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A Novel Core Biopsy Technique for AnteriorCruciate Ligament Preserves Ligament Structural

Integrity: A Porcine StudyMichiel Mylle, M.D., Steven Claes, M.D., Peter Verdonk, M.D., Ph.D., and

Johan Bellemans, M.D., Ph.D.

Purpose: The objective of this study was to validate a new technique to safely obtain core biopsy specimens of theanterior cruciate ligament (ACL) without jeopardizing the ACL’s biomechanical properties. Methods: Eleven pairs offresh porcine femur-ACL-tibia complexes were tested in a loading frame. The ACL of one knee was biopsied usinga spring-loaded core biopsy device, whereas the contralateral ACL was tested as the control. Biomechanical properties ofthe biopsied and control ACLs were compared. Results: The ultimate load to failure was 1,202 N � 171.1 N and 1,193 N� 228.7 N (P¼ .8984) for biopsied and non-biopsied ACLs, respectively. No significant differences were noted for maximalelongation at failure, maximal strain, absorbed energy, and stiffness between biopsied and non-biopsied ACLs.Conclusions: The results of this study indicate that a new ACL core biopsy technique can be performedwhile preserving theligament’s structural integrity. Clinical Relevance: The presented core biopsy technique could be regarded as a dedicatedtool to elucidate the poorly understood (patho)biological processes occurring in both the native and reconstructed ACLs.

ccording to the literature, 2.6% to 7.7% of an-

Aterior cruciate ligament (ACL)ereconstructedpatients have graft failure develop with recurrentinstability and will require revision ACL reconstruc-tion.1-3 A recent report of the Multicenter ACL RevisionStudy (MARS) study group identified that the cause ofACL graft failure can be traumatic (32%), technical(24%), biological (7%), a combination (37%), infection(<1%), and not known (<1%).4 Unlike reasons fortechnical failure of ACL reconstruction, the underlyingmechanisms leading to a biological graft failure (i.e.,non-traumatized though nonfunctional graft) remainlargely unclear. The diagnosis of biological graft failureis therefore predominantly based on exclusion of other

From the Department of Orthopedic Surgery & Traumatology, Universityospitals Leuven (M.M., S.C., J.B.), Leuven, and Department of Orthopedicurgery & Traumatology, University Hospital Gent (P.V.), Gent, Belgium.The authors report the following potential conflict of interest or source ofnding: P.V. receives support from Smith & Nephew, DePuy Synthes, Activeplants, and Orteq Sports Medicine.Received April 11, 2013; accepted September 3, 2013.Address correspondence to Michiel Mylle, M.D., Department of Orthopedic

urgery & Traumatology, University Hospitals Leuven, Weligerveld 1, B-3212ellenberg, Belgium. E-mail: myllemichiel@gmail.com� 2013 by the Arthroscopy Association of North America0749-8063/13237/$36.00http://dx.doi.org/10.1016/j.arthro.2013.09.003

Arthroscopy: The Journal of Arthroscopic and Related

reasons for failure,5 thus likely creating an underesti-mation of this enigmatic mode of graft failure.Animal studies have shown that after a period of

time, the implanted tendon grafts undergo a process ofseveral phases to become ligamentous “ACL-like”structures.6 The process of “ligamentization” of tendongrafts is indeed believed to consist of an initial phase ofnecrosis followed by revascularization, cellular repo-pulation, and remodeling.7 Today, however, there isgrowing evidence that the results of animal studiescannot be directly applied to human graft biology.8

According to one of our articles,8 humans and pigsare indeed different in a biological way, for example, inthe timeline of ligamentization. However, there is goodevidence that the ex vivo anatomy and biomechanics9

are quite similar. For this study, only time 0 ex vivobiomechanical aspects were of interest.All current knowledge on human ACL graft biology is

derived from superficial biopsy specimens obtained witha basket forceps during arthroscopy.7 This means that allavailable information on the biology of the humanreconstructed ACL is based on biopsy specimens ofa peripheral portion of the graft. The question remainswhether such biopsy specimens can be representative ofthe entire 3-dimensional graft structure. In this view, wepreviously concluded that the development of a corebiopsy technique is a prerequisite to better understandthe biology of the healing ACL graft in the human knee.8

Surgery, Vol -, No - (Month), 2013: pp 1-6 1

Fig 1. FAT complex on loading frame. The ACL was alignedin the direction of elongation.

