Mechanisms of anterior cruciate ligament neovascularization and ligamentization

Mechanisms of Anterior Cruciate Ligament Neovascularizationand Ligamentization

Pierce E. Scranton, Jr., M.D., William L. Lanzer, M.D., Marina S. Ferguson, M.T.,Thomas R. Kirkman, B.A., and Daniel S. Pflaster, M.S.

Summary: Columbia-Rambouillet cross-bred sheep were used to study therevascularization and ligamentization process of anterior cruciate ligament (ACL)reconstruction over a 6-month period using basic histology, immunohistochemis-try, and electron microscopy. The reconstruction technique studied was a quadruple-hamstring, interference screw fixation technique. Further, these specimens, afterretrieval at 6, 12, and 26 weeks, were compared with human arthroscopic ‘secondlooks’ and with 10 en bloc specimens obtained when a cruciate-sacrificing totalknee replacement was performed. The study showed that, with this reconstructiontechnique, Sharpey’s fibers were seen at 6 weeks in both sheep and humanspecimens. The intratunnel specimens showed proliferative chondrification, thenossification of the matrix. Intra-articular neovascularization, ligamentization, andjunction ossification occurred. Myoblasts or smooth muscle cells appear to mediatethe ligamentization as evidenced in electron microscopy by proliferate collagenmanufacture. These myoblasts were seen in both the healing sheep and humansecond looks, but not seen in mature ACL grafts or in normal ACLs. At 6 monthspostoperatively, the sheep ACL reconstruction appeared clinically, histologically,and immunohistochemically indistinguishable from the normal sheep ACL. Acorrelation of this work with published animal studies in which biomechanicaltesting was performed and with human ‘second looks’ would imply that an ACLreconstruction may be vulnerable during this period of neovascularization andligamentization. Key Words: Ligament—Revascularization—Myoblasts—Maturation.

One of the goals in reconstructive anterior cruciateligament (ACL) surgery is biological fixation.

Sharpey and Ellis described the histology of a normaltendon’s attachment to bone as ‘‘perforating fibers,projecting like nails driven perpendicularly or slant-ingly through a board.’’1 When biological fixation inthe bony tunnel occurs, this will facilitate the transfor-mation of graft into a viable, functioning ligament. It isa matter of debate at what point this process translates

into a ligamentous reconstruction strong enough toallow an individual to resume unprotected athleticactivity.

The ACL and tunnel-graft interface have beenextensively studied in live humans, cadaveric speci-mens, and animal models. Research into ACL graftneovascularization, ligamentization, biomechanicalstrength, fixation strength, and fibroblast repopulationhas been performed both in human arthroscopic sec-ond looks, and in animal models, using histological,immunohistochemistry, and scanning electron micro-scopic techniques.2-7 Grafts studied have includedbone–patella tendon–bone, hamstring(s), fascia lata,Achilles tendon, artificial or synthetic ligaments, andbriefly, xenografts.2,8-11 Finally, histological studieswith both arthroscopic second-look specimens as well

From the University of Washington, Seattle (P.E.S., W.L.L.);BioSupport Inc, Redmond, Washington (M.S.F., T.R.K.); and Smith& Nephew Brace & Support, Vista, California (D.S.P.), U.S.A.

Address correspondence and reprint requests to Pierce E.Scranton, M.D., 1600 E. Jefferson, Suite 400, Seattle, WA 98122,U.S.A.

r 1998 by the Arthroscopy Association of North America0749-8063/98/1407-1849$3.00/0

702 Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 14, No 7 (October), 1998: pp 702–716

as short- and long-term clinical follow-up have shownthe superiority of autograft tissue compared withallograft or artificial ligaments with respect to thespeed of effective neovascularization and the subse-quent ligamentization process.2,6,11-15

A rigidly fixed, isometrically placed ACL graft willundergo neovascularization and ligamentization muchmore rapidly than a loosely fixed graft.2 If the graft isnot completely in contact with its bony tunnel and,instead, bathed during the healing process with oldblood degradation products and synovial fluid, aninflammatory response can delay or impair healing.The inflammatory process associated with allografts,artificial ligaments, or an artificial stint (ligamentaugmentation devise), would be expected to interferewith natural healing. To optimize fixation with ahamstring autograft, a variety of endoscopic tech-niques in ACL reconstruction have been devel-oped.7,16,17These procedures involve the use of eithertitanium or biodegradable screws, or suspension tech-niques to provide fixation of the hamstring construct intibial and femoral tunnels. The clinical results of thesenew ACL reconstructive procedure in humans appearsto be very successful.7,16,17However, the histology andimmunohistochemistry of the healing process in thebony tunnel and the speed of neovascularization andligamentization has not yet been evaluated. Factorsinvolved in the mediation of these processes areincompletely defined. It is, therefore, the purpose ofthis study to evaluate the histology of the tunnel-grafthealing process, as well as the immunohistochemistryand electron microscopic findings of the neovascular-ization and ligamentization processes of intra-articularhamstring autograft over time. Finally, it is our pur-pose to correlate this observational study in sheep withhuman second looks, biopsy results, and previouslypublished biomechanical studies in order to infer howlong a patient with a reconstructed ACL might requirethat knee to be protected.

MATERIALS AND METHODS

Six Columbia-Rambouillet cross-bred sheep wereused for the study. These animals were humanely caredfor in compliance with regulations advanced in ‘‘Prin-ciples of Laboratory Animal Care Guide for the Careand Use of Laboratory Animals.’’ During the studythey were housed at BIOSUPPORT, INC., Redmond,Washington, an AAALAC accredited facility.

It was elected to reconstruct the ACL of the rightknee of each sheep. The surgical technique wasidentical to that advanced by Pinczewski using two

blunt-threaded titanium RCI 73 25 mm screws (Smith &Nephew Endoscopy, Mansfield, MA).17These provided aninterference fit for a quadrupled hamstring graft thathad been passed through the tibial and femoral tunnels,drilled with a 6.5-mm router. These operations wereperformed using an open approach rather than thearthroscopic-assisted technique that is used in humans.

Surgery was performed on the sheep by the seniorauthor on May 17, 1995. Each sheep was sedated usingintramuscular xylazine 0.2 mg/kg and intravenousketamine 10 mg/kg. Surgery was performed with theanimals under general endotracheal anesthesia withisoflurane.

An arthrotomy on each sheep’s right knee wasperformed, extending the incision distally down thetibia. Unlike humans with their proximal per anserineinsertion of the hamstring tendons, the sheep ham-string tendons insert much more distally. In each case,two distally inserting tendons were identified, har-vested, and then pulled through the tibial and femoralrouter holes that had been drilled through the anatomicorigin and insertion of the original sheep ACL. Thesterile interference screw was driven into each drillhole, fixing theACL tendon graft in place.A roentgeno-gram confirmed our direct observation of appropriatescrew location. The wounds were closed using No. 0and 00 absorbable sutures. Perioperatively, the animalsreceived intramuscular cephalosporin 20 mg/kg beforesurgery and again for 5 days after surgery. They alsoreceived buprenorphine 0.005 mg/kg twice daily fortwo days.

The study design was to harvest two successfullyreconstructed knees each at 6, 12, and 26 weeks. If thereconstruction was unsuccessful, more animals wouldbe included in the study. Animals were humanelysacrificed by overdosing with intravenous sodiumpentobarbital. All reconstructed knee joints were har-vested by the senior author. Normal contralateral kneejoints were also harvested for ligament comparison.The excised total joint was placed in a large containerof 10% neutral buffered formalin for no less than 1week to ensure adequate fixation. Decalcificationoccurred in 10% formic acid for several weeks. Thespecimens were trimmed throughout the decalcifica-tion process. The screw was removed at the end of thedecalcification period and 3-mm serial segments of thetunnel and surrounding material were submitted forprocessing, embedding, and sectioning. The stainsused were the routine H&E and Masson’s trichromespecial stain for collagen.

Electron microscopy was performed at the Biogenet-ics Research Laboratory using transmission electron

703ACL NEOVASCULARIZATION AND LIGAMENTIZATION

microscopy to evaluate the cells staining positive foractin in the immunoistochemical portion of the study.These cells transmission electon micrographs werecompared with fibroblasts seen in the sheep controlnormalACL, harvested at the same time the reconstruc-tion was harvested.

Immunohistochemical Reagents: Antibodiesand Antiserum

To identify cells with contractile proteins, which, inthe context of remodeling bone and cartilage, mightrepresent either myofibroblasts or vascular smoothmuscle cells, immunohistochemical testing was per-formed. A mouse monoclonal antibody to smoothmuscle cell alpha-actin was obtained from Boehringer-Mannheim (Indianapolis, IN; catalogue no. 1148-818)and used at a titer of 1:1,500.

