CLINICAL ORTHOPAEDICS AND RELATED RESEARCHNumber 391S, pp. S116–S123© 2001 Lippincott Williams & Wilkins, Inc.

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Chondrocytes undergo apoptosis in response tomechanical injury in vitro. The current clinicalstudy correlates arthroscopic and magnetic res-onance imaging results with biopsy specimens ofcartilage from patients with knee injury. Twentypatients were evaluated at a mean 2.7 months af-ter acute knee injury. The mean age of the pa-tients was 32 years and the mean weight was 83kg. Cartilage lesions were graded separately onmagnetic resonance images and arthroscopy in ablinded manner. During arthroscopy, a 1.8 mmdiameter biopsy specimen was obtained from theedge of cartilage lesion. The biopsy specimen un-derwent histologic examination by safranin Ostaining and detection of chondrocyte apoptosisby the presence of deoxyribonucleic acid frag-mentation. There was a positive correlation in50% (10 of 20) when the presence or absence ofcartilage lesions by magnetic resonance imagingwas correlated with arthroscopy. All cases ofpartial thickness or full-thickness cartilage lossthat were seen by arthroscopy also were detectedby magnetic resonance images. Apoptotic cellswere significantly more numerous in biopsyspecimens from lesions compared with controlbiopsy specimens. The findings of reduced cell

viability attributable to apoptosis may have pro-found implications for cartilage repair. Thisopens potential therapeutic avenues for thetreatment of posttraumatic cartilage lesionsthrough apoptosis prevention.

Cartilage injury is one of the most significantfactors leading to secondary osteoarthritis. Theconsequences in vivo and the long-term effectshave yet to be documented adequately. The cur-rent study was designed to explore the arthro-scopic, histologic, and magnetic resonanceimaging (MRI) findings after acute joint traumaand determine correlations between the above.

Several studies have shown cartilage matrixdamage and chondrocyte death in response tomechanical injury.3,11 Recently, it has beenshown that chondrocytes undergo apoptosiswhen subjected to mechanical trauma.4,10,21

Cartilage from various sources displayed a sim-ilar response to mechanical injury. Three differ-ent modes of injury were modeled in the abovestudies: injurious compression, blunt impact,injury leading to loss of cartilage, and chronicinjury attributable to instability. Three specieswere tested: rabbit, bovine, and human. In addi-

In Vivo Changes After Mechanical Injury

Clifford W. Colwell, Jr., MD; Darryl D. D’Lima, MD; Heinz R. Hoenecke, MD; Jan Fronek, MD; Pamela Pulido, RN;

Beverly A. Morris, RN; Christine Chung, MD; Donald Resnick, MD; and Martin Lotz, MD

From the Division of Orthopaedic Surgery, ScrippsClinic, La Jolla, CA.Funded by the ALSAM Foundation: Skaggs Institute forResearch.Reprint requests to Clifford W. Colwell, Jr, MD, ScrippsClinic, MS126, 11025 North Torrey Pines Road, Suite140, La Jolla, CA 92037.

List of Abbreviations Used

DNA deoxyribonucleic acid

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tion, the apoptotic response was seen in experi-ments ranging from full-thickness cartilage tointact joints. These studies have been done invitro under artificial conditions or in vivo in an-imals. The theory that cartilage injury results inapoptosis requires validation with clinical data.The current study determined whether cartilagelesions associated with acute joint injuryshowed evidence of apoptosis.

Data exist to support the theory that apop-tosis can be inhibited in vitro and in vivo.20

This opens possibilities of alternative thera-peutic approaches to chondroprotection. Chon-drocyte and matrix responses to mechanicalinjury are significant factors in determiningthe development, history, and treatment ofcartilage lesions. A recent approach to thetreatment of cartilage lesions is chondropro-tection. Chondroprotection involves the use oftherapeutic measures, usually pharmacologicagents, that maintain matrix integrity and re-tard the development and progression of carti-lage lesions. Several chