The American Journal of Sports Medicine http://ajs.sagepub.com/
Ligamentization of Autogenous Hamstring Grafts After Anterior Cruciate Ligament Reconstruction: Midterm Versus Long-term Results Shikui Dong, Guoming Xie, Yang Zhang, Peng Shen, Xiaoqiao Huangfu and Jinzhong Zhao Am J Sports Med published online June 1, 2015 DOI: 10.1177/0363546515584039 The online version of this article can be found at: http://ajs.sagepub.com/content/early/2015/05/30/0363546515584039
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Ligamentization of Autogenous Hamstring Grafts After Anterior Cruciate Ligament Reconstruction Midterm Versus Long-term Results Shikui Dong,* MD, Guoming Xie,* MD, Yang Zhang,* MD, Peng Shen,* MD, Xiaoqiao Huangfu,* MD, and Jinzhong Zhao,*y MD Investigation performed at the Department of Sports Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China Background: In previous studies, unimodal, small-diameter collagen fibrils have been commonly observed as the final collagen ultrastructure of the implanted grafts used in anterior cruciate ligament (ACL) reconstruction. However, the native ACL and hamstring tendon show bimodal collagen fibril distribution, consisting of both large- and small-diameter collagen fibrils. Hypothesis: Bimodal collagen fibril distribution of the graft is a common phenomenon after ACL reconstruction with hamstring tendon grafts and is time dependent. Study Design: Controlled laboratory study. Methods: A total of 52 patients who underwent double-bundle ACL reconstruction using autogenous hamstring tendons and who also underwent second-look arthroscopic surgery were enrolled. The patients were divided into 2 groups according to the time interval between the 2 operations: the midterm group (27 patients), with a 13- to 30-month time interval between operations, and the long-term group (25 patients) with a 31- to 62-month interval. During the second-look arthroscopic procedures, ACL graft biopsies were performed. Normal ACL tissues were harvested from 9 patients who underwent total knee replacement, and biopsy specimens of the to-be-grafted semitendinosus tendon tissues were also harvested from another 9 patients who underwent ACL reconstruction with hamstring tendons, which were designated as normal controls. Graft vascularity, cellularity, metaplasia, cellular metabolism, and collagen fibril distribution were analyzed. Results: Large-diameter (.100 nm) collagen fibrils were detected in 81.5% of the specimens in the midterm group and in 68.0% of the specimens in the long-term group. A typical bimodal distribution mode was observed in 62.6% of the specimens in the midterm group and in 52.0% of the specimens in the long-term group. There was no significant difference between groups with respect to the presence of large-diameter collagen fibrils, bimodal distribution, graft vascularity, cellularity, metaplasia, or cellular metabolic status. Conclusion: Graft ultrastructural maturation, characterized by large-diameter collagen fibrils and a bimodal collagen fibril distribution, is a common phenomenon and is not time dependent in the midterm to long term. Clinical Relevance: After hamstring tendon ACL reconstruction, the implanted grafts can transform into ACL-like tissue with a similar ultrastructure and metabolism, implying their usefulness as grafts. Keywords: anterior cruciate ligament; ligamentization; hamstring tendon; collagen fibril distribution
y Address correspondence to Jinzhong Zhao, MD, Department of Sports Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, China (email: scope [email protected]). *Department of Sports Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China. The authors declared that they have no conflicts of interest in the authorship and publication of this contribution.
Anterior cruciate ligament (ACL) reconstruction is a common procedure in the field of orthopaedic sports medicine.31 Full restoration of the native ACL function depends on its reconstruction with grafts that provide sufficient initial strength, ideal tendon-bone healing, and ideal ligamentization of the graft tissue.23 Numerous studies have examined the restoration of ACL anatomy and the ideal ACL reconstruction techniques.15 Numerous studies have also examined the enhancement of tendon-bone healing, to achieve a firm tendon-bone connection, during and
The American Journal of Sports Medicine, Vol. XX, No. X DOI: 10.1177/0363546515584039 Ó 2015 The Author(s)
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TABLE 1 Patient Details for the Midterm and Long-term Groups Patient Sex, n Group Midterm Long-term
Female
Male
Age, Mean 6 SD (Range), y
9 7
18 18
29.0 6 9.3 (17-49) 29.6 6 8.8 (17-48)
Limb, n Right 16 10
Left
Duration, Mean 6 SD, mo
Lysholm Score, Mean 6 SD
11 15
23.4 6 5.9 39.3 6 7.9
94.9 6 3.8 95.8 6 2.9
after ACL reconstruction.11,19,30,33,36 However, whether the graft tissues finally transform into ultrastructurally similar ACL tissue or just function as a fibril connection between the femur and tibia, without complete transformation, remains to be explored.10 ACL grafts are known to undergo a biological transformation process after being implanted into the knee joint; thereafter, the graft remodels itself into a viable ACLlike tissue, a process termed ‘‘ligamentization.’’3 Several studies have reported that the maturation period varies from 9 to 18 months postoperatively.1,9,37,38 However, even if the matured free-tendon graft looks similar, grossly and light microscopically, to an intact ACL, its ultrastructure continues to change over time. In previous studies, an ultrastructural ligamentization endpoint has not been observed.1,12,27,28,29 The final, commonly observed collagen ultrastructure of the implanted graft is a small-diameter (\100 nm), unimodal collagen fibril distribution pattern.1,8,37 On the other hand, the native ACL and hamstring tendon show bimodal collagen fibril distribution patterns and consist of large-diameter (.100 nm) collagen fibrils in addition to small-diameter collagen fibrils; the large-diameter collagen fibrils are believed to contribute to the tissue’s tensile strength.1,8,23,30,34-36 However, in our pilot study of hamstring tendon graft ligamentization, bimodal collagen fibril distribution of the implanted tissue was observed (unpublished observation). Whether this ultrastructural maturation of the graft is a common phenomenon and whether it is time dependent remain to be explored. Thus, we conducted a study to evaluate implanted graft ultrastructural maturation. Our hypothesis was that graft maturation, with bimodal collagen fibril distribution as a main characteristic, is a common phenomenon after hamstring tendon ACL reconstruction and is time dependent. This hypothesis implies that better morphological results can be observed in patients at later follow-up time points.
METHODS This was a retrospective evaluation of prospectively collected data. The study was approved by our local ethics committee, and only patients who provided informed consent were included in this study. Patients who underwent arthroscopic, double-bundle ACL reconstruction with 8 strands of autogenous hamstring tendons and who also underwent second-look arthroscopic
Lachman Test Result, n 0
11
24 21
3 4
surgery were considered for enrollment. Second-look arthroscopic surgery was indicated for patients undergoing hardware removal. Before second-look arthroscopic surgery, magnetic resonance imaging and clinical examinations were performed. The patients with imaging-indicated graft failure (graft absence, discontinuity, extreme volume loss) or clinical failure (side-to-side anterior knee laxity .5 mm or a positive pivot-shift test result) were excluded from this study. During second-look arthroscopic surgery, patients with .50% anteromedial (AM) bundle graft tissue loss were excluded. Finally, 52 patients were enrolled, between February 2013 and April 2014, after the exclusion of 2 patients because of inadequate volume of the AM bundle and 2 patients because of clinical failure with a side-toside anterior knee laxity of .5 mm and a positive pivot-shift test result. The patients were divided into 2 groups according to the time interval between ACL reconstruction and their second-look arthroscopic procedures: The midterm group (27 patients) had a 13- to 30-month time interval (mean, 23.4 6 5.9 months) between ACL reconstruction and second-look arthroscopic surgery, whereas the longterm group (25 patients) had an interval of 31 to 62 months (mean, 39.3 6 7.9 months) (Table 1). There were no statistical differences between the 2 groups with respect to their clinical results, such as the Lysholm score and Lachman test. During the second-look arthroscopic procedures, biopsies of the ACL graft were performed. Normal ACL tissues were harvested from 9 patients aged 64 to 78 years who underwent total knee replacement (normal ACL control group). In another 9 patients who also underwent ACL reconstruction with hamstring tendons (normal semitendinosus [ST] control group), biopsies of the to-be-grafted ST tissues were performed.
Surgical and Biopsy Techniques Double-Bundle, 8-Strand Hamstring ACL Reconstruction. All 52 patients underwent anatomic double-bundle ACL reconstruction through 2 tibial and 2 femoral tunnels. Four-stranded autogenous ST grafts, with a mean diameter of 7.5 mm, were used for the AM bundle, and 4stranded autogenous gracilis tendon (GT) grafts, with a mean diameter of 6 mm, were used for the posterolateral bundle. On the femoral side, suspension fixation was adopted using 12 mm–long mini-plates (Aesculap). On the tibial side, suspension fixation was also adopted using 14 mm– diameter mini-buttons (Aesculap) with 1 bioabsorbable interference screw (Smith & Nephew) as a backup.
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Full weightbearing was allowed immediately after surgery. Range of motion exercises began in the third postoperative week, and proprioception exercises began in the seventh postoperative week. Running and agility training began in the fourth postoperative month. Second-Look Arthroscopic Surgery and Biopsy. During second-look arthroscopic surgery, the volume of the AM bundle was checked. When the AM bundle was suitable for biopsy, the synovial layer over the AM bundle was separated to expose the graft fibers, which were examined for tension and continuity. On the superficial side of the AM bundle, 1 strand of graft fiber, approximately 0.5 mm wide with firm connections to both the tibia and femur, was separated from the other graft tissue. The biopsy was performed at the midportion of this graft strand to harvest a 3-mm segment. Control Biopsy. In the 9 control ACL patients who underwent total knee replacement, the ACL tissues were harvested. A 3 3 2–mm ACL fiber segment was obtained from the midportion of the AM bundle of the ACL for the histological examination. In the 9 other patients who underwent hamstring tendon ACL reconstruction (normal ST control group), the ST was first trimmed, and then a 3 3 2–mm specimen was obtained from the trimmed section.
Histological Evaluation Each biopsied tissue sample was dissected into a shorter section and a longer section; the shorter one was used for the electron microscopy evaluation, and the longer one was prepared for the light microscopy evaluation. Before light microscopy observation, the tissue was fixed in 10.0% neutral buffered formalin, alcohol dehydrated, hyalinized in chloroform, and embedded in paraffin. Sections 5 mm thick were cut longitudinally and stained with hematoxylin and eosin. Before transmission electron microscopy, the specimens were fixed with precooled (4°C) 2.5% glutaraldehyde, postfixed in 1.0% osmium tetroxide, alcohol dehydrated, acetone infiltrated, and embedded in Spurr resin. Sections 5 mm thick were cut sequentially and stained with 1.0% toluidine and then observed using light microscopy (DM4000B; Leica) to select the zone of interest. Sequential sections 70 nm thick were then cut and stained with 3.0% uranyl acetate and lead citrate and were observed using a transmission electron microscope (Tecnai G20; FEI). Graft vascularity, cellularity, metaplasia, cellular metabolic status, and collagen fibril distribution were analyzed in specimens from the 4 groups. The results from the midterm graft group were compared with those from the longterm group; the results of the normal ACL and ST groups were used as controls. Vascularity. Vascularity was evaluated using the presence of arterioles or venules in the midsubstance of a slice (light microscopy, 3100). Vascularity was graded as grade 0, vascularity similar to frank granulation tissue; grade 1, hypertrophic vascularity; grade 2, moderate arterioles or venules; grade 3, minimal vascularity; or grade 4, no evidence of vascularity.
Cellularity. Cellularity was assessed based on the composition of the differently shaped nuclei (light microscopy, 3200). The nuclear shapes were recorded as linear (L) (the length is 4 times the width), spindled (S) (the length is 2-4 times the width), oblong (O) (intermediate between spindled and round), and round (R). Progression of the nuclear shape from linear to round was considered to reflect decreasing cell maturation. The degree of cellularity was assessed based on the predominant nuclear shape in the sample, arranged from most common to least common. Five grades were recognized: grade 0, round nuclei were the most common; grade 1, spindled nuclei were the most common, followed by round nuclei; grade 2, spindled nuclei were the most common, followed by oblong nuclei; grade 3, spindled nuclei were the most common, followed by linear nuclei; and grade 4, linear nuclei were the most common, followed by spindled nuclei. Metaplasia. Metaplasia was evaluated under light microscopy, with 5 descriptive grades representing the degree of metaplasia (acellular, avascular, irregularly oriented collagen fibril bundles): grade 0, no metaplasia present; grade 1, minimal areas of metaplasia; grade 2, focal areas of metaplasia; grade 3, bubbly areas of metaplasia; and grade 4, large areas of metaplasia.12 Cellular Metabolism. Cellular metabolism was evaluated, under transmission electron microscopy, based on the cytoplasm-to-nuclear ratio and the quantity of synthesisrelated organelles, including abundant, rough endoplasmic reticula; mitochondria; Golgi bodies; and secretory vesicles. The 5 cellular metabolism grades were as follows: grade 0, minimal cytoplasm and organelles; grade 1, little cytoplasm and few organelles; grade 2, moderate cytoplasm and organelles; grade 3, major cytoplasm and organelles; and grade 4, abundant cytoplasm and organelles. Collagen Fibril Distribution. Collagen fibril distribution was evaluated under transmission electron microscopy as representing the number of large-diameter (.100 nm) collagen fibrils observed and their overall distribution. The morphometric analysis of a collagen fibril’s diameter was obtained from a transverse section at 311,000 magnification. According to the degree of similarity of the number of large-diameter fibrils and the overall distribution between the implanted graft and the normal ACL or ST tissues, the grades were set as follows: grade 0, unimodal; grade 1, bimodal, focally existing; grade 2, bimodal, locally distributed; grade 3, bimodal, maldistributed; and grade 4, bimodal, well distributed. The best possible grade for ligamentization was a 4 for vascularity, cellularity, metabolism, and collagen fibril distribution. Grafts receiving grades 3 and 4 in these areas were considered to be satisfactory. Metaplasia indicated a low degree of maturation, with grade 0 metaplasia representing a satisfactory graft. Finally, the satisfaction rates for the items in the midterm and long-term groups were compared with those of the normal ACL and ST controls. Two trained observers, blinded to the patient’s clinical presentation, independently analyzed the histological data. The grades offered by the 2 observers were averaged for each of the histological features to obtain the final outcome.
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Figure 1. Representative vascularity in longitudinal sections. Light microscopy, hematoxylin and eosin stain, 3100. Vessels are marked by arrows. ACL, anterior cruciate ligament; LTG, long-term group; MTG, midterm group; ST, semitendinosus tendon.
Statistical Analysis
TABLE 2 Vascularity Evaluationa
The histological features with satisfactory rates in the midterm and long-term groups were compared using a 2-tailed Fisher exact test. Statistical analyses were performed using SPSS software (v 20.0; SPSS Inc); P \ .05 was considered significant.
RESULTS Vascularity The satisfactory rates for vascularity (grades 3 and 4) were 51.8% in the midterm group and 72.0% in the long-term group (Table 2). Those with mature vascular composition looked similar to normal ACL or ST tissues, with minimal numbers (or none) of arterioles and venules in the midsubstance of the specimens (Figure 1). Statistical analysis did not reveal a significant difference between the midterm and long-term groups.
Biopsy Samples, n (%) Grade
Midterm Group
0 1 2 3 4
0 1 12 7 7
(0.0) (3.7) (44.4) (25.9) (25.9)
Long-term Group 0 2 5 7 11
(0.0) (8.0) (20.0) (28.0) (44.0)
ACL 0 0 0 4 5
(0.0) (0.0) (0.0) (44.4) (55.6)
ST 0 0 0 0 9
(0.0) (0.0) (0.0) (0.0) (100.0)
a
P = .163 between the midterm and long-term groups. ACL, anterior cruciate ligament; ST, semitendinosus tendon.
significant differences between the midterm and longterm groups (P = .217) (Table 3 and Figure 3).
Cellular Metabolism Cellularity and Metaplasia The cellularity satisfactory rates (grades 3 and 4 predominating) were 85.2% in the midterm group and 84.0% in the long-term group, similar to the normal ACL or normal ST tissues (Figure 2). Statistical analysis did not show a significant difference between the midterm and long-term groups (P = .370) (Table 3). Grade 0 metaplasia, representing the absence of avascular, acellular zones and the presence of regularly oriented fiber bundles, was defined as fine; the metaplasia satisfaction rate was 77.8% in the midterm group and 84.0% in the long-term group. Again, there were no
The cellular metabolism satisfactory rates were 85.2% and 88.0% in the midterm and long-term groups, respectively. Specimens with grades 3 and 4 demonstrated cells with spindled or oblong nuclei, with few wavy indentations of the nuclear surface. These activated cells had higher cytoplasm-to-nucleus ratios, more cytoplasmic processes, and higher numbers of synthesis-related organelles (such as abundant, rough endoplasmic reticula; mitochondria; and secretory vesicles) compared with the other grades. These features were similar to normal ACL tissues. In the midterm or long-term groups, dispersed, light-staining chromatin; membrane-wrapped, small collagen fibrils and large
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Figure 2. Representative cellularity in longitudinal sections. Light microscopy, hematoxylin and eosin stain, 3200. ACL, anterior cruciate ligament; LTG, long-term group; MTG, midterm group; ST, semitendinosus tendon.
Figure 3. Representative metaplasia in longitudinal sections of the midterm and long-term groups. Light microscopy, hematoxylin and eosin stain, 3100. *Metaplasia area. TABLE 3 Cellularity and Metaplasia Evaluationsa
TABLE 4 Cellular Metabolism Evaluationa
Biopsy Samples, n (%) Midterm Group Cellularity Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Metaplasia Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 a
0 0 4 2 21
(0.0) (0.0) (14.8) (7.4) (77.8)
Long-term Group
0 0 4 7 14
(0.0) (0.0) (16.0) (28.0) (56.0)
Biopsy Samples, n (%)
ACL
0 0 0 1 8
(0.0) (0.0) (0.0) (11.1) (88.9)
ST
0 0 0 0 9
(0.0) (0.0) (0.0) (0.0) (100.0)
Grade
Midterm Group
0 1 2 3 4
0 0 4 7 16
(0.0) (0.0) (14.8) (25.9) (59.3)
Long-term Group 0 0 3 6 16
(0.0) (0.0) (12.0) (24.0) (64.0)
ACL 0 0 0 2 7
(0.0) (0.0) (0.0) (22.2) (77.8)
ST 9 0 0 0 0
(100.0) (0.0) (0.0) (0.0) (0.0)
a
21 1 4 1 0
(77.8) (3.7) (14.8) (3.7) (0.0)
21 2 1 1 0
(84.0) (8.0) (4.0) (4.0) (0.0)
9 0 0 0 0
(100.0) (0.0) (0.0) (0.0) (0.0)
9 0 0 0 0
(100.0) (0.0) (0.0) (0.0) (0.0)
ACL, anterior cruciate ligament; ST, semitendinosus tendon.
P . .999 between the midterm and long-term groups. ACL, anterior cruciate ligament; ST, semitendinosus tendon.
collagen fibrils; large collagen fibrils assembled with smaller fibrils; and large, secreted collagen fibrils located in the compartment were observed (Figures 4 and 5). These
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Figure 4. Representative cellular ultrastructure in longitudinal sections. Transmission electron microscopy: (A, F, I) 35800; (B, E) 33200; (C, H) 37400; (D) 32100; and (G) 33400. ACL, anterior cruciate ligament; LTG, long-term group; MTG, midterm group; ST, semitendinosus tendon.
Figure 5. Representative active collagen synthesis in transverse sections in the midterm and long-term groups. (A, B, D) Small collagen fibrils inside the cellular process (thin arrowhead) and collagen fibril release (thick arrowheads in A). (C) Large collagen fibrils inside the cellular processes (thin arrowhead), similar to those outside the cellular process (thick arrowhead). (E) Large collagen fibrils gathered near the cellular process (arrowhead). (F) An assembly of large collagen fibrils with small collagen fibrils (arrowhead). Transmission electron microscopy: (A, F) 36400; (B, D, E) 311,000; and (C) 330,000. LTG, long-term group; MTG, midterm group. phenomena indicated a high cellular metabolic level. Statistical analysis revealed no significant differences between the midterm and long-term groups, but the midterm and
long-term groups and the normal ACL group had higher levels of cellular metabolism than did the normal ST group (Table 4).
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Figure 6. Representative collagen fibril distribution in transverse sections. Transmission electron microscopy, 311,000. ACL, anterior cruciate ligament; LTG, long-term group; MTG, midterm group; ST, semitendinosus tendon.
TABLE 5 Collagen Fibril Distribution Evaluationa Biopsy Sample, n (%) Grade
Midterm Group
0 1 2 3 4
5 3 2 12 5
(18.5) (11.1) (7.4) (44.4) (18.5)
Long-term Group 8 2 2 9 4
(32.0) (8.0) (8.0) (36.0) (16.0)
ACL 0 0 0 0 9
(0.0) (0.0) (0.0) (0.0) (100.0)
ST 0 0 0 0 9
(0.0) (0.0) (0.0) (0.0) (100.0)
a
P = .575 between the midterm and long-term groups. ACL, anterior cruciate ligament; ST, semitendinosus tendon.
Collagen Fibril Distribution The presence of large-diameter (.100 nm) collagen fibrils was detected in 81.5% of the specimens in the midterm group and in 68.0% of the specimens in the long-term group. The simultaneous presence of large- and smalldiameter collagen fibrils resulted in a bimodal collagen fibril distribution (Figure 6). The rate of satisfactory results (grades 3 and 4, which were considered the typical bimodal distribution mode and approached the normal ACL or ST appearance) was 62.9% in the midterm group and 52.0% in the long-term group. There were no
significant differences between the midterm and longterm groups (Table 5). Furthermore, in the midterm group, the time interval was 13 to 18 months in 3 specimens. One 13-month specimen showed a grade 3 bimodal collagen distribution pattern (Figure 6B), one 14-month specimen showed a unimodal collagen distribution pattern (Figure 6E), and one 18-month specimen showed a grade 3 bimodal collagen distribution pattern (Figure 5C).
