Post on 11-Aug-2020
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Title 1
'Biomechanical Properties of Bovine Tendon Xenografts Treated with a Modern 2
Processing Method'. 3
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Authors & Contributions Statement 5
Mr Henry B Colaço*: study design, data collection/analysis, manuscript preparation 6
Mr Breck R Lord**: data collection, data analysis 7
Ms Diane L Back***: study design, manuscript preparation 8
Mr Andrew J Davies***: study design, manuscript preparation 9
Prof Andrew A Amis**: study design, manuscript preparation, approval 10
Mr Adil Ajuied***: study design, data analysis, manuscript preparation, approval 11
All authors have read and approved the final submitted manuscript. 12
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*Royal Free London NHS Foundation Trust, London, UK 14
**Imperial College London, London, UK 15
***Guy's and St Thomas' Hospital NHS Foundation Trust, London, UK 16
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Corresponding Author 18
Mr Henry B Colaço 19
22 Ballingdon Road, London, SW11 6AJ, UK 20
tel: +44(0)7958 567 359 21
e-mail: henry@henrycolaco.com 22
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Keywords: Xenograft; Tendon; Knee Ligaments; ACL; Sterilization 24
Word Count: 2,359 (Introduction through Discussion) 25
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Abstract 26
Xenograft tendons have been used in few human studies, with variable results. With 27
the advent of novel tissue processing techniques, which may mitigate against an 28
immune-mediated rejection response without adversely affecting mechanical 29
properties, there may now be a clinical role for xenograft tendons, particularly in knee 30
ligament reconstruction. We hypothesize that ‘BioCleanse®’ processed bovine 31
extensor digitorum medialis (EDM) tendons exhibit favorable time-zero pre-32
implantation biomechanical characteristics when compared to both unprocessed 33
bovine EDM tendons and BioCleanse® processed human cadaveric allograft tibialis 34
anterior tendons. 35
In this in vitro case controlled laboratory study, three groups of tendons underwent a 36
5-stage static loading test protocol: 15 BioCleanse® bovine (BCB), 15 fresh frozen 37
unprocessed bovine (FFB), and 12 BioCleanse® human allograft (BCA) tendons. 38
Cross-sectional area of the grafts was measured using an alginate molding technique, 39
and tendons were mounted within an Instron® 5565 Materials Testing System using 40
cryogenic clamps. 41
BCB tendons displayed a higher ultimate tensile stress (p<0.05), with equivalent 42
ultimate failure load, creep, and modulus of elasticity when compared to the FFB 43
tendons (p>0.05). BCB tendons had an equivalent cross-sectional area to the BCA 44
tendons (p>0.05) whilst exhibiting a greater failure load, ultimate tensile stress, less 45
creep and a higher modulus of elasticity (p<0.05). 46
The BioCleanse® process did not adversely affect the time-zero biomechanical 47
properties of bovine xenograft EDM tendons. BioCleanse® processed bovine 48
xenograft EDM tendons exhibited superior biomechanical characteristics when 49
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compared with BioCleanse® processed allograft tibialis anterior tendons. These 50
findings support further investigation of xenograft tendons in orthopaedic soft tissue 51
reconstructive surgery. 52
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Keywords: Knee ligaments; Xenograft; Sterilization; Biomechanics of tendon 54
4
Introduction 55
Current graft options for knee ligament reconstruction include autograft, allograft, and 56
synthetic graft (Strobel et al., 2006; Bonanzinga et al., 2014; Legnani et al., 2010). 57
These exhibit different biomechanical properties, complication profiles, handling 58
characteristics, availability, host integration, cost, and the ability to replicate native 59
anatomy (Barrera Oro et al., 2011). The limit of available autograft represents a 60
significant challenge in the context of revision soft tissue reconstructions and the 61
multi-ligament knee reconstruction (Levy et al., 2009; Lind et al., 2009). One possible 62
solution would be to implant tendon grafts of animal origin (xenograft) (Laurencin 63
