffold. For translation to human trials, the initial scaffolds will be the same diameter as those used in this study, but the length will be increased to increase the volume. When the human-sized scaffolds for the first in human trial were evaluated, they had no loss of sterility, likely due to the fact that the diameter of the scaffolds remained identical to this preclinical study. Another limitation was the freezing and thawing of the knee that occurred before the mechanical testing, which could potentially have influenced the biomechanical performance compared to in vivo. However, a previous study evaluating changes in the tensile properties of ligaments suggests these are minimal.³¹ Furthermore, a consistent freeze-thaw protocol was used for all specimens to minimize the variability due to this handling.

Conclusion

Tissue engineering strategies employing an ECM-based scaffold have the potential to become a valuable clinical treatment. This study evaluates a critical step in the translation of these scaffolds to clinical use, namely the effects of terminal sterilization on the ability of the scaffold to support in vivo ligament healing in a validated large animal model. Electron beam irradiation at 15 kGy effectively sterilizes the scaffold without significantly harming the in vivo function of the scaffold. This result contributed to the generation of scaffolds suitable for the use in the first in human trial for bridge-enhanced ACL repair.

Table 4.

Linear stiffness, yield load, and maximum load ratios, as well as AP laxity at 30°, 60° and 90° differences as compared to the control values (untreated leg), n = 8. No comparisons are considered significant with all P-values > 0.39 using a t-test. Effect analysis did not yield a substantial difference between the means with a Cohen's d< 0.473 for all comparisons.

Surgical Group	Linear Stiffness Ratio	95% CI	Yield Load Ratio	95% CI	Max Load Ratio	95% CL	AP30°	95% CI	AP60°	95% CI	AP90°	95% CI
ASEPTIC	0.258	0.181 0.334	0.171	0.095 0.246	0.165	0.092 0.238	9.03	6.93 11.12	11.55	8.68 14.1	8.4	6.36 10.44
EBEAM	0.215	0.129 0.299	0.142	0.075 0.21	0.138	0.072 0.203	9.5	7.99 11.01	10.95	9.13 12.76	7.94	6.49 9.39
P-value	0.391		0.512		0.523		0.67		0.68		0.67
d-value	0.473		0.340		0.351		0.232		0.223		0.234

95% CI – 95% confidence interval

ACL – Anterior cruciate ligament

AP – Anterior-posterior knee laxity at a given knee flexion angle