DISCUSSION ST and GT grafts have become standard for ACL reconstruction because of their excellent morphological, histological, and biomechanical parameters.2,6,16,18,20,23,26 The implanted free-tendon grafts undergo a biological transformation and may eventually become similar to the normal ACL.3 However, there is no consensus regarding the timing of this process. Currently, opinions about ligamentization time are based on light microscopy examinations, with the reported time to graft maturation ranging from 9 to 18 months postoperatively.1,12,27,28 Most researchers agree, however, that while a graft’s gross or light microscopic appearance is similar to the native ACL 1 to 2 years after reconstruction, the graft never completely transforms into normal ACL tissue.1,8,37,38 In the present study, the electron microscope
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examination results were in agreement with those of previous reports. Most of the specimens, 85.2% in the midterm group and 88.0% in the long-term group, had a more active metabolic status than did the normal ST and reached a status similar to the cells in the normal ACL; both synthesis and excretion were at high levels. Thus, at the indicated time points, most of the implanted hamstring grafts in both groups had lost their ‘‘tendon-specific’’ biological features. They also exhibited ‘‘ligamentous’’ histological properties, with the higher metabolic state offering the strongest evidence. Previous studies indicated that the large-diameter collagen fibrils gradually decrease over time and eventually disappear. Zaffagnini et al37 observed that a few largediameter collagen (.90 nm) fibrils remained visible 1 year postoperatively in hamstring autografts and that the grafts were composed of only small collagen fibrils at 4 and 10 years postoperatively. In a study of patellar tendon grafts by Abe et al,1 the collagen fibrils in the grafts had a uniformly small diameter (\100 nm) 6 to 15 months postoperatively. Large-diameter collagen fibrils are normally reduced within 9 postoperative months and disappear completely approximately 1 year after surgery. Shino et al32 observed a similar phenomenon in free-tendon allografts, wherein the large-diameter (.90 nm) collagen fibrils gradually decreased over the first 12 postoperative months and completely disappeared thereafter. Additionally, the loss of the large-diameter collagen fibrils in the ACL grafts led to a change in the collagen distribution pattern from a bimodal to unimodal pattern. This phenomenon has been reported to occur even earlier in animal models. Jackson et al17 reported the depletion of large-diameter fibrils in goat ACL autografts as early as 12 weeks postoperatively and their complete absence in the superficial regions of the graft within 52 weeks. Similar results for collagen changes from bimodal to unimodal distributions were observed in other animal models, and the heterogeneous composition of varied-diameter collagen fibers present in the intact ACL was never restored.17,21,34 However, in the present study, unlike in previously published studies, a distinctly higher percentage of implanted grafts had large-diameter collagen fibrils (81.5% and 68.0% in the midterm and long-term groups, respectively). The presence of grade 4 large-diameter collagen fibrils, which imply substantial numbers of largediameter collagen fibrils distributed uniformly among the small collagen fibrils, mimicked the original ACL microstructure. Biopsy specimens with grades 3 and 4 showed typical bimodal distributions, which were also observed in normal ligament and tendon tissues. The percentage of specimens with grades 3 and 4 in the midterm and longterm groups was approximately 62.9% and 52.0%, respectively, with the mean graft ages being 23.4 months and 39.3 months, respectively. As previously mentioned, grafts of a similar age have been reported to be mostly homogeneous, composed of small-diameter collagen fibrils.1,37,38 Interestingly, there were 2 exceptions in the Shino et al32 free-tendon allograft study. One 12-month and one 59month biopsy specimen had significant numbers of largediameter fibrils (approximately 125 nm), and these largediameter collagen fibrils were attributed to a delay in the
remodeling process (and degradation of the large-diameter fibrils) in the original allograft. The reason for the common appearance of large-diameter collagen fibrils in the grafts reported in the present study is unknown. This phenomenon may be the result of the special ACL reconstruction method used, namely, anatomic, double-bundle, 8-strand hamstring tendon ACL reconstruction. To the best of our knowledge, this study is the first to investigate the ligamentization of a human autologous hamstring graft in living patients after clinically successful ACL reconstruction with this technique. Further studies are needed to explore the real cause of this type of graft maturity. The endpoint of graft maturation has never been detected, and immature features have been observed in long-term animal model studies. Rougraff et al27 reported the presence of areas of degeneration and hypercellularity 3 years after ACL reconstruction. In the present study, we found that even in the long-term group (.30 months postoperatively), 16.0% of the specimens demonstrated an immature cellular mode (grade 2), 28.0% had higher vascularity (grades 1 and 2), and 16.0% exhibited varied degrees of metaplasia (grades 1, 2, and 3), indicative of a maturation gap between the graft and the original ACL. There were no significant differences between the midterm and long-term groups, indicating that time may not be crucial for the completion of graft maturation, after completion of the initial short-term period. In our research, we observed previously assembled, largediameter, membrane-wrapped collagen fibrils in the cytoplasm of some specimens, along with small-diameter collagen fibrils. Simultaneously, there were also some large-diameter collagen fibrils being assembled in the adjacent zones, and with further magnification, large-diameter collagen fibrils were confirmed to be fused with some microfibrils that were much thinner than normal, small-diameter collagen fibrils in the same specimen. The collagen structure inside the cells was similar in size and density to that outside the cell, a biological phenomenon also observed by Birk et al.5 Thus, we hypothesize that large-diameter collagen fibrils can be directly synthesized inside the cell or assembled from small-diameter collagen fibrils outside the cell. Most scholars suggest that the distribution of collagen is directly related to the tissue’s level of stress and strain.7,13,14,24,25,35 There is also a positive correlation between the connective tissue’s ultimate tensile strength and the mean diameter of the constituent collagen fibrils. Furthermore, the distribution of the collagen fibril diameter may be directly related to the mechanical properties of the tissue. For example, larger diameter fibrils provide increased strength in human aortic valves, and fibril diameters increase with loading in mouse tendons.4,22 Hence, autografts with large-diameter collagen fibrils are reasonably deduced to be stronger than those with smalldiameter collagen fibrils, although this is difficult to prove in living patients. The main limitation of this study is that it was an examination of biopsy samples instead of the whole graft. Although functional damage to ACL grafts has not been previously observed after microbiopsies, the specimen sizes
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in the present study were deliberately smaller. At the same time, to decrease any potential deleterious effects, all biopsy specimens were collected from the superficial region of the midzone of the reconstructed AM grafts. Thus, we cannot conclusively state that the biopsy sample is representative of the entire graft. The invasive nature of specimen collection directly resulted in a small sample size, decreasing the efficiency of statistical analysis. This was an unavoidable limitation because we were studying biopsy specimens collected from living patients. The other limitation is that only patients defined as having undergone successful ACL reconstruction during second-look arthroscopic surgery were enrolled. Thus, the same results cannot be expected in patients having undergone unsuccessful ACL reconstruction. Furthermore, the normal ACL tissues used for control were obtained from elderly patients who underwent total knee replacement and may not be representative of younger patients undergoing ACL reconstruction. In addition, this was a study of midterm to long-term ligamentization of ACL grafts; we did not study short-term ligamentization. Finally, interobserver and intraobserver statistics were not obtained.
CONCLUSION The results of the present study proved that graft ultrastructural maturation, mainly characterized by largediameter collagen fibrils and a bimodal collagen fibril distribution, is a common phenomenon in the midterm and long term after ACL reconstruction, using hamstring tendons. This study also indicates that graft ligamentization is not time dependent; there were no significant changes in graft ligamentization from the midterm to long term.