and El-Amin, 2008). This is an established concept in soft tissue reconstruction, 64
which has been applied with varying degrees of success over the last 60 years, 65
although there remains a paucity of research in this field (Allen et al., 1987, Dahlstedt 66
et al., 1989, Stone et al., 2007a). 67
The potential benefits of xenografts include avoidance of donor site morbidity, a 68
reduction of surgical anaesthetic time, a potentially lower cost profile, elimination of 69
the risk of human blood-borne diseases (e.g. HIV, Hep C), and an abundance of 70
source material resulting in a more robust supply chain (Cooper et al., 2010, Nagda et 71
al., 2010, Simonds et al., 1992, Asselmeier et al., 1993, Patel and Trampuz, 2004, 72
Kainer et al., 2004). Commercially available xenograft products for orthopaedic 73
surgery range from osteoconductive hydroxyapatite and collagen-based bone graft 74
substitutes, to soft tissue extracellular matrix grafts from dermis and small intestine 75
submucosa (Mucalo, 2007; Neen et al., 2006, Soler et al., 2007, Iannotti et al., 2006). 76
At present, there are no widely available commercial xenogeneic tendons or ligaments 77
suitable for implantation in humans. 78
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If unprocessed bovine or porcine xenografts, which express α-gal and other epitopes 79
(e.g. Neu-Gc) known collectively as ‘non-gal epitopes’, are implanted into humans or 80
non-human primates, the graft will trigger an acute immune mediated rejection 81
response and result in catastrophic failure (Stone et al., 2007b, Galili, 1993; Galili, 82
2013; Galili, 2012). Xenograft tissue also carries with it the inherent risks of immune 83
mediated rejection and transmission of zoonoses (e.g. Bovine Spongiform 84
Encephalopathy (BSE) (Laurencin and El-Amin, 2007). For bovine tissue, the 85
foremost risk can be mitigated by sourcing all cattle from BSE-free countries, as 86
recognized by the World Organization for Animal Health (OIE). The challenge lies in 87
developing processing techniques that effectively sterilise the tissue and overcome 88
immune-mediated rejection, whilst maintaining favourable biomechanical properties 89
and graft-host integration. 90
Previous methods of terminal sterilization such as ethylene oxide, gamma irradiation, 91
and glutaraldehyde have been shown to adversely affect the handling and 92
biomechanical properties of graft tissue (Allen et al., 1987; Jackson et al., 1990; 93
Gibbons et al., 1991; Rasmussen et al., 1994). Lyophilization can have similar 94
adverse effects and does not guarantee removal of pathogens (Kamiński et al., 2009; 95
Galili, 2013; Crawford et al., 2004). Although a large cohort study recently identified 96
a number of patient and graft-related factors, including processing method, that were 97
associated with revision risk after allograft ACL reconstruction, the use of allograft 98
for ACL and multiligament knee reconstruction remains standard practice (Tejwani et 99
al). 100
Several novel proprietary methods have been developed for treating allograft, which 101
involve physical and chemical processes to achieve decontamination and sterilization 102
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(Vangsness et al., 2003; Vangsness and Dellamaggiora, 2009; McAllister et al., 103
2007). These processes have the potential to play a role in processing xenograft tissue 104
for implantation in humans if the biomechanical properties of the tissue are not 105
adversely affected, and if antigen expression is reduced below a threshold where no 106
significant immune response is triggered (Stone et al., 1997; Stone et al., 2007a). 107
The purpose of this study was to compare the pre-implantation tensile biomechanical 108
properties of chemically processed bovine extensor digitorum medialis (EDM) 109
tendons against unprocessed fresh-frozen bovine tendons, and chemically processed 110
tibialis anterior allograft tendons. It was hypothesized that chemically processed 111
bovine extensor tendons would have similar biomechanical properties to unprocessed 112
bovine and chemically processed human allograft tendons. The clinical relevance of 113
the study is that if the bovine xenograft tendons exhibit similar or superior mechanical 114
properties to human allograft tendons, and the BioCleanse® process does not 115
adversely affect the time zero biomechanical properties, we will have identified a 116
potential tendon graft source for further study in animal and human trials. 117
7
Methods 118
Following institutional approval, bovine extensor digitorum medialis (EDM) tendons 119
were harvested aseptically from certified BSE-free cattle with a minimum weight of 120
295kg and age under 30 months, confirmed by dentition (Maverick Biosciences Pty 121
Ltd., Dubbo, East NSW, Australia). Fifteen fresh frozen bovine (FFB) tendons 122
underwent no further processing, and were stored at -30°C. A further 15 bovine EDM 123
tendons (BCB: BioCleanse bovine) and 12 human tibialis anterior allograft (BCA: 124
BioCleanse allograft) tendons were processed using the proprietary BioCleanse® 125
process (RTI, Alachua, Florida, USA). All tendons were imported frozen on dry ice 126
and stored at -30°C until testing, at which time the grafts were placed in a 23°C water 127
bath to achieve controlled thawing in watertight sealed packs. 128
Tendon specimens were prepared to 90mm length, allowing secure cryogenic clamp 129
fixation of a 30mm segment at each end, leaving the central 30mm section, similar to 130
the length of the native anterior cruciate ligament (ACL), to undergo mechanical 131
tensile testing. The mean cross sectional area (CSA) of each tendon was calculated 132
prior to testing using a ‘replica molding’ technique (Goodship and Birch, 2005). An 133
alginate dental impression material (Blueprint cremix, Dentsply DeTrey, Germany) 134
was used to form a cast and axial digital photographs taken of 3 equally spaced 135
sections using a mounted Canon EOS600D digital SLR with an 18-megapixel sensor 136
and fixed focal length Canon EF 50mm f/1.8 II standard lens. The mean CSA was 137
calculated using image analysis software (Image J 1.48, National Institute of Health, 138
Bethesda, Maryland, US). This method is accurate to within 0.8%, and reproducible 139
(coefficient of variation = 1.42%), allowing calculation of ultimate tensile stress and 140
Young’s modulus of elasticity. 141
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The custom soft-tissue cryogenic clamps (Riemersma and Schamhardt, 1982), used 142
corrugated plates and injection of liquid carbon dioxide (CO2) to freeze the tissue and 143
prevent slippage. The rounded edges of the clamps reduced the stress riser at the 144
interface between the clamp and the central 30mm length of tendon to be tested. An 145
initial clamp temperature of approximately -30°C was reached by expansion of liquid 146
CO2. At this temperature, tissue thermal transition is acceptable, even at a distance of 147
5 mm from the device, which minimizes alteration of viscoelastic properties (Rincón 148
et al., 2001; Lam et al., 1990; Woo et al., 1987). 149
Tensile mechanical testing was performed using a single-axis materials testing 150
machine (Model 5565, Instron, High Wycombe, UK) accurate to +/- 0.02 mm. The 151
upper cryogenic clamp was attached to a 5kN load cell (accuracy < 0.025 %) mounted 152
to the mobile crosshead, with the lower clamp secured to a static base plate. The 153
automated test protocol was set using Instron® Bluehill® 2 modular applications 154
software with data capture every 100ms. Testing was conducted at room temperature 155
(23°C). Each tendon was secured within the clamps using moderate compression 156
before increasing the tension manually using fine position control to achieve a starting 157
load of 0 to 0.005 N. 158
The test protocol was divided into five phases (Table 1): each tendon underwent an 159
initial preload to allow uncrimping of collagen fibers (phases 1&2), followed by a 160
steady ramp to 500 N (phase 3), which exceeded the maximum load experienced in 161
ACL graft during accelerated rehabilitation (Noyes and Grood, 1976; Woo et al., 162
1991). The load was maintained at 500N for two minutes (phase 4) to allow for 163
measurement of creep (Jaglowski et al., 2014). Mean creep strain rate (S s-1) was 164
calculated for each tendon by dividing change in strain (S), where S = creep 165