2 M. MYLLE ET AL.

Ideally, the technique must meet following require-ments: safe, reproducible, minimally invasive, andproviding representative tissue samples with dimen-sions enabling histologic analysis. Given its widespreadclinical use in other medical disciplines,10,11 a spring-loaded needle core biopsy device was chosen asa possible candidate to fulfill all those requirements. Forsafety evaluation with regard to the structural integrityof the ACL after biopsy, a study was set up to comparethe biomechanical properties of a biopsied ACL with thecontrol native ACL in the contralateral knee.The purpose of this study was to validate a new

technique to safely obtain core biopsy specimens of theACL without jeopardizing the ACL’s biomechanicalproperties. We hypothesized that there would be nodifference in biomechanical properties between thebiopsied and non-biopsied ACLs.

MethodsPrior sample size calculation showed that 22 paired

(11 � 2) fresh cadaveric porcine knees were needed toobtain a power of 0.8. Pigs were chosen because thephysical dimensions and biomechanical properties ofthe porcine ACL have been shown to closely mimicthose of the human ACL,9,12 with the advantage ofbeing easily available in a fresh state. The pigs werekilled at a mean age of 180 days, according to the rulesfor human consumption regulated by Belgian law. Thefirst author was present during packaging of the spec-imens to guarantee the correct pairing of the kneesfrom the same cadaver. The paired knees were thensealed in a plastic bag and stored in the refrigerator at3�C, with the experiments being performed the dayafter slaughter.At the start of the experiments, both the femur and

tibia were cut with an oscillating saw at a level 15 cmaway from the joint line. The knees were then dissectedand stripped of all surrounding soft tissues except forthe ACL, which was carefully isolated. The femur-ACL-tibia (FAT) complex was kept moist with saline solutionspray before and during testing. Knees with a grossdeformity or the slightest visible damage to the ACLwere excluded. Ethical approval for this study wasgranted through our university’s committee for medicalethics.

Testing ProtocolThe FAT complex was subsequently potted firmly in

custom-made metal cups by use of a resin consisting of2-hydroxypropyl methacrylate (VersoCit-2; Struers A/S, Ballerup, Denmark). To avoid slippage of the femuror tibia, an additional bolt was placed througha custom-made hole in the cups. Both metal cups werethen mounted onto the loading frame (MTS BIONIX858 Axial Torsional test system; MTS Systems, EdenPrairie, MN) so that the FAT complex was placed on the

loading frame with the knee at 30� of flexion, theso-called tibial orientation described by Woo et al.13

(Fig 1). Preloading to 5 N was performed to allowa method of standardization of the specimens beforetesting. The dimensions of the ACL (length and ante-roposterior and mediolateral diameter at the mid-substance) were measured 3 times with a Verniercaliper (� 0.05 mm). The mean anteroposterior andmediolateral width and length of the ACL were 4.96mm, 6.77 mm, and 39.93 mm, respectively. Pairedspecimens were randomized by the toss of a coin toeither undergo biopsy or act as a control. All experi-ments were performed at room temperature.In the biopsy group, a single core biopsy was obtained

with the use of the Monopty disposable biopsy instru-ment (Bard Peripheral Vascular, Tempe, AZ). TheMonopty instrument is a fully automated biopsy devicethat automatically triggers a rapid-firing side-notch corebiopsy needle. It uses a 2-stage biopsy action: a springaction thrusts the inner trocar forward, followed almostinstantaneously by a similar forward thrust of the outercutting cannula. Thus the tissue specimen is trapped inthe side notch of the trocar when the cutting cannula isadvanced.11 For the aim of this study, the Monopty121610 instrument measuring 16 gauge � 10 cm long

Fig 2. The Monopty Core biopsy instrument, generally usedin other medical disciplines, is an ideal instrument for takingbiopsy specimens for histologic investigation.

Fig 4. Typical specimen obtained using core biopsy.

