Anterior cruciate ligament fascia lata allograft reconstruction: Progressive histologic changes toward maturity

Arthroscopy: The Journal of Arthroscopic and Related Surgery 9(5):509-518

Published by Raven Press, Ltd. © 1993 Arthroscopy Association of North America

Anterior Cruciate Ligament Fascia Lata Allograft Reconstruction: Progressive Histologic Changes Toward Maturity James K. Horstman, M.D., Faisal Ahmadu-Suka, D.V.M, Ph.D., and R. W. Norrdin, D.V.M, Ph.D.

Summary: Biopsy samples were obtained arthroscopically from 21 patients who had undergone anterior cruciate ligament (ACL) reconstruction using roiled, freeze-dried fascia lata allograft in order to evaluate progressive histologic changes toward maturation. The~study period was 3-20 months postoperation. The mean age (+SEM) was 31.9 - 10.3. Histomorphometry was used for quantitative evaluation. Arthroscopic examination showed fully synovialized allografts in all patients. Varying degrees of degenerative tissues were observed histologically. There was a significant, direct correlation between the percentage of polarized tissue and the maturity of the biopsy specimen (r = 0.9; p < 0.04). The mean area of polarization in the postrehabilitation period (10-20 months) was significantly higher (p < 0.01) than in the rehabilitation period (3-20 months). Overall, there was a progressive decrease in cellularity and vascularity as the allograft matured. Compared with the biopsy samples of normal ACLs, the allograft was still undergoing maturation 20 months postoperatively. Key Words: Anterior cruciate ligament--Reconstruction--Fascia lata Histology.

allografts has been inconsistently described in the literature (15-20,22). Histologic conclusions from these reports were based on subjective evaluations. The objectives of this study are to identify the histologic changes that occur over time in a uniform group of allograft recipients who are clinically asymptomatic and to attempt to define when the allograft has reached maturity. Histomorphometric (objective) analysis of the histologic changes was used in order to minimize the use of subjective analysis.

The long-term consequences of chronic anterior cruciate ligament" (ACL) deficiency have been well documented (1-4). To minimize the problems associated with chronic instability, various reconstruction techniques with autogenous tissue grafts have been developed (5-13). Several of these techniques have demonstrated excellent long-term results. However, a number of adverse complications associated with autocraft harvest have been identified. With the advent of allografts (14-21) used for ACL reconstruction (ACLR), it was hoped that many of these complications could be prevented. Long-term results of allograft ACLR have been favorable, but the histologic process of "ligamentization" in these

MATERIALS AND METHODS Twenty-one patients who had undergone ACLR by the senior author (J.K.H.) gave their consent to participate in this study. These patients had received nonirradiated rolled freeze dried fascia lata (FDFL) allografts. There were 12 men and nine

From the Orthopaedic Research Center of the Rockies, Fort Collins, Colorado, U.S.A. Address correspondence and reprint requests to Dr. James K. Horstman, Orthopaedic Research Center of the Rockies, 2500 E. Prospect Road, Fort Collins, CO 80525, U.S.A.

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women with a mean age (_+SEM) of 31.9 --- 10.3 years (range 15-49). Core biopsy samples were obtained from each patient. Two sets of controls were used. Three human cadaveric ACLs (Mile High Tissue Bank, Denver, CO) comprised the first control group. The second control group included five samples of reconstituted rolled freeze dried fascia lata (FDFL) allografts fixed in 10% buffered neutral formalin. Patient charts were reviewed for any postoperative complications or diagnoses (before biopsy) that may affect the histologic appearance of the graft. Lachman's test was performed on each patient. Three patients were diagnosed as having chondromalacia patella (24, 29, and 43 days before biopsy). All three patients and one other were excluded from the study due to extensive degeneration of the biopsy sample and/or small sample size. One patient had a I + Lachman's test on physical examination and was excluded for the above reasons. All remaining patients in the study were clinically asymptomatic and all had clinically stable knees (negative Lachman's tests). With the exclusion of the above five patients, a total of 16 patients were thus used for statistical analysis. ACL Reconstruction Fluoroarthroscopic ACLRs were performed using a rolled F D F L allograft (23). Graft widths varied from 10 to 13 cm. The grafts were placed isometrically and tensioned with a 5-1b weight. They were fixed proximally with a threaded Delryn anchor, which was screwed into a tapped femoral hole. Distal fixation was accomplished with a screw and spiked ligament washer or with a staple (Richards). All patients were braced in full extension on the operating table. Range of motion was begun 3-5 days postoperatively. Full weight bearing without crutches was begun 7-10 days postoperatively. All patients completed the same aggressive physical therapy protocol, stressing early weight bearing and a near full range of motion by week 4. There were no postoperative complications, and no episodes of instability, swelling, catching, etc. that would have necessitated a "second look." All patients returned to cutting sports 10-12 months postoperatively.

were infiltrated with 2% xylocaine with epinephrine. A 1.8-mm needle arthroscope (Medical Dynamics) was introduced through the anterolateral portal. Observations were made regarding the integrity and synovialization of the ACL. Through the anteromedial portal, a 14-gauge Tru-cut biopsy needle (Baxter, Valantia, CA) was introduced and two core biopsy samples were obtained from the midportion of the graft. After drainage of the knee, Steri Strips (3M) were applied to the incisions. A light compression dressing and a Cryo Cuff (Aircast, Summit, NJ) were applied. There were no postoperative infections. The excision of biopsy samples did not compromise the integrity of the graft as could be determined by clinical follow-up.

Histopathology and Histomorphometry Paraffin sections were obtained and stained with hematoxylin-eosin (HE) and Masson trichrome. Glass slides were randomly coded and covered to facilitate blind examination. The slides were examined by the authors (F.A.S. and R.W.N.) and histologic findings were recorded. A 25-point ocular integration grid was calibrated with an ocular micrometer, and the surface areas of the section were estimated by the total point count with each hit equivalent to 0.0231 mm (2). Histomorphometry (24,25) was performed under a 10× objective lens. The histomorphometric variables quantitated included areas of degeneration, mature connective tissue (CT), active fibroplasia, intermediate fibroplasia, blood vessels, areolar CT, and synovium. Each field was also examined under polarized light, and the total area of polarization was calculated. The percent area of polarization was calculated by dividing the total area of polarization by the total surface area (from total point count above). The slides were decoded after completion of the histologic and morphometric evaluations.

Statistical Analysis Regression and correlation analysis, the unpaired Student's t test, and descriptive statistics were used. The significance level was set at p < 0.05. RESULTS

Biopsy Procedure The biopsy samples were obtained arthroscopically, under local anesthetic, in an outpatient setting. Sixty ml of saline was injected into the joint after being prepared with Betadine and sterile draping. The anteromedial and anterolateral scope portals Arthroscopy, Vol. 9, No. 5, 1993

Arthroscopic examination performed at the time of biopsy showed taut, fully synovialized ACLRs in all patients (26). There was no clinical or histological evidence of graft rejection in any of the patients examined via biopsy. The mean area of the biopsy

ACL FASCIA LATA ALLLOGRAFT RECONSTRUCTION

sample ranged from 4.8 to 8.7 mm (2). Histopathologic examination of the biopsy sections showed the presence of varying degrees of degenerative tissue. This tissue was thought to represent remnant fascia lata allograft (27). Because the freeze-dried fascia lata allograft is regarded as nonviable at the time of implantation and mainly serves as a scaffold for ingrowth of new tissue, the areas of degeneration (fascia lata remnant) in the 16 patients were deducted from the total area. This allowed for a more appropriate evaluation of progressive changes within viable areas of newly formed tissue. Subsequent analysis thus refers to viable tissue areas.

Subjective Histologic Evaluation Biopsy samples in the 3- to 4-month group showed single or multiple layers of synovial cells. Beneath the synovium were areas of extensive cellular proliferation, generally around vascular bundles. Because of the relationship of these cellular proliferations to the vascular bundles, these cells were assumed to be pericytes, some of which had differentiated into fibroblasts. The fibroblast in this region had started to produce collagen that was granular in appearance. The nuclei in this region were plump and irregularly arranged. Such areas represent active fibroplasia (Fig. 1). Deep to the area of active fibroplasia (progressing toward the center of the section) were areas described as intermediate fibroplasia. These areas were characterized by decreased cellularity and longitudinally oriented collagen fibers. The nuclei in this region were more