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elongation/original length, over the 120 second period. Finally the specimen was 166
loaded to failure (phase 5). The ultimate tensile stress (UTS) was calculated for each 167
tendon, by dividing the ultimate failure load (UFL, N) by CSA (mm2); so UTS = UFL 168
/ CSA (MPa). Young’s modulus of elasticity (E) was calculated for each tendon from 169
the linear, elastic portion of the force versus extension curve during phase 5 ramp to 170
failure loading, which was converted to a stress versus strain curve by dividing the 171
force by the CSA and the extension by the original gauge length of the specimen. 172
Statistical analysis was performed using IBM® SPSS® Statistics v22.0.0.0 (IBM, 173
Armonk, NY, USA). One-way analysis of variance (ANOVA) test was applied, and 174
post hoc analysis was performed using the Tukey Honestly Significantly Different 175
(HSD) test to identify significant differences between pairs of compared groups. A 176
significance level of P<0.05 was applied throughout. 177
As the effect of the BioCleanse® process on bovine tendons was not yet known, an 178
accurate a priori power calculation could not be made with certainty. The number of 179
tendons required to adequately power the study ( = 0.8) was therefore estimated 180
from a review of similar published data (Schimizzi et al., 2007; Reid et al., 2010; 181
Conrad et al., 2013). A post-hoc power analysis confirmed the assumption that the 182
study was adequately powered (>0.8) to reach significance. 183
10
Results 184
The cross-sectional areas (CSA) of both BioCleanse® groups were smaller than the 185
unprocessed bovine group (p< 0.01), but a difference was not found between BCB 186
and BCA (p>0.05) (Table 2). Levene’s test for equality of variances was >0.05, which 187
means that we could assume homogeneity of variances, or an equal variance within 188
each group when compared with the other two groups. 189
The ultimate failure load (UFL) was higher in both bovine groups compared with 190
allograft (p< 0.05 and p< 0.01 for BCB and FFB, respectively), but a difference was 191
not found between BCB and FFB (p >0.05) (Table 2). The ultimate tensile stress 192
(UTS) was higher in the processed bovine group than both other groups (p<0.05 and 193
p<0.01 for FFB and BCA, respectively). , but a difference was not found between 194
FFB and BCA (p >0.05) (Table 2). 195
The allograft tendons exhibited more creep and a lower Young’s modulus of elasticity 196
(E) than both bovine groups. Analysis found that the BCA tendons crept faster than 197
BCB (p< 0.05) and FFB (p< 0.01), but did not find a difference in creep between 198
BCB and FFB (p >0.05), and that while E was significantly lower in BCA than BCB 199
(p< 0.01) and FFB tendons (p< 0.05), a difference was not found between BCB and 200
FFB (p >0.05) (Table 2). 201
Interestingly, the BioCleanse® treated bovine tendons exhibited a closer correlation 202
between CSA and ultimate failure load (r2 = 0.745) (Figure 1), when compared with 203
fresh frozen bovine (r2 = 0.28735), and BioCleanse® treated human tendons (r2 = 204
0.220) (Table 2, Figure 1). A post-hoc data analysis revealed the power to be 0.86 205
(>0.8), which confirmed pre-test estimates that the sample size would be sufficient to 206
adequately power the study. 207
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Discussion 208
The most important finding of this study was that the chemically treated tendons had 209
similar or superior mechanical properties to their fresh-frozen controls. The processed 210
bovine tendons (BCB) had greater ultimate tensile stress, with equivalent ultimate 211
failure load, creep, and modulus of elasticity when compared with the fresh frozen 212
bovine tendons (FFB). The processed bovine (BCB) tendons had similar cross-213
sectional area, higher ultimate tensile stress, failure load, and modulus of elasticity, 214
with less creep, when compared with the processed human allograft (BCA) tibialis 215
anterior tendons. Thus, the ‘time zero’ mechanical properties of the treated tendons 216
are suitable for a ligament graft. 217
The effects of the BioCleanse process on the time-zero biomechanical properties of 218
human allograft have been investigated previously. Schimizzi et al. (2007) compared 219
BioCleanse treated allograft human tibialis anterior tendons against gamma-irradiated, 220
and fresh-frozen tendons, and found no significant difference in failure load and 221
failure stress. 222
Jones et al. (2007) compared BioCleanse treated human bone-patellar tendon-bone 223
(BTB) grafts against fresh frozen BTB grafts, again using a cyclic testing protocol 224
followed by ramp to failure. They found no difference in ultimate failure load, 225
ultimate stress, cyclic creep, or stiffness. 226
Reid et al. (2010) compared three groups of human bone-patellar tendon-bone 227
allografts: BioCleanse, low-dose irradiated (50kGy) fresh frozen, and Clearant 228
(Clearant Inc., Los Angeles, California, USA) treated specimens, half of which were 229
terminally irradiated with 50 kGy gamma radiation. They found no significant 230
differences between the groups for stiffness under cyclic loading, or ultimate stress. 231
12
Conrad et al. (2013) compared BioCleanse treated human allograft Achilles tendons 232
against tendons treated with 1.5-2.5 MRads gamma radiation, and fresh-frozen 233
tendons. The BioCleanse group exhibited similar ultimate failure load and stiffness, 234
but a lower Young’s modulus and ultimate stress. 235
This study compared chemically processed tendon with frozen human allograft 236
tendons and not with fresh specimens. That was because it is not possible to source, 237
transport and store them prior to use without freezing. Freezing of fresh tendons is a 238
standard method of tissue preparation and storage for allograft (Schimizzi et al., 239
2007; Reid et al., 2010; Conrad et al., 2013). Studies have found minimal effects of 240
deep-freezing and thawing on the biomechanical characteristics of human and animal 241
tendons, including multiple freeze-thaw cycles (Huang et al, 2011; Moon et al, 2006; 242
Jung et al, 2011; Lee et al, 2009). 243
One limitation of our study is that although the temperature of the cryogenic clamps 244
was known, the temperature at the mid-substance of the tendon was not measured 245
during testing. However, although tissue temperature can alter biomechanical 246
properties, the thermal transition profile was acceptable and unlikely to affect results 247
properties (Rincón et al., 2001; Lam et al., 1990; Woo et al., 1987). 248
Although this study has demonstrated acceptable mechanical properties of the 249
chemically treated xenografts when compared to untreated xenografts and human 250
tibialis anterior allografts, this does not mean that they are suitable for clinical use. It 251
is well-known that ligament constructs other than autografts suffer from differing 252
tissue reactions and changes of strength post-implantation, and may also provoke 253
unwanted host reactions (Wong and Griffiths, 2014); those aspects require testing in 254
animals, ideally non-human primates, prior to human trials. 255
13
Conclusion 256
This study has established that the time-zero biomechanical characteristics of 257
BioCleanse® processed bovine tendons are suitable for a ligament graft. This 258
supports further investigation of the application of xenograft tendon in orthopaedic 259
soft tissue reconstructive surgery prior to clinical use. 260
261
Acknowledgements 262
Funding was received in the form of donated tendons from Regeneration 263
Technologies Inc., Alachua, FL. 264
The Instron machine was donated by the Arthritis Research UK charity. 265
No authors have any relevant financial disclosures or conflicts of interest do declare. 266
14
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Phase Start load (N) End load (N) Time (s) Load rate
1 Ramp to preload 0 10 10 1 N/s
2 Preload 10 10 60 0
3 Ramp to 500N 10 500 10 49 N/s
4 Creep 500 500 120 0
5 Ramp to failure 500 (failure) (variable) 10 mm/s
427
Table 1. Tensile testing load protocol. 428
429
430
BCB FFB BCA
CSA (mm2) 24 ± 6 33* ± 5 24 ± 4
UFL (N) 2,132 ± 473 2,286 ± 573 1,559* ± 176
UTS (MPa) 88.1* ± 10.2 70.2 ± 13.2 68.9 ± 9.3
Creep (S s-1) 0.035 ± 0.011 0.028 ± 0.010 0.046* ± 0.020
Young’s modulus (E; MPa) 725.5 ± 139.8 642.8 ± 182.4 484.2* ± 99.1
431
Table 2. Results of tensile testing. * denotes significant difference (p<0.05) 432
433
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Fig 1. 435