ImmunohistochemistrySingle-label immunoperoxidase staining of 6-µm

bone sections was performed as described previ-ously.18 Briefly, tissue sections were deparaffinizedwith xylene and then rehydrated with graded alcohols.The slides were blocked with 3% hydrogen peroxidethen washed with phosphate-buffered saline. Theywere then incubated for 30 minutes with the primaryantibody, anti–smooth muscle alpha-actin. The second-ary biotinylated antimouse antibody (Vector Laborato-ries, Burlingame, CA) was applied for 30 minutes,followed by an avidin-biotin-peroxidase conjugate(ABC Elite; Vector Laboratories) for 30 minutes.3,3’-diamino-benzidine with nickel chloride was usedas a chromogen, yielding a black reaction product. Cellnuclei were counterstained with methyl green. Nega-tive controls included substitution of primary antibodywith irrelevant antibodies at the same titer.

Human StudiesHuman specimens of ACL tissue were available for

study from the following sources: (1) A cored tibialtunnel from a noncompliant patient who had anintrasubstance ACL tear 6 weeks postoperatively andrequired revision. (2) A 9-weeks postoperative intrasu-bstance ACL failure from a patient thrown from ahorse who required revision. (These patients had graftsplaced in the anatomical foot print of their old ACLand the second surgery was performed 1 week and 2weeks after injury, respectively.) (3) Two patients, 12and 15 months postoperatively, who underwent arthro-scopic procedures for new injuries to the meniscuswherein, with informed consent, biopsy specimens ofthe intact reconstructed ACL were obtained. (4) Ten enbloc ACLs were procured from patients between theages of 41 and 62 years of age undergoing cruciate-

sacrificing total knee replacement. They each hadposttraumatic or degenerative arthritis and their normal-appearing ACL was harvested en bloc for purposes ofexamining normal human ACLs immunohistochemi-cally for cells containing actin.

RESULTS

All six sheep had uneventful, uncomplicated rightknee surgery. There were no infections. All six sheep atthe time of retrieval yielded viable reconstructionspecimens with an otherwise normal appearing joint(Fig 1). Comparison postoperative roentgenograms inthe anteroposterior and lateral planes were taken at thetime of sacrifice to confirm screw position (Fig 2).There was no evidence roentgenographically or clini-cally of screw loosening in any specimen.

Histological study revealed there were areas insidethe femoral tunnel in various stages of repair. The6-week sheep reconstruction (Fig 3) in the femoraltunnel taken in cross-section shows the transition ofthe inflammatory phase to the reparative phase withthe intense biological activity. The old tendon rem-nants are visible at the interface of the screw and bonetunnel. Chondrification, neo-ossification and prolifer-ate osteoblastic activity are seen. The healing processof this 6-week reconstruction also shows prominentSharpey’s fibers, which anchor the fibroproliferativeresponse to the bone (Fig 4). Intra-articularly, theautograft is still avascular.

The ligament at the junction of the tunnel andintra-articular portion at 3 months showed focal areasof acellular collagen scaffold being invaded by col-umns of chondrocytes (Figs 5 and 6). The sectionillustrated was taken from the screw tunnel intra-articular junction. Microfibrils form integral compo-nents of the elastic fiber in ligaments. These microfi-brils also support cell adhesion, thus ensuring themaintenance of the alignment of the collagen scaffoldin this biomechanical environment. Tensile stressesmay play a significant role in the alignment of thescaffolding, as evidenced by the linear nature ofchondrocyte differentiation.

In the midsubstance intra-articular portion of thegraft at 3 months, there are focal areas of remodeling.This vascular remodeling is driven by a wide band ofthrombus that advances into the necrotic tendon (Fig7). A closer look at this junction shows vascularchannels adjacent to necrotic tendon (Fig 8). Anti–smooth muscle actin applied by the Avidin Biotintechnique reveals significant numbers of smooth musclecells present in the necrotic matrix, in advance of theneovascularization (Fig 9). Scanning electron micros-

704 P. E. SCRANTON ET AL.

copy shows that these cells are actively manufacturingcollagen (Fig 10). In contrast, using electron micros-copy, the cells present in the control ACL appearbiologically ‘‘quiet.’’The remodeling of theintra-articulartendon appears to occur in a distinct progression.Necrotic tendon is metabolized by macrophages andoccasional multinucleated giant cells (Fig 11). At this3-month period, the ligament differentiation appears tobe devoid of inflammation, which would prolong orprevent ligamentization. The intra-tunnel interferencefixation technique appears to result in a noninflamma-tory fixation with no evidence of loosening, andosteoblastic activity and Sharpey fiber formation ispresent immediately adjacent to the screw (Fig 12).