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8. Cho S, Muneta T, Ito S, Yagishita K, Ichinose S. Electron microscopic evaluation of two-bundle anatomically reconstructed anterior cruciate ligament graft. J Orthop Sci. 2004;9(3):296-301. 9. Chun CH, Han HJ, Lee BC, Kim DC, Yang JH. Histological findings of anterior cruciate ligament reconstruction with Achilles allograft. Clin Orthop Relat Res. 2004;421:273-276. 10. Claes S, Verdonk P, Forsyth R, Bellemans J. The ‘‘ligamentization’’ process in anterior cruciate ligament reconstruction: what happens to the human graft? A systematic review of the literature. Am J Sports Med. 2011;39(11):2476-2483. 11. Ekdahl M, Wang JH, Ronga M, Fu FH. Graft healing in anterior cruciate ligament reconstruction. Knee Surg Sports Traumatol Arthrosc. 2008;16(10):935-947. 12. Falconiero RP, DiStefano VJ, Cook TM. Revascularization and ligamentization of autogenous anterior cruciate ligament grafts in humans. Arthroscopy. 1998;14(2):197-205. 13. Goh KL, Holmes DF, Lu Y, et al. Bimodal collagen fibril diameter distributions direct age-related variations in tendon resilience and resistance to rupture. J Appl Physiol. 2012;113(6):878-888. 14. Herchenhan A, Bayer ML, Svensson RB, Magnusson SP, Kjaer M. In vitro tendon tissue development from human fibroblasts demonstrates collagen fibril diameter growth associated with a rise in mechanical strength. Dev Dyn. 2013;242(1):2-8. 15. Hofbauer M, Muller B, Murawski CD, van Eck CF, Fu FH. The concept of individualized anatomic anterior cruciate ligament (ACL) reconstruction. Knee Surg Sports Traumatol Arthrosc. 2014;22(5):979-986. 16. Ibrahim SA, Al-Kussary IM, Al-Misfer AR, Al-Mutairi HQ, Ghafar SA, El Noor TA. Clinical evaluation of arthroscopically assisted anterior cruciate ligament reconstruction: patellar tendon versus gracilis and semitendinosus autograft. Arthroscopy. 2005;21(4):412-417. 17. Jackson DW, Grood ES, Goldstein JD, et al. A comparison of patellar tendon autograft and allograft used for anterior cruciate ligament reconstruction in the goat model. Am J Sports Med. 1993;21(2):176-185. 18. Khan RM, Prasad V, Gangone R, Kinmont JC. Anterior cruciate ligament reconstruction in patients over 40 years using hamstring autograft. Knee Surg Sports Traumatol Arthrosc. 2010;18(1):68-72. 19. Kyung BS, Kim JG, Chang M, et al. Anatomic double-bundle reconstruction techniques result in graft obliquities that closely mimic the native anterior cruciate ligament anatomy. Am J Sports Med. 2013;41(6):1302-1309. 20. Li S, Su W, Zhao J, et al. A meta-analysis of hamstring autografts versus bone–patellar tendon–bone autografts for reconstruction of the anterior cruciate ligament. Knee Surg Sports Traumatol Arthrosc. 2011;18(5):287-293. 21. Liu SH, Yang RS, al-Shaikh R, Lane JM. Collagen in tendon, ligament, and bone healing: a current review. Clin Orthop Relat Res. 1995;318:265-278. 22. Michna H. Morphometric analysis of loading-induced changes in collagen-fibril populations in young tendons. Cell Tissue Res. 1984;236(2):465-470. 23. Middleton KK, Hamilton T, Irrgang JJ, Karlsson J, Harner CD, Fu FH. Anatomic anterior cruciate ligament (ACL) reconstruction: a global perspective. Part 1. Knee Surg Sports Traumatol Arthrosc. 2014;22(7):1467-1482. 24. Parry DA, Barnes GR, Craig AS. A comparison of the size distribution of collagen fibrils in connective tissues as a function of age and a possible relation between fibril size distribution and mechanical properties. Proc R Soc Lond B Biol Sci. 1978;203(1152):305-321. 25. Patterson-Kane JC, Wilson AM, Firth EC, Parry DA, Goodship AE. Comparison of collagen fibril populations in the superficial digital flexor tendons of exercised and nonexercised thoroughbreds. Equine Vet J. 1997;29(2):121-125. 26. Pinczewski L, Roe J, Salmon L. Why autologous hamstring tendon reconstruction should now be considered the gold standard for anterior cruciate ligament reconstruction in athletes. Br J Sports Med. 2009;43(5):325-327. 27. Rougraff B, Shelbourne KD, Gerth PK, Warner J. Arthroscopic and histologic analysis of human patellar tendon autografts used for ante-
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Ligamentization of Autogenous Hamstring Grafts After Anterior Cruciate Ligament Reconstruction: Midterm Versus Long-term Results Shikui Dong, Guoming Xie, Yang Zhang, Peng Shen, Xiaoqiao Huangfu and Jinzhong Zhao Am J Sports Med published online June 1, 2015 DOI: 10.1177/0363546515584039 The online version of this article can be found at: http://ajs.sagepub.com/content/early/2015/05/30/0363546515584039
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Ligamentization of Autogenous Hamstring Grafts After Anterior Cruciate Ligament Reconstruction Midterm Versus Long-term Results Shikui Dong,* MD, Guoming Xie,* MD, Yang Zhang,* MD, Peng Shen,* MD, Xiaoqiao Huangfu,* MD, and Jinzhong Zhao,*y MD Investigation performed at the Department of Sports Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China Background: In previous studies, unimodal, small-diameter collagen fibrils have been commonly observed as the final collagen ultrastructure of the implanted grafts used in anterior cruciate ligament (ACL) reconstruction. However, the native ACL and hamstring tendon show bimodal collagen fibril distribution, consisting of both large- and small-diameter collagen fibrils. Hypothesis: Bimodal collagen fibril distribution of the graft is a common phenomenon after ACL reconstruction with hamstring tendon grafts and is time dependent. Study Design: Controlled laboratory study. Methods: A total of 52 patients who underwent double-bundle ACL reconstruction using autogenous hamstring tendons and who also underwent second-look arthroscopic surgery were enrolled. The patients were divided into 2 groups according to the time interval between the 2 operations: the midterm group (27 patients), with a 13- to 30-month time interval between operations, and the long-term group (25 patients) with a 31- to 62-month interval. During the second-look arthroscopic procedures, ACL graft biopsies were performed. Normal ACL tissues were harvested from 9 patients who underwent total knee replacement, and biopsy specimens of the to-be-grafted semitendinosus tendon tissues were also harvested from another 9 patients who underwent ACL reconstruction with hamstring tendons, which were designated as normal controls. Graft vascularity, cellularity, metaplasia, cellular metabolism, and collagen fibril distribution were analyzed. Results: Large-diameter (.100 nm) collagen fibrils were detected in 81.5% of the specimens in the midterm group and in 68.0% of the specimens in the long-term group. A typical bimodal distribution mode was observed in 62.6% of the specimens in the midterm group and in 52.0% of the specimens in the long-term group. There was no significant difference between groups with respect to the presence of large-diameter collagen fibrils, bimodal distribution, graft vascularity, cellularity, metaplasia, or cellular metabolic status. Conclusion: Graft ultrastructural maturation, characterized by large-diameter collagen fibrils and a bimodal collagen fibril distribution, is a common phenomenon and is not time dependent in the midterm to long term. Clinical Relevance: After hamstring tendon ACL reconstruction, the implanted grafts can transform into ACL-like tissue with a similar ultrastructure and metabolism, implying their usefulness as grafts. Keywords: anterior cruciate ligament; ligamentization; hamstring tendon; collagen fibril distribution
y Address correspondence to Jinzhong Zhao, MD, Department of Sports Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, China (email: scope [email protected]). *Department of Sports Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China. The authors declared that they have no conflicts of interest in the authorship and publication of this contribution.
Anterior cruciate ligament (ACL) reconstruction is a common procedure in the field of orthopaedic sports medicine.31 Full restoration of the native ACL function depends on its reconstruction with grafts that provide sufficient initial strength, ideal tendon-bone healing, and ideal ligamentization of the graft tissue.23 Numerous studies have examined the restoration of ACL anatomy and the ideal ACL reconstruction techniques.15 Numerous studies have also examined the enhancement of tendon-bone healing, to achieve a firm tendon-bone connection, during and
The American Journal of Sports Medicine, Vol. XX, No. X DOI: 10.1177/0363546515584039 Ó 2015 The Author(s)
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TABLE 1 Patient Details for the Midterm and Long-term Groups Patient Sex, n Group Midterm Long-term
Female
Male
Age, Mean 6 SD (Range), y
9 7
18 18
29.0 6 9.3 (17-49) 29.6 6 8.8 (17-48)
Limb, n Right 16 10
Left
Duration, Mean 6 SD, mo
Lysholm Score, Mean 6 SD
11 15
23.4 6 5.9 39.3 6 7.9
94.9 6 3.8 95.8 6 2.9
after ACL reconstruction.11,19,30,33,36 However, whether the graft tissues finally transform into ultrastructurally similar ACL tissue or just function as a fibril connection between the femur and tibia, without complete transformation, remains to be explored.10 ACL grafts are known to undergo a biological transformation process after being implanted into the knee joint; thereafter, the graft remodels itself into a viable ACLlike tissue, a process termed ‘‘ligamentization.’’3 Several studies have reported that the maturation period varies from 9 to 18 months postoperatively.1,9,37,38 However, even if the matured free-tendon graft looks similar, grossly and light microscopically, to an intact ACL, its ultrastructure continues to change over time. In previous studies, an ultrastructural ligamentization endpoint has not been observed.1,12,27,28,29 The final, commonly observed collagen ultrastructure of the implanted graft is a small-diameter (\100 nm), unimodal collagen fibril distribution pattern.1,8,37 On the other hand, the native ACL and hamstring tendon show bimodal collagen fibril distribution patterns and consist of large-diameter (.100 nm) collagen fibrils in addition to small-diameter collagen fibrils; the large-diameter collagen fibrils are believed to contribute to the tissue’s tensile strength.1,8,23,30,34-36 However, in our pilot study of hamstring tendon graft ligamentization, bimodal collagen fibril distribution of the implanted tissue was observed (unpublished observation). Whether this ultrastructural maturation of the graft is a common phenomenon and whether it is time dependent remain to be explored. Thus, we conducted a study to evaluate implanted graft ultrastructural maturation. Our hypothesis was that graft maturation, with bimodal collagen fibril distribution as a main characteristic, is a common phenomenon after hamstring tendon ACL reconstruction and is time dependent. This hypothesis implies that better morphological results can be observed in patients at later follow-up time points.
METHODS This was a retrospective evaluation of prospectively collected data. The study was approved by our local ethics committee, and only patients who provided informed consent were included in this study. Patients who underwent arthroscopic, double-bundle ACL reconstruction with 8 strands of autogenous hamstring tendons and who also underwent second-look arthroscopic
Lachman Test Result, n 0
11
24 21
3 4
surgery were considered for enrollment. Second-look arthroscopic surgery was indicated for patients undergoing hardware removal. Before second-look arthroscopic surgery, magnetic resonance imaging and clinical examinations were performed. The patients with imaging-indicated graft failure (graft absence, discontinuity, extreme volume loss) or clinical failure (side-to-side anterior knee laxity .5 mm or a positive pivot-shift test result) were excluded from this study. During second-look arthroscopic surgery, patients with .50% anteromedial (AM) bundle graft tissue loss were excluded. Finally, 52 patients were enrolled, between February 2013 and April 2014, after the exclusion of 2 patients because of inadequate volume of the AM bundle and 2 patients because of clinical failure with a side-toside anterior knee laxity of .5 mm and a positive pivot-shift test result. The patients were divided into 2 groups according to the time interval between ACL reconstruction and their second-look arthroscopic procedures: The midterm group (27 patients) had a 13- to 30-month time interval (mean, 23.4 6 5.9 months) between ACL reconstruction and second-look arthroscopic surgery, whereas the longterm group (25 patients) had an interval of 31 to 62 months (mean, 39.3 6 7.9 months) (Table 1). There were no statistical differences between the 2 groups with respect to their clinical results, such as the Lysholm score and Lachman test. During the second-look arthroscopic procedures, biopsies of the ACL graft were performed. Normal ACL tissues were harvested from 9 patients aged 64 to 78 years who underwent total knee replacement (normal ACL control group). In another 9 patients who also underwent ACL reconstruction with hamstring tendons (normal semitendinosus [ST] control group), biopsies of the to-be-grafted ST tissues were performed.
Surgical and Biopsy Techniques Double-Bundle, 8-Strand Hamstring ACL Reconstruction. All 52 patients underwent anatomic double-bundle ACL reconstruction through 2 tibial and 2 femoral tunnels. Four-stranded autogenous ST grafts, with a mean diameter of 7.5 mm, were used for the AM bundle, and 4stranded autogenous gracilis tendon (GT) grafts, with a mean diameter of 6 mm, were used for the posterolateral bundle. On the femoral side, suspension fixation was adopted using 12 mm–long mini-plates (Aesculap). On the tibial side, suspension fixation was also adopted using 14 mm– diameter mini-buttons (Aesculap) with 1 bioabsorbable interference screw (Smith & Nephew) as a backup.
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Full weightbearing was allowed immediately after surgery. Range of motion exercises began in the third postoperative week, and proprioception exercises began in the seventh postoperative week. Running and agility training began in the fourth postoperative month. Second-Look Arthroscopic Surgery and Biopsy. During second-look arthroscopic surgery, the volume of the AM bundle was checked. When the AM bundle was suitable for biopsy, the synovial layer over the AM bundle was separated to expose the graft fibers, which were examined for tension and continuity. On the superficial side of the AM bundle, 1 strand of graft fiber, approximately 0.5 mm wide with firm connections to both the tibia and femur, was separated from the other graft tissue. The biopsy was performed at the midportion of this graft strand to harvest a 3-mm segment. Control Biopsy. In the 9 control ACL patients who underwent total knee replacement, the ACL tissues were harvested. A 3 3 2–mm ACL fiber segment was obtained from the midportion of the AM bundle of the ACL for the histological examination. In the 9 other patients who underwent hamstring tendon ACL reconstruction (normal ST control group), the ST was first trimmed, and then a 3 3 2–mm specimen was obtained from the trimmed section.
Histological Evaluation Each biopsied tissue sample was dissected into a shorter section and a longer section; the shorter one was used for the electron microscopy evaluation, and the longer one was prepared for the light microscopy evaluation. Before light microscopy observation, the tissue was fixed in 10.0% neutral buffered formalin, alcohol dehydrated, hyalinized in chloroform, and embedded in paraffin. Sections 5 mm thick were cut longitudinally and stained with hematoxylin and eosin. Before transmission electron microscopy, the specimens were fixed with precooled (4°C) 2.5% glutaraldehyde, postfixed in 1.0% osmium tetroxide, alcohol dehydrated, acetone infiltrated, and embedded in Spurr resin. Sections 5 mm thick were cut sequentially and stained with 1.0% toluidine and then observed using light microscopy (DM4000B; Leica) to select the zone of interest. Sequential sections 70 nm thick were then cut and stained with 3.0% uranyl acetate and lead citrate and were observed using a transmission electron microscope (Tecnai G20; FEI). Graft vascularity, cellularity, metaplasia, cellular metabolic status, and collagen fibril distribution were analyzed in specimens from the 4 groups. The results from the midterm graft group were compared with those from the longterm group; the results of the normal ACL and ST groups were used as controls. Vascularity. Vascularity was evaluated using the presence of arterioles or venules in the midsubstance of a slice (light microscopy, 3100). Vascularity was graded as grade 0, vascularity similar to frank granulation tissue; grade 1, hypertrophic vascularity; grade 2, moderate arterioles or venules; grade 3, minimal vascularity; or grade 4, no evidence of vascularity.
Cellularity. Cellularity was assessed based on the composition of the differently shaped nuclei (light microscopy, 3200). The nuclear shapes were recorded as linear (L) (the length is 4 times the width), spindled (S) (the length is 2-4 times the width), oblong (O) (intermediate between spindled and round), and round (R). Progression of the nuclear shape from linear to round was considered to reflect decreasing cell maturation. The degree of cellularity was assessed based on the predominant nuclear shape in the sample, arranged from most common to least common. Five grades were recognized: grade 0, round nuclei were the most common; grade 1, spindled nuclei were the most common, followed by round nuclei; grade 2, spindled nuclei were the most common, followed by oblong nuclei; grade 3, spindled nuclei were the most common, followed by linear nuclei; and grade 4, linear nuclei were the most common, followed by spindled nuclei. Metaplasia. Metaplasia was evaluated under light microscopy, with 5 descriptive grades representing the degree of metaplasia (acellular, avascular, irregularly oriented collagen fibril bundles): grade 0, no metaplasia present; grade 1, minimal areas of metaplasia; grade 2, focal areas of metaplasia; grade 3, bubbly areas of metaplasia; and grade 4, large areas of metaplasia.12 Cellular Metabolism. Cellular metabolism was evaluated, under transmission electron microscopy, based on the cytoplasm-to-nuclear ratio and the quantity of synthesisrelated organelles, including abundant, rough endoplasmic reticula; mitochondria; Golgi bodies; and secretory vesicles. The 5 cellular metabolism grades were as follows: grade 0, minimal cytoplasm and organelles; grade 1, little cytoplasm and few organelles; grade 2, moderate cytoplasm and organelles; grade 3, major cytoplasm and organelles; and grade 4, abundant cytoplasm and organelles. Collagen Fibril Distribution. Collagen fibril distribution was evaluated under transmission electron microscopy as representing the number of large-diameter (.100 nm) collagen fibrils observed and their overall distribution. The morphometric analysis of a collagen fibril’s diameter was obtained from a transverse section at 311,000 magnification. According to the degree of similarity of the number of large-diameter fibrils and the overall distribution between the implanted graft and the normal ACL or ST tissues, the grades were set as follows: grade 0, unimodal; grade 1, bimodal, focally existing; grade 2, bimodal, locally distributed; grade 3, bimodal, maldistributed; and grade 4, bimodal, well distributed. The best possible grade for ligamentization was a 4 for vascularity, cellularity, metabolism, and collagen fibril distribution. Grafts receiving grades 3 and 4 in these areas were considered to be satisfactory. Metaplasia indicated a low degree of maturation, with grade 0 metaplasia representing a satisfactory graft. Finally, the satisfaction rates for the items in the midterm and long-term groups were compared with those of the normal ACL and ST controls. Two trained observers, blinded to the patient’s clinical presentation, independently analyzed the histological data. The grades offered by the 2 observers were averaged for each of the histological features to obtain the final outcome.