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regularly arranged and some had started to elongate. These areas were less vascular and represented intermediate fibroplasia (Fig. 2). The innermost layers of the viable tissue were generally mature CT. These layers were least cellular with welloriented longitudinal collagen fibers that were either wavy (crimp) or straight (Figs. 3-5). The nuclei were elongated or spindle shaped. These areas resembled fascia lata or human ACL, but were slightly more cellular. In general, degenerative tissue, if present, occupied the innermost layer, bordering areas of mature CT. From 6 months onward there was a progressive maturation of the CT toward normal ACL. Gradual decreases in cellularity, vascularity, and fibroplasia were all noted. The collagen fibers were oriented in different directions and the degree of directional disparity decreased over time. Areas of mature CT had both wavy and straight collagen patterns in all sections. This mixed appearance did not change with time. In general, the percentage of mature CT progressively increased with time. Histomorphometry (Objective Evaluation) Table 1 shows the area of polarization and the same area expressed as a percentage of the mean area of polarization of normal fascia lata allograft (75.8%). Polarization (Fig. 4) was used as an index of collagen maturity. There was a significant direct correlation between the mean area of polarization and the number of postoperative months (r = 0.9; p < 0.04) (Fig. 6). The slope of the regression line

FIG. 1. Three months postoperatively. Proliferation o f fibroblasts around a blood vessel (pericyte origin). Such an area represents active fibroplasia. On the opposite side of the blood vessel is an area of mature CT (original magnification x40).

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FIG. 2. Four months postoperatively. Area of intermediate fibroplasia, characterized by distinct collagen fibers, oriented in different directions and with nuclei of fibroblasts/fibrocytes randomly arranged (original magnification x40).

(rate o f change o f polarization per month) was 2.6 -+ 0.08 with a 95% confidence interval of 0.1-5.1. Using polarization as an index of collagen maturity, a crude estimate of time for the transplanted allograft to reach maturity (in relation to fascia lata) was 29.1 months. Because there was no significant difference between the mean areas of polarization of fascia lata allograft (control 2) and human A C L (Table 1), it can be assumed that 29.1 months is also a crude estimate of the time required for the fascia lata allograft to mature to the level of normal human

ACL. Similar correlation was also found between polarization expressed as a percentage of the mean area of polarization of fascia lata allograft and months postoperative (r = 0.9; p < 0.04). F r o m Table 2 it can be seen that there was no significant correlation between postoperative months and mature CT (under ordinary light). Similarly, the area of active fibroplasia showed no significant correlation (r = 0.8; p < 0.1). H o w e v e r , a negative slope of - 1.03 +-- 0.5 in the area o f active fibroplasia was one of the best estimates o f an ex-

FIG. 3. Four months postoperatively. Mature CT with crimp (wavy) collagen configuration (original magnification x40).

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FIG. 4. Nine months postoperatively. Mature CT with wavy collagen under polarized light (original magification x40).

pected trend in this study. The areas of intermediate fibroplasia, blood vessels, areolar CT, and synovium showed poor correlation with postoperative months. Tables 3 and 4 show the changes in the variables with increasing time. The biopsy samples of 10 patients were classified in the rehabilitation period (3I0 months). The biopsy samples of six patients were classified in the postrehabilitation period (13-18 months). Such grouping was of clinical significance and also allowed for an adequate number of observations within each category in order to facilitate statistical analysis. The Student's t test was used to compare the two'postoperative periods. From Table 3 it can be seen that the mean areas of degeneration, mature CT, active and intermediate fibroplasia, blood vessels, and s y n o v i u m showed no significant difference between the rehabilitation and postrehabilitation periods. The mean area of areolar CT in the rehabilitation period was 0.04 ± 0.04 versus 2.0 +-- 0.7 in the postrehabilitation period, and the difference is significant (p < 0.005). From Table 4, mean areas of polarization in the rehabilitation and postrehabilitation periods were 26.6 +- 4.5 and 51.3 ± 6.6, respectively, and the difference was significant (p < 0.01). The mean areas of polarization expressed as a percentage of the mean area of polarization of control fascia lata allograft were 47.8 ---6.5 for the rehabilitation period versus 82.7 ± 8.3 for the postrehabilitation period, and the difference was significant (p < 0.005). The

FIG. 5. Nine months postoperatively. Mature CT with straight collagen configuration and well-oriented fibroblast/fibrocyte nuclei (original magnification x40). Arthroscopy, Vol. 9, No. 5, 1993

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T A B L E 1. Histomorphometric quantitation of variables: percent area of polarization (mean +- SEM)

Mean months 3.5 (n = 4) 6.0 (n = 3) 9.3 (n = 3) 13.3 (n = 3) 17.0 (n = 3) Total = 16

Polarization 37.9 26.1 44.2 61.8 63.6

-+ 7.8 -+ 3.7 +- 11.9 + 10.5 -+ 9.2

Constant ( m e a n polarization of fascia lata, control 1)

Biopsy polarization x 100 fascia lata polarization

75.8 75.8 75.8 75.8 75.8

50.0 34.5 58.3 81.6 83.9

-+ 10.0 - 4.8 --- 15.7 -+ 13.8 +- 12.2

Control 1 (fascia lata), n = 5, 75.8 - 6.0. Control 2 ( h u m a n A C L ) , n = 5, 55.8 -+ 22,4.

mean area between active and intermediate areas of fibroplasia were 37.4 +-- 2.3 and 25.9 --- 5.6 for the rehabilitation and postrehabilitation periods, respectively, and the difference was significant (p < 0.025). Similarly, the mean of the sum of the areas of the latter variables were 72.2 +- 6.5 and 40.5 --8.6 for the rehabilitation and postrehabilitation periods, respectively, and the difference was significant (p < 0.005). DISCUSSION The use of allografts for reconstruction of the torn ACL is appealing to orthopedic surgeons (15, 17,18). Although fresh autogenous grafts are most frequently used for ACL reconstructions, several studies have shown no clinically significant differences between autografts and allografts (18,20,21). Donor site morbidity and many associated postoperative problems can be eliminated or minimized through the use of allografts. The potential for disease transmission, with special reference to human immunodeficiency virus (HIV) infection, is the main concern of surgeons and allograft recipients. With adequate screening of donors and more reliable laboratory tests for the detection of HIV infection, as well as improved processing techniques, such concern has considerably diminished (28,29). Several factors must be considered in using allografts or autogenous grafts as substitutes for the ACL. Biomechanical properties of the grafts, before transplantation, must ideally be as close to normal human ACL as possible. Noyes et al. reported biomechanical properties of ACL grafts commonly used in reconstructions (30). In this report, the maximum load to failure of a 16-mm wide fascia lata graft (fresh cadaveric) was 36% of normal ACL. An Arthroscopy, Vol. 9, No. 5, 1993

1 l-cm wide rectangular fascia lata allograft that is rolled into a tube will have a calculated strength to failure of 247% of a normal ACL (using Noyes' data) (30). The biomechanical properties of the transplanted graft deteriorate in the immediate postoperative period (16). The graft must therefore have adequate tensile strength in the immediate postoperative period to allow for some degree of function, including physical therapy, before the onset of revascularization and remodeling. Isometric placement of the graft, tensioning, and fixation are also vital to long-term functional stability of the reconstructed knee (31). The histological appearance of the ACL allograft should resemble normal ACL as it progresses toward maturity. The normal ACL is histologically similar in appearance to fascia lata, tendons, and other ligaments. In the normal ACL, the synovium consists of a single layer of synovial cells; bundles of collagen.fibers run in the same direction; and the nuclei of fibrocytes that produce the collagen are elongated and regularly arranged in parallel rows. The general appearance is of a low cell-to-collagen matrix ratio. Blood vessels are scarce or inapparent. From this histologic description of normal ACL, our study shows that with both histomorphometric (objective) and subjective evaluations there was a progressive metamorphosis of the rolled F D F L aUograft ACLR toward normal ACL. The cellularity of the ACL allograft decreased with time. The areas of active fibroplasia plotted against postoperative months showed an expected negative slope of -1.03 --- 0.5 (r = 0.8; p < 0.1). Furthermore, the mean of areas of active (Fig. 1) and intermediate (Fig. 2) fibroplasia (index of cellularity) diminished with time (p < 0.025). Even 3 months postoperatively there were areas of mature CT, characterized by crimp (wavy) (Figs. 3 and 4) as well as straight (Fig. 5) and dense collagen bundles with well-organized parallel rows of fibrocytes/ fibroblasts. The relative proportion of dense mature collagen bundles, irrespective of whether they exhibited straight or crimp configuration, progressively increased with time. Although histologic index of maturity of the ACL substitute may or may not correlate with functional knee stability (32,33), all of the patients in this biopsy study had stable knees, as determined by the Lachman's test. Polarization (Fig. 4) used as an index of collagen maturity was one of the most reliable modes of estimating the progressive changes in the fascia lata ACLR in this investigation (Fig. 6). Although the

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6O

] FIG. 6. A plot of the mean percent area of polarization (index of collagen maturity) versus months postoperative showing progressive maturity of newly formed collagen.