The histological appearance of the control ligamentand the reconstructed tendon at 6 months is quitesimilar (Fig 13). Cell density is equivalent. Theintracellular matrix has a homogenous appearancewith fibers aligned in a linear array with a similarcrimp pattern. At 6 months, there was no evidence offurther neovascularization or smooth muscle cells ormyofibroblasts in the ligament matrix.

Human specimens obtained from failed and intacthamstring ligament reconstruction were available forstudy. Two had intrasubstance failure; intra-articularligament disruption at 6 and 9 weeks, respectively. Thefirst specimen was a cored tibia, intratunnel specimenfrom a failed hamstring ACL reconstruction at 6 weekswhen a noncompliant patient played sports. Thiscross-section specimen (Fig 14) shows abundant intra-tunnel Sharpey’s fibers. The second specimen is anACL hamstring reconstruction intra-articular failurethat occurred at 9 weeks when the patient attempted to

FIGURE 1. Gross anatomicspecimen harvested 26 weeks af-ter reconstruction. Note the neo-vascularization as indicated by thecapillary visible on the surface ofthe anterior ligament (arrow). Theremaining joint is normal in ap-pearance.

FIGURE 2. Anteroposterior roentgenogram of the sheep knee priorto harvest. There was no evidence of screw loosening or migration.


leap onto a horse that moved. Areas similar to the12-week sheep specimens of neovascularity adjacentto necrotic intra-articular graft are seen (Figs 15 and16) A tissue section taken from this same area stainedfor anti–smooth muscle actin had smooth muscle cellssimilar to the cells seen in the sheep specimens (Figs17 and 18).

Two additional human specimens underwent biopsyexamination from the midsubstance of the recon-structed ACL at 12 and 15 months after surgery, whena second operation for a torn cartilage became neces-sary. Their findings were identical; Fig 19 shows theintact ACL reconstruction and Fig 20 shows a longitu-dinal section of the intra-articular biopsy specimen

FIGURE 3. Intense biologicalactivity of a cross-section of thetunnel in the 6-week specimenis shown by a large cluster ofossifying chondrocytes (C), andthe distinct network of theSharp-ey’s fibers (arrow) thatconnectislands of metabolically activebone (B). Remnants of the ten-don are seen at the bottom left(T) (Masson’s trichrome, origi-nal magnification3100).

FIGURE 4. Cross-section of thesheep ACL reconstruction intra-tunnel at the junction of theintra-articular ligament at 6weeks. The blue Sharpey’s fi-bers (arrow) protrude from thebone (B) into the new fibrousmatrix (Masson’s trichrome,original magnification3400).


with normal cell density and a normal crimp pattern inthe collagen. No actin was present in this specimen,indicating the myoblastic process seen in the healingspecimens earlier appeared to be absent in ‘‘mature’’specimens. In the 10 human ACLs harvested at thetime of total knee replacement, none exhibited anyevidence of actin in cells in the ACL substance, except

for the expected actin in the smooth muscle cells ofblood vessels.

DISCUSSION

It has been estimated that over 75,000 individuals ayear tear their ACL.19 Many of these patients do not

FIGURE 5. At 3 months, theautografted tendon shows in thelongitudinal section at the tun-nel intra-articular junctionwhere chondrocytes transitioninto an area of cellular collagenscaffold (H&E, original magni-fication3100).

FIGURE 6. The alignment ofchondrocytes within collagen fi-bers in the longitudinal sectionin response to tensile and shearforces (Masson’s trichrome,original magnification3400).


require reconstructive surgery. For those who experi-ence symptomatic instability with daily activities ofliving, who have strenuous avocations, or wish tocontinue vigorous competitive athletics, an ACL recon-struction is the treatment of choice. Ideally, the recon-struction should efficiently offer the patient a stableknee with as little risk and morbidity as possible.