NOVEL ACL CORE BIOPSY TECHNIQUE 3

with a penetration depth of 22 mm and a sample notchlength of 17 mm was used. Biopsy specimens weresystematically obtained from the mid-portion of theACL while the trocar rested against the cartilage of themedial femoral condyle to induce some reproducibility(Figs 2-4).Finally, an uniaxial tensional load on the FAT

complex was generated by the loading frame by dis-placing the femur at a rate of 0.33 mm/s, whereas thetibia was held stationary, until a point well beyond theACL had snapped. The ACL was aligned in the directionof elongation according to the recommendations ofWoo et al.13 and Paschos et al.14

The load cell was connected to a computer, recordingthe load-displacement data at 100 Hz. The highest pointof the force-displacement curve was considered theultimate failure point. Strain was calculated as the ratioof displacement to the initial length of the ACL andstress as the ratio of load to the initial cross-sectionalarea. Energy absorption (in newton millimeters) wasobtained by taking the area under the force-displacement curve up to the failure point (Fig 5).

Fig 3. The biopsy specimen was placed on the load cell afterpreloading.

Stiffness (in newtons per millimeters) was calculatedfrom the slopes of the linear portion of the force-displacement curve obtained from testing to failure.Accordingly, the Young modulus (in megapascals) wasobtained from the stress-strain curve.

Statistical AnalysisA sample size calculation was performed to detect

a change in maximum load based on a 2-sided paired ttest with a equal to .05. Assuming an SD equal to 250N15 and a correlation of 0.5 between paired data, a totalof 11 paired specimens were needed to obtain a powerof 0.8 to detect a difference in ultimate failure load of235 N. Data are expressed as mean � standard devia-tion. A paired Wilcoxon signed rank test was used tocompare the measurements between control andbiopsy groups. Analyses have been performed with SASsoftware (SAS System for Windows, version 9.2; SASInstitute, Cary, NC).

Fig 5. Force-displacement curve. The highest point on thecurve was taken as the failure point.

4 M. MYLLE ET AL.

ResultsSeven variables were analyzed and compared

between biopsied and control knees: ultimate loadto failure, elongation at failure, maximum strain,maximum stress, energy absorption, Young modulus,and stiffness. The values for these parameters aresummarized in Table 1 and reported as mean � stan-dard deviation.After statistical analysis, no significant difference

could be found between the control group and thebiopsy group for any of the tested variables. Even moreimportantly, the mean differences for ultimate load (9N), elongation (0.15 mm), stress (3.64 MPa), strain(0.005), stiffness (3.8 N/mm), Young modulus (2.3MPa), and energy absorption (243 Nmm) between the2 conditions seemed of no clinical relevance at all. Thedifference in percentage is shown in Fig 6.

DiscussionThe purpose of this study was to document the safety

of a new core biopsy technique for obtaining centraltissue samples of the ACL. The results confirm thatusing our technique, the mechanical properties of thenative ACL are unaffected.Given this result, the proposed technique enables the

safe procurement of a piece of tissue obtained from thecore an ACL while avoiding mechanical damage. Giventhis biopsy device’s widespread use in other surgicaldisciplines, the tissue sample size obtained by thisdevice is definitely large enough for histologic investi-gation.10,11 In this view, this study might offer a toolthat is able to safely deliver tissue samples from the verycore of a reconstructed human ACL to study its liga-mentization process without causing damage to thegraft’s biomechanical properties.Ntoulia et al.16 investigated the graft revascularization

process in humans after ACL reconstruction withmagnetic resonance imaging (MRI). Although MRImight be a noninvasive procedure to investigate therevascularization phase of the ligamentization process,there is no proven correlation between these MRIfindings and histologic properties. Furthermore, revas-cularization forms only one aspect in the healingprocess of an ACL graft.