I i

i

6

8

l'0

1'2

!'6

1'8

MEAN NUMBER OF MONTH8 POSTOPI~RATIVE

mean area of polarization of fascia lata allograft (control 1) was higher than the mean of human ACL (control 2), the difference was riot significant (Table 1). This showed that the mature CT content of fascia lata and the human ACL are similar. This was further supported in the finding of no significant difference in mature CT (under ordinary light) between fascia lata and human ACL (Table 1). The apparent disparity under polarized light may be associated with advanced age of the patients in human ACL (control 2) group. Estimated parameters using fascia lata will thus be histologically applicable to human ACL. The significantly higher mean areas of polarization in the postrehabilitation period compared with the rehabilitation period (p < 0.01) and the significant correlation between months postoperative and mean areas ofp01arization (r = 0.9; p < 0.04) (Fig. 1) indicated progressive maturity of the

fascia lata ACL substitute. Similar observation was also made using polarization of the biopsy expressed as a percentage of the mean area of polarization of fascia lata (r = 0.9; p < 0.04). The estimate of the time needed for the substituted ACL allograft to reach histologic maturity in this study was -29.1 months. In histologic evaluation of changes in human allograft ACL substitute, Shino et al. estimated that the allograft reached maximum histologic maturity in - 18 months (19). There were areas of degeneration in biopsy samples from 3 to 20 months postoperatively. These areas were acellular and in most instances the original collagen framework was discernable. In an experimental animal study, remnants of degenerated bovine xenographs were detected in the ACLreconstructed knees of dogs up to 1 year postreconstruction (27). Shino et al. arthroscopically docuArthroscopy, Vol. 9, No. 5, 1993

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T A B L E 2.

Histomorphometric quantitation of variables: percent area of viable tissue (mean +- SEM)

Mean

F i bropl a s i a

months

Mature CT

3.5 (n = 4) 6.0 (n = 3) 9.3 (n = 3) 13,3~(n = 3) 17.0 (n = 3) Total = 16 Control 1 (fascia lata) (n = 5) Control 2 (human A C L ) (n = 3)

15.8 32.5 23.0 59.7 20.8

-+ ± ± ± -+

8.3 19.8 5.5 17.4 8.4

Active 35.2 27.6 20.9 16.6 22.1

Blood

Intermediate

_+ 8.2 ± 13.8 ± 6.7 ± 6.1 ± 4.8

44.2 36.2 50.1 19.2 45.7

± ---+ ± ±

8.5 5.3 15.0 11.1 5.5

vessels 2.5 3.3 5.8 2.9 6.2

-+ ± ± ± ±

0.9 1.8 4.7 1.3 1.9

Areolar CT 0.1 ± 0.1 0.0 0.0 1.0 + 0.5 3.0 --- 0.9

Synovium 2.1 0.5 0,1 0.7 2.1

-+0.9 - 0.2 --- 0.1 - 0.4 -+ 1.3

97.1 - 2.1

0.3 -+ 0,7

0.0

0.06 ± 0.04

2.5 --- 1.7

0.0

96.7 ± 2.2

0.8 + 0.7

0.6 -+ 0.4

0.5 --- 0.5

1.3 ± 0.7

0.1 -+ 0.06

(NS) NS, not significant; CT, c o n n e c t i v e tissue.

mented the presence of a small area of necrosis on the anterolateral aspect of ACL-substituted allograft (17). These areas of degeneration appeared to be confined to the innermost (central) aspect of the graft. Such observations of centrally located degeneration of the substituted ACLs have been reported in animal experiments by several investigators (14,20,27,34,35). These areas of degeneration apparently had no functional significance in the stability of the reconstructed knees in this study. This remnant acellular, avascular tissue would be expected to proceed to normal cellularity and vascularity if the observation period were extended. Areas of acellularity may persist, however, and might possibly represent unstressed areas between developing anteromedial and anterolateral bundles. Cross-sectional analysis of the whole ACL might confirm this hypothesis. Due to the limitations of soft tissue histomorphometry (25), subjective histologic evaluation is appropriate as a complement to the objective method (histomorphometry). Synovial presence that may be missed with histomorphometry may be evaluated by careful examination of the entire tissue section. Multiple layers of the synovium in most T A B L E 3.

of the sections may be indicative of ongoing maturation and remodeling. On the other hand, injury of any degree to the substituted fascia lata ACL may elicit proliferative cellular and vascular reactions that may affect the evaluation of the stage maturation. Such an injury may have significant histologic manifestations and yet be of little or no concern to the patient. The variability of the histomorphometric quantitation may in part be explained by this factor. Shino et al. followed patients that have had allograft reconstructions of the ACL using various tendons. Biopsy samples obtained arthroscopically between 6 and 90 months postoperatively showed that by 18 months maximum histologic maturity of the ACL substitute was attained (19). After 18 months, the substituted ACL maintained the same histologic characteristics for periods up to 90 months postoperatively. Our study showed progressive histologic maturity of the fascia lata ACL substitute from 3 to 20 months postoperatively. Even at 20 months, ACLRs were relatively hypercellular and hypervascular. This suggests that the graft is still remodeling and that histologic maturity has not been attained. Using polarization as an in-

Histomorphometric comparison o f variables: percent area over rehabilitation and postrehabilitation periods (mean +- SEM)

Months postoperative

Degeneration

Mature CT

Active fibroplasia

Intermediate fibroplasia

Blood vessels

Areolar CT

Synovium

3-10 (n = 10) a REH 13-18 (n = 6) b

29.6 -+ 5.7 39.6 -+ 7.1 c

23.0 -+ 6.5 40.2 -+ 12.2 c

28.6 -+ 5.4 19.3 -+ 3.7 c

43.6 - 5.4 32.5 _+ 8.V

3.8 ± 1.4 4.5 + 1.3 c

0.04 -+ 0.04 2.0 ± 0.7 d

1.0 ± 0.4 1.4 +- 0.8 c

a Rehabilitation period. b Postrehabilitation period. c N o t significant. d p < 0.005.

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T A B L E 4. Histomorphometric comparison o f variables: percent area over rehabilitation and postrehabilitation periods (mean +- SEM) Months postoperative 3-10 (n = 10)a 13-18 (n = 6) b Total = 16

Polarization

Biopsy polarization × 100 (Fascia lata polarization)

Mean of active and intermediate fibroplasia

Sum of active and intermediate fibroplasia

26.6 --+ 4.5 51.3 -+ 6.6 (p < 0.01)

47.8 +- 6.5 82.7 -+ 8.3 (p < 0.005)

37.4 - 2.3 25.9 -+ 5.6 (p < 0.025)

72.2 -+ 6.5 40.5 +- 8.6 (p < 0.005)

a Rehabilitation period; bPostrehabilitation period.

dex of maturity, we estimated that these grafts may not reach "histologic maturity" until 29 months. Obviously, return to cutting sports is possible much earlier than this, indicating that the graft attains an acceptable strength level before reaching a histologically mature pattern. It would be desirable to correlate these two seemingly separate events so that surgeons could more accurately identify when an athlete with ACLR could safely return to cutting sports. It would also be of great interest to more closely examine the quality of the collagen in allograft ACLRs. Earlier studies have shown that with autogenous ACLRs, the normal large-diameter collagen fibers are variably replaced with smaller diameter fibers (36). We do not know if this also occurs with allografts. Collagen typing would clarify whether this is a different collagen than that found in the normal ACL. The problem of graft rejection and immunology has been previously addressed (18,37). Freezedried, sterilely harvested soft tissue allografts appear not to incite a clinically detectable immunological response (26.). In support of this observation, there was no histologic evidence of graft rejection in any of our biopsy specimens. CONCLUSIONS

This study has demonstrated how an ACL allograft matures histologically in a homogeneous group of patients. For the first time in a study of this nature, histomorphometry was used to objectively identify events in the maturation process. Degenerative CT was present up to 20 months postoperatively and represents the remnants of the implanted allograft. The process of allograft maturation or ligamentization extends beyond 20 months, as evidenced by relative increase in cellularity and vascularity, in comparison with normal ACL. A c k n o w l e d g m e n t : W e t h a n k J a y M a r t i n e z for t e c h n i c a l assistance with the arthroscopy equipment, Angela

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