The issues of efficiency, risk, morbidity, and a safereturn to unprotected activity are still subject to debate.They may be dependent on the type of graft selectedfor reconstruction, the surgical technique, and postop-erative patient compliance. Purely from a cost stand-point, ignoring the risk of immunogenicity and/or viraltransmission of disease, allografts add significant

FIGURE 7. H&E stainingshows the longitudinal sectionof acellular necrotic tendon onthe bottom and the wide band ofbright red blood that pours outof the advancing vascular front.The arrow points to smoothmuscle cells that lead in thereorganization of the tendon. Afew granulocytes remain scat-tered in the denuded tendon(original maginification310).

FIGURE 8. A 400 magnifica-tion close-up of the same areaof neovascularization seen inFig 7 in longitudinal relief us-ing Masson’s trichrome to showwhere intense proliferative vas-cularization is invading the oldnecrotic tendon.


expense to an ACL reconstruction. This is also true ofartificial ligaments, the cost and long-term efficacy ofwhich make them less desirable than autografts. ACLautografts from bone–patella tendon–bone constructs

or hamstring tendons have proven efficacy and areclearly preferable both from the standpoint of cost andrisk, as well as biological revascularization and liga-mentization.2,5,6,11,12,15,20

FIGURE 9. A smooth muscleantibody immunocytochemicalstain of the advancing vascularfront of the tendon revealssmooth muscle cells (arrow)projecting like fingers into thewide band of blood (see Fig 7)(original magnification3400).

FIGURE 10. Image at 30,000magnification of the cells seenin Fig 7 showing dilated roughendoplasmic reticulism withactive protein synthesis and col-lagen production. Prominentcollagen bundles surround theactive cell.


To minimize morbidity, adequate autograft fixationmust be achieved to allow physiological joint mobiliza-tion and minimize quadriceps atrophy while protectingthe graft. Kurosaka et al.21 showed the importance offixation technique. Graft constructs in his study weretested in cadaver knees and failed at the fixation site.This indicates that in actual patients, graft protectionmust be adequate until biological fixation occurs.Kurosaka showed that interference screws were signifi-cantly superior to all other fixation techniques both in

terms of maximal tensile strength and in the preventionof elongation. Graft constructs with sutures tied over abutton actually elongated up to 25 mm before failure.This superiority of interference screw fixation hasbeen confirmed by many other investigators.14,22-24

Therefore, secure fixation is important both for graftprotection and the preservation of physiological kneefunction while biological fixation occurs. Secure fixa-tion with physiological function will actually enhancebiological fixation. Woo et al.11 showed that immobili-

FIGURE 11. Three-month in-terface of the tunnel and tendon-ligament composite. The longi-tudinal section of necrotictendon(left) is being phagocytized bymultinucleated giant cells (ar-row). The bed of resultant fi-brotic tissue (right) will be re-placed by ligament (H&E, originalmagnification3200).

FIGURE 12. A cross-section ofthe screw interface of the sheepACL reconstruction at 6 months.This interface shows the lack offibrosis and inflammation andthe remarkably active bone.Sharpey’s fibers (arrow) whirlfrom bone (B) that is separatedfrom metal by very few smoothmuscle cells (immunocytochem-istry for smooth muscle actin notshown). The lack of inflamma-tion is consistent with biocom-patible titanium screws (Masson’strichrome, original maginification3400).


zation disrupts or disorganizes the linearity of ligamentcollagen in normal and injured knees. Arnoczky et al.2

showed that intimate contact and rigid immobilizationof a graft facilitated biological fixation and healingstrength. Finally, the application of physiological ten-sion and motion has been shown to aid in repair,causing fibroblast and collagen fibrils to align parallelto the direction of force.11,15,25

If an endoscopically placed autograft results in lessmorbidity, it would appear to be more desirable than atwo-incision technique. The issue then arises as to

when ligamentization and biological fixation haveadvanced to the extent that it is safe for a patient with afully rehabilitated knee to resume unprotected activity.Both animal and human studies have been used in aneffort to infer when an ACL reconstruction is matureenough to withstand normal physiological stress.2-

4,6,26-28 However, there is broad variation in theseinferences because of the variations in graft materialand in the difficulty in extrapolating animal studies tohuman studies.