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Figure 1. Representative vascularity in longitudinal sections. Light microscopy, hematoxylin and eosin stain, 3100. Vessels are marked by arrows. ACL, anterior cruciate ligament; LTG, long-term group; MTG, midterm group; ST, semitendinosus tendon.
Statistical Analysis
TABLE 2 Vascularity Evaluationa
The histological features with satisfactory rates in the midterm and long-term groups were compared using a 2-tailed Fisher exact test. Statistical analyses were performed using SPSS software (v 20.0; SPSS Inc); P \ .05 was considered significant.
RESULTS Vascularity The satisfactory rates for vascularity (grades 3 and 4) were 51.8% in the midterm group and 72.0% in the long-term group (Table 2). Those with mature vascular composition looked similar to normal ACL or ST tissues, with minimal numbers (or none) of arterioles and venules in the midsubstance of the specimens (Figure 1). Statistical analysis did not reveal a significant difference between the midterm and long-term groups.
Biopsy Samples, n (%) Grade
Midterm Group
0 1 2 3 4
0 1 12 7 7
(0.0) (3.7) (44.4) (25.9) (25.9)
Long-term Group 0 2 5 7 11
(0.0) (8.0) (20.0) (28.0) (44.0)
ACL 0 0 0 4 5
(0.0) (0.0) (0.0) (44.4) (55.6)
ST 0 0 0 0 9
(0.0) (0.0) (0.0) (0.0) (100.0)
a
P = .163 between the midterm and long-term groups. ACL, anterior cruciate ligament; ST, semitendinosus tendon.
significant differences between the midterm and longterm groups (P = .217) (Table 3 and Figure 3).
Cellular Metabolism Cellularity and Metaplasia The cellularity satisfactory rates (grades 3 and 4 predominating) were 85.2% in the midterm group and 84.0% in the long-term group, similar to the normal ACL or normal ST tissues (Figure 2). Statistical analysis did not show a significant difference between the midterm and long-term groups (P = .370) (Table 3). Grade 0 metaplasia, representing the absence of avascular, acellular zones and the presence of regularly oriented fiber bundles, was defined as fine; the metaplasia satisfaction rate was 77.8% in the midterm group and 84.0% in the long-term group. Again, there were no
The cellular metabolism satisfactory rates were 85.2% and 88.0% in the midterm and long-term groups, respectively. Specimens with grades 3 and 4 demonstrated cells with spindled or oblong nuclei, with few wavy indentations of the nuclear surface. These activated cells had higher cytoplasm-to-nucleus ratios, more cytoplasmic processes, and higher numbers of synthesis-related organelles (such as abundant, rough endoplasmic reticula; mitochondria; and secretory vesicles) compared with the other grades. These features were similar to normal ACL tissues. In the midterm or long-term groups, dispersed, light-staining chromatin; membrane-wrapped, small collagen fibrils and large
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Figure 2. Representative cellularity in longitudinal sections. Light microscopy, hematoxylin and eosin stain, 3200. ACL, anterior cruciate ligament; LTG, long-term group; MTG, midterm group; ST, semitendinosus tendon.
Figure 3. Representative metaplasia in longitudinal sections of the midterm and long-term groups. Light microscopy, hematoxylin and eosin stain, 3100. *Metaplasia area. TABLE 3 Cellularity and Metaplasia Evaluationsa
TABLE 4 Cellular Metabolism Evaluationa
Biopsy Samples, n (%) Midterm Group Cellularity Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Metaplasia Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 a
0 0 4 2 21
(0.0) (0.0) (14.8) (7.4) (77.8)
Long-term Group
0 0 4 7 14
(0.0) (0.0) (16.0) (28.0) (56.0)
Biopsy Samples, n (%)
ACL
0 0 0 1 8
(0.0) (0.0) (0.0) (11.1) (88.9)
ST
0 0 0 0 9
(0.0) (0.0) (0.0) (0.0) (100.0)
Grade
Midterm Group
0 1 2 3 4
0 0 4 7 16
(0.0) (0.0) (14.8) (25.9) (59.3)
Long-term Group 0 0 3 6 16
(0.0) (0.0) (12.0) (24.0) (64.0)
ACL 0 0 0 2 7
(0.0) (0.0) (0.0) (22.2) (77.8)
ST 9 0 0 0 0
(100.0) (0.0) (0.0) (0.0) (0.0)
a
21 1 4 1 0
(77.8) (3.7) (14.8) (3.7) (0.0)
21 2 1 1 0
(84.0) (8.0) (4.0) (4.0) (0.0)
9 0 0 0 0
(100.0) (0.0) (0.0) (0.0) (0.0)
9 0 0 0 0
(100.0) (0.0) (0.0) (0.0) (0.0)
ACL, anterior cruciate ligament; ST, semitendinosus tendon.
P . .999 between the midterm and long-term groups. ACL, anterior cruciate ligament; ST, semitendinosus tendon.
collagen fibrils; large collagen fibrils assembled with smaller fibrils; and large, secreted collagen fibrils located in the compartment were observed (Figures 4 and 5). These
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Figure 4. Representative cellular ultrastructure in longitudinal sections. Transmission electron microscopy: (A, F, I) 35800; (B, E) 33200; (C, H) 37400; (D) 32100; and (G) 33400. ACL, anterior cruciate ligament; LTG, long-term group; MTG, midterm group; ST, semitendinosus tendon.
Figure 5. Representative active collagen synthesis in transverse sections in the midterm and long-term groups. (A, B, D) Small collagen fibrils inside the cellular process (thin arrowhead) and collagen fibril release (thick arrowheads in A). (C) Large collagen fibrils inside the cellular processes (thin arrowhead), similar to those outside the cellular process (thick arrowhead). (E) Large collagen fibrils gathered near the cellular process (arrowhead). (F) An assembly of large collagen fibrils with small collagen fibrils (arrowhead). Transmission electron microscopy: (A, F) 36400; (B, D, E) 311,000; and (C) 330,000. LTG, long-term group; MTG, midterm group. phenomena indicated a high cellular metabolic level. Statistical analysis revealed no significant differences between the midterm and long-term groups, but the midterm and
long-term groups and the normal ACL group had higher levels of cellular metabolism than did the normal ST group (Table 4).
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Figure 6. Representative collagen fibril distribution in transverse sections. Transmission electron microscopy, 311,000. ACL, anterior cruciate ligament; LTG, long-term group; MTG, midterm group; ST, semitendinosus tendon.
TABLE 5 Collagen Fibril Distribution Evaluationa Biopsy Sample, n (%) Grade
Midterm Group
0 1 2 3 4
5 3 2 12 5
(18.5) (11.1) (7.4) (44.4) (18.5)
Long-term Group 8 2 2 9 4
(32.0) (8.0) (8.0) (36.0) (16.0)
ACL 0 0 0 0 9
(0.0) (0.0) (0.0) (0.0) (100.0)
ST 0 0 0 0 9
(0.0) (0.0) (0.0) (0.0) (100.0)
a
P = .575 between the midterm and long-term groups. ACL, anterior cruciate ligament; ST, semitendinosus tendon.
Collagen Fibril Distribution The presence of large-diameter (.100 nm) collagen fibrils was detected in 81.5% of the specimens in the midterm group and in 68.0% of the specimens in the long-term group. The simultaneous presence of large- and smalldiameter collagen fibrils resulted in a bimodal collagen fibril distribution (Figure 6). The rate of satisfactory results (grades 3 and 4, which were considered the typical bimodal distribution mode and approached the normal ACL or ST appearance) was 62.9% in the midterm group and 52.0% in the long-term group. There were no
significant differences between the midterm and longterm groups (Table 5). Furthermore, in the midterm group, the time interval was 13 to 18 months in 3 specimens. One 13-month specimen showed a grade 3 bimodal collagen distribution pattern (Figure 6B), one 14-month specimen showed a unimodal collagen distribution pattern (Figure 6E), and one 18-month specimen showed a grade 3 bimodal collagen distribution pattern (Figure 5C).
DISCUSSION ST and GT grafts have become standard for ACL reconstruction because of their excellent morphological, histological, and biomechanical parameters.2,6,16,18,20,23,26 The implanted free-tendon grafts undergo a biological transformation and may eventually become similar to the normal ACL.3 However, there is no consensus regarding the timing of this process. Currently, opinions about ligamentization time are based on light microscopy examinations, with the reported time to graft maturation ranging from 9 to 18 months postoperatively.1,12,27,28 Most researchers agree, however, that while a graft’s gross or light microscopic appearance is similar to the native ACL 1 to 2 years after reconstruction, the graft never completely transforms into normal ACL tissue.1,8,37,38 In the present study, the electron microscope
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examination results were in agreement with those of previous reports. Most of the specimens, 85.2% in the midterm group and 88.0% in the long-term group, had a more active metabolic status than did the normal ST and reached a status similar to the cells in the normal ACL; both synthesis and excretion were at high levels. Thus, at the indicated time points, most of the implanted hamstring grafts in both groups had lost their ‘‘tendon-specific’’ biological features. They also exhibited ‘‘ligamentous’’ histological properties, with the higher metabolic state offering the strongest evidence. Previous studies indicated that the large-diameter collagen fibrils gradually decrease over time and eventually disappear. Zaffagnini et al37 observed that a few largediameter collagen (.90 nm) fibrils remained visible 1 year postoperatively in hamstring autografts and that the grafts were composed of only small collagen fibrils at 4 and 10 years postoperatively. In a study of patellar tendon grafts by Abe et al,1 the collagen fibrils in the grafts had a uniformly small diameter (\100 nm) 6 to 15 months postoperatively. Large-diameter collagen fibrils are normally reduced within 9 postoperative months and disappear completely approximately 1 year after surgery. Shino et al32 observed a similar phenomenon in free-tendon allografts, wherein the large-diameter (.90 nm) collagen fibrils gradually decreased over the first 12 postoperative months and completely disappeared thereafter. Additionally, the loss of the large-diameter collagen fibrils in the ACL grafts led to a change in the collagen distribution pattern from a bimodal to unimodal pattern. This phenomenon has been reported to occur even earlier in animal models. Jackson et al17 reported the depletion of large-diameter fibrils in goat ACL autografts as early as 12 weeks postoperatively and their complete absence in the superficial regions of the graft within 52 weeks. Similar results for collagen changes from bimodal to unimodal distributions were observed in other animal models, and the heterogeneous composition of varied-diameter collagen fibers present in the intact ACL was never restored.17,21,34 However, in the present study, unlike in previously published studies, a distinctly higher percentage of implanted grafts had large-diameter collagen fibrils (81.5% and 68.0% in the midterm and long-term groups, respectively). The presence of grade 4 large-diameter collagen fibrils, which imply substantial numbers of largediameter collagen fibrils distributed uniformly among the small collagen fibrils, mimicked the original ACL microstructure. Biopsy specimens with grades 3 and 4 showed typical bimodal distributions, which were also observed in normal ligament and tendon tissues. The percentage of specimens with grades 3 and 4 in the midterm and longterm groups was approximately 62.9% and 52.0%, respectively, with the mean graft ages being 23.4 months and 39.3 months, respectively. As previously mentioned, grafts of a similar age have been reported to be mostly homogeneous, composed of small-diameter collagen fibrils.1,37,38 Interestingly, there were 2 exceptions in the Shino et al32 free-tendon allograft study. One 12-month and one 59month biopsy specimen had significant numbers of largediameter fibrils (approximately 125 nm), and these largediameter collagen fibrils were attributed to a delay in the
remodeling process (and degradation of the large-diameter fibrils) in the original allograft. The reason for the common appearance of large-diameter collagen fibrils in the grafts reported in the present study is unknown. This phenomenon may be the result of the special ACL reconstruction method used, namely, anatomic, double-bundle, 8-strand hamstring tendon ACL reconstruction. To the best of our knowledge, this study is the first to investigate the ligamentization of a human autologous hamstring graft in living patients after clinically successful ACL reconstruction with this technique. Further studies are needed to explore the real cause of this type of graft maturity. The endpoint of graft maturation has never been detected, and immature features have been observed in long-term animal model studies. Rougraff et al27 reported the presence of areas of degeneration and hypercellularity 3 years after ACL reconstruction. In the present study, we found that even in the long-term group (.30 months postoperatively), 16.0% of the specimens demonstrated an immature cellular mode (grade 2), 28.0% had higher vascularity (grades 1 and 2), and 16.0% exhibited varied degrees of metaplasia (grades 1, 2, and 3), indicative of a maturation gap between the graft and the original ACL. There were no significant differences between the midterm and long-term groups, indicating that time may not be crucial for the completion of graft maturation, after completion of the initial short-term period. In our research, we observed previously assembled, largediameter, membrane-wrapped collagen fibrils in the cytoplasm of some specimens, along with small-diameter collagen fibrils. Simultaneously, there were also some large-diameter collagen fibrils being assembled in the adjacent zones, and with further magnification, large-diameter collagen fibrils were confirmed to be fused with some microfibrils that were much thinner than normal, small-diameter collagen fibrils in the same specimen. The collagen structure inside the cells was similar in size and density to that outside the cell, a biological phenomenon also observed by Birk et al.5 Thus, we hypothesize that large-diameter collagen fibrils can be directly synthesized inside the cell or assembled from small-diameter collagen fibrils outside the cell. Most scholars suggest that the distribution of collagen is directly related to the tissue’s level of stress and strain.7,13,14,24,25,35 There is also a positive correlation between the connective tissue’s ultimate tensile strength and the mean diameter of the constituent collagen fibrils. Furthermore, the distribution of the collagen fibril diameter may be directly related to the mechanical properties of the tissue. For example, larger diameter fibrils provide increased strength in human aortic valves, and fibril diameters increase with loading in mouse tendons.4,22 Hence, autografts with large-diameter collagen fibrils are reasonably deduced to be stronger than those with smalldiameter collagen fibrils, although this is difficult to prove in living patients. The main limitation of this study is that it was an examination of biopsy samples instead of the whole graft. Although functional damage to ACL grafts has not been previously observed after microbiopsies, the specimen sizes
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in the present study were deliberately smaller. At the same time, to decrease any potential deleterious effects, all biopsy specimens were collected from the superficial region of the midzone of the reconstructed AM grafts. Thus, we cannot conclusively state that the biopsy sample is representative of the entire graft. The invasive nature of specimen collection directly resulted in a small sample size, decreasing the efficiency of statistical analysis. This was an unavoidable limitation because we were studying biopsy specimens collected from living patients. The other limitation is that only patients defined as having undergone successful ACL reconstruction during second-look arthroscopic surgery were enrolled. Thus, the same results cannot be expected in patients having undergone unsuccessful ACL reconstruction. Furthermore, the normal ACL tissues used for control were obtained from elderly patients who underwent total knee replacement and may not be representative of younger patients undergoing ACL reconstruction. In addition, this was a study of midterm to long-term ligamentization of ACL grafts; we did not study short-term ligamentization. Finally, interobserver and intraobserver statistics were not obtained.
CONCLUSION The results of the present study proved that graft ultrastructural maturation, mainly characterized by largediameter collagen fibrils and a bimodal collagen fibril distribution, is a common phenomenon in the midterm and long term after ACL reconstruction, using hamstring tendons. This study also indicates that graft ligamentization is not time dependent; there were no significant changes in graft ligamentization from the midterm to long term.
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