Table 1. Means and Standard Deviations for All Biomechanical P

Statistic Maximum Load (N) Elongation (mm) Stress (MPa) Strain

Biopsy groupMean 1,202 10.66 44.81 0.2SD 171.1 3.33 9.5 0.0

Control groupMean 1,193 10.51 48.45 0.2SD 228.7 3.38 10.87 0.0

P value .8984 .8984 .2402 .9

Core biopsy specimens as a diagnostic tool in biolog-ical failure of the graft or mucoid degeneration could beused in the individual patient. Their potential applica-tions in clinical practice are therefore multifold.Ménétrey et al.5 concluded that the pathologic entity

of biological graft failure, though accounting for at least7% of the failures after ACL reconstruction,4 is stillpoorly understood. In this field the implementation ofthis new, minimally invasive biopsy technique in sus-pected cases might show important pathobiologicalclues surrounding this enigmatic biological graft failure,providing a direct diagnosis of this entity rather thana diagnosis by exclusion and allowing possible preven-tive or therapeutic measures.In the same view, remnant-preserving techniques for

ACL reconstruction have recently gained interest withtheir assumed benefit related to an improved or accel-erated ligamentization process.17 It has been suggestedthat the revascularization phase of the ACL graft mightstart earlier and that the proprioceptive function of theligament could be better preserved with this technique.Löcherbach et al.,18 for example, concluded that thistechnique could form a possible advantage for ACLreconstruction, although currently, no reliable methodexists to investigate graft revascularization and healingafter ACL surgery. Moreover, in this field the possibilityto obtain safe and representative core biopsy specimensof the ACL graft has the potential to show the addedvalue of such a remnant-preserving technique at thebiological level.Mucoid degeneration of the human ACL is still

an intriguing clinical entity that is more frequentlyencountered than previously thought.19 Typical clinicalfindings are posterior knee pain and restriction of kneeflexion and extension. The diagnosis is made based onclinical presence and MRI. Current treatment witharthroscopic partial excision has been shown to beeffective and safe, although postoperative laxityremains a subject of debate. This pathology is histo-logically characterized by mucoid degeneration of theACL tissue with accumulation of glycosaminoglycansbetween the collagen fibrils.20 Makino et al.21 foundthat mucoid degeneration could be histologicallyconfirmed in only 6 of 10 patients with clinical and MRIcriteria for mucoid degeneration. Therefore a more

arameters in Biopsy and Control Groups

(Ratio) Stiffness (N/mm) Modulus (MPa) Energy Absorption (Nmm)

71 185.5 293.5 7,08895 46.51 103 2,665

66 181.7 295.8 6,84581 35.87 86.65 3396658 .8311 .9658 .8984

Fig 6. Difference between biopsy andcontrol groups (percent). (abs, absorp-tion; max, maximum.)

NOVEL ACL CORE BIOPSY TECHNIQUE 5

efficient method to diagnose this pathologic entity moreaccurately would be useful. In this way the presentedcore biopsy technique could earn its place in mucoiddegeneration as a diagnostic tool but also for furtherresearch into the exact histologic processes and causa-tive mechanisms.The quest to develop an off-the-shelf synthetic graft

for ACL reconstruction has delivered variable results.The ideal graft is an ACL scaffold that meets the func-tional mechanical demands immediately followed bya gradual degradation while host tissue grows in.Finally, attempts to enhance biological healing

processes have gained much interest in the orthopaediccommunity.22 The use of platelet-rich plasma andvascular endothelial growth factor has been proposed toimprove the biological healing of ACL grafts. However,their effect on clinical outcome remains unclear.23,24

Whereas the biology of ACL graft healing remainsa hot topic, the results of this study could offer a -dedicated tool to gain a better understanding of theprocess of biological graft failure after ACL reconstruc-tion, the enigmatic entity of mucoid degeneration of anative ACL, and the biological effects of growthfactor application (e.g., platelet-rich plasma) orremnant-preserving ACL reconstruction techniques ongraft healing.

LimitationsOur study has some limitations. First, an animal

model was used for our analyses. Even though pigshave been shown to be a good animal model for studieswith ACLs, the results may not automatically apply tohumans.Second, the biopsy would have ideally been per-

formed in living animals. A healing effect of the biopsy

specimen could occur in vivo with less damage to theACL, and therefore we require further assurance of itssafety in use.Finally, the biopsy specimens in this study were ob-

tained from native porcine ACLs, whereas biopsyspecimens from in situ ACL grafts undergoing theprocess of ligamentization possibly would mimic themechanical properties of the human healing graft evenbetter. However, this study design would entail anextremely complex setup involving porcine ACLreconstruction procedures and subsequent slaughteringand biomechanical testing weeks after the operation.

ConclusionsThe results of this study indicate that a new ACL core

biopsy technique can be performed while preservingthe ligament’s structural integrity.

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