Animal studies investigating biomechanical liga-

FIGURE 13. Normal controlligament (left) and reconstructedACL (right) display cellular den-sity and fiber orientation thatare essentially identical at 6months (H&E, original magini-fication3400).

FIGURE 14. A intra-tunnelcross-section of a human ACLreconstruction taken from thetibia at six weeks. Note theabundant Sharpey’s fibers (Mas-son’s trichrome, original magni-fication3400).


ment strength over time have all concluded that thereis significant decay of ligament strength until at least 6months postoperatively.2,3,26 We did not duplicatethese studies because their data are generally inagreement. In a retrospective human study, Glasgow etal.4 and Howell and Taylor27 did not see detrimentaleffects in an earlier return to athletics. From a histologi-cal standpoint, however, graft maturity timing isunclear. Fascia lata grafts do not achieve histologicalmaturity until 29 months.10 In humans, bone–patellatendon–bone ACL reconstruction goes through four

stages of ligamentization.29 The third stage of matura-tion occurs between 1 and 3 years after reconstructionwith some areas of necrosis and neovascularizationstill seen in the ligament. Given the known temporarydecay in autograft ligament strength and the findingsof areas of necrosis and neovascularization present insecond looks, it would appear to be prudent that therebe some period of ligament protection. In a caninestudy, Rodeo et al.6 examined the biological fixation ofan extensor tendon when passed through a tibialtunnel. They concluded that at least 8 weeks ofprotection was necessary in humans before a recon-struction could withstand stress. They did not specu-late on a time frame for a safe return to competitiveathletics. Finally Clancey et al.26 reported on thestrengths of medial one-third patellar tendon ACLreconstructions in rhesus monkeys. On the basis ofstudying these reconstructed knees, they inferred that,in human reconstructions, it was wise to wait at least 1year before the resumption of competitive activity.

This discussion brings us to the purpose of thiswork. We have attempted to define, over time, theprocess of ACL autograft biological fixation andligamentization in an animal model. We have tried tocorrelate this observational study to human secondlooks and our clinical experience so that we can betterpredict when it is safe to resume unprotected activity.Goats, sheep, monkeys, dogs, and rabbits all have been

FIGURE 15. An intraoperative view of the 9-week intrasubstancefailure of the reconstructed ACL.

FIGURE 16. A longitudinal in-tra-articular section of a 9-weekhuman ACL failure. Note theintense neovascularization adja-cent to necrotic hamstring ten-don (H&E, original magnifica-tion 3100).


extensively used in animal models to assist investiga-tors in learning what happens to an ACL reconstruc-tion.2,3,6,9,15,26,27,30-32

There are obvious limitations in animal models, butthere are also areas of definite correlation. The disabil-ity animals experience with ACL deficiency is signifi-cant and well documented.31 The ACL is necessary,

even in quadrupeds, for stable normal knee jointfunction.

Our observational study used the sheep model. Our‘‘n’’ was only two per time frame but, in each case, thereconstructions were successful and, except for thearthrotomy scar, the joint otherwise appeared un-harmed, normal, and the reconstruction intact. We did

FIGURE 17. H&E stain of thecentral necrotic area of the liga-ment substance from Fig 15(original magnification3400).

FIGURE 18. An SMA immu-nocytochemical stain of the vi-able cells containing actin inthis region from Fig 17.


not extend the study to 1 year because all previousanimal model studies showed no significant differencein the histological or mechanical properties of thereconstruction ligament between 6 months and 1year.3,26,28

Our study used adult sheep that had an anatomichamstring ACL reconstruction with interference screwfixation. There were no infections and all reconstruc-tions yielded viable specimens at the time of harvest.We could not control the animal’s limb protection or

‘‘compliance,’’ and therefore only one limb was oper-ated on so that each sheep could favor and protect thatlimb until the operative pain had subsided. In all cases,within a matter of days, each sheep was fully weight-bearing in its pen, subjecting the limb to physiologicalforces.

Arnoczky et al.2 showed that intimate contact andrigid immobilization facilitated biologicalal fixationand ultimately healing strength. We believe that thequadruple tendon, tightly packed in the bony tunneland fixed with a blunt-threaded screw, fulfills Arnocz-ky’s criteria of intimate contact and rigid immobiliza-tion. In this technique, human cadaveric hamstringpullout studies for the screw are 336 N6 95 N.32

These animal and human reconstructions were ana-lyzed histologically and immunocytochemically. Theresults of these observations can be summarized asfollows: At 6 weeks, in the tunnel, necrotic tendon isseen, but Sharpy’s fibers and neochondrification andneo-ossification are already occurring. Intra-articu-larly, the tendon appears avascular. At 12 weeks, in thetunnel, the chondrocytes now appear to be sitting on abase of bone, aligned with the mechanical forces andparallel columns of neocollagen. These areas were notdissimilar to the ligament ‘‘base’’ or origin seen in thehuman en bloc ACLs harvested at total knee artho-plasty. Intra-articularly, in the mid substance of theligament, the avascular tendon is being transformed bygiant cells and myoblasts into a new ligament. At 26

FIGURE 19. A 15-month second look of a patient’s secondarthroscopy after an ACL reconstruction for problems associatedwith a torn lateral meniscus.

FIGURE 20. Biopsy specimenof mid-substance ACL from thepatient in Fig 19 showing anormal cell density pattern andcollagen fibers orientation (H&E,original magnification3400).


weeks, the process appears to be complete and theneoligament seems similar in morphology, cell densitypopulation, and collagen alignment to a control ACL.At this time, the myoblasts are not present in theanimal model, nor in the human second looks or the 10human control ACLs.

We report for the first time in both human and sheepspecimens, the invasion of smooth muscle cells as anintegral step in this cellular reparative state of trans-plant tendon healing. These cells are not seen innormal ACLs or in mature, healed autograft ACLs.Kielty and Shuttleworth25 showed that smooth musclecells produce fibrils as seen in ligaments. This processpromotes biological healing without the influence ofan anti-inflammatory synovial environment. Thus thetensile forces and intrafiber shear forces may exert aninfluence on these mesenchymal cells to accelerate theproduction of glycoproteins and the rapid invasion ofchondroblasts. This encourages more rapid ligamenti-zation of the tendon.

The Sharpey fiber formation was seen in bothanimal and human specimens. It is an indication ofbiological anchoring of the graft to the host bone.Because of this stability, shear forces continuallyinfluence the fiber maximally at the interface anddecreasingly toward the hypothetical isometric fibersin the center of the graft. Intense metabolic activity isseen between 6 weeks to 3 months with intratunnelossification and intra-articular vascularity. This waveof apparent neovascularization supplies both smoothmuscle cells and chondroblasts, which ligamentize thetendon. From 3 months to 6 months, remodelingcontinues in accordance with the biomechanical forcesthat allow maturation to occur. The resulting matureligament structure closely approximates the normalACL in both cell population density and fiber orienta-tion and fibroblast population in both the sheep modeland in human second looks.

This study shows that in the sheep model by 3months the processes of revascularization and ligamen-tization are incomplete. This was also seen in the twofailed human specimens as well, with areas of necroticligament and neovascularization. However, at 6 monthspostoperatively in the sheep model and later in thehuman specimens, there does not appear to be distin-guishable anatomic or histological differences be-tween an ACL reconstruction and controls or normals.Other animal models show no difference between 6months and 1 year in testing tensile strength.26,28 Somuch work has already been performed in the area oftensile strength in both human and animal ligaments,that we believe further sheep reconstructed-ACL ten-

sile-strength studies are unnecessary.13,26,28,32-35Thestudy of Brown et al.34 defines the breaking force of aquadruple hamstring construct to be 4,589 N. Ouranimal model showed the ligamentization process wasincomplete at 12 weeks, and we studied two patientswho had failures at 6 and 9 weeks, which correlateshistologically to this animal model. These studies aswell as previous published work lead us to believe thatan ACL-reconstruction patient with a fully rehabili-tated and stable knee should be protected until liga-ment maturation occurs. Noyes and Barber-Westin36

recommended protecting the knee of an athlete whohad undergone an ACL reconstruction for at least 4months. On the basis of the biology of the healingmechanisms we have seen in this animal model and inhumans, we agree with this recommendation. Furtherstudies are anticipated to better define the time frame.

Acknowlegment: The authors express their appreciationto Michael Grabowski, M.D., for assistance in preparation ofthe manuscript and to Leo Pinczewski, M.D., for providingone of the pathological specimens.

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Mechanisms of anterior cruciate ligament neovascularization and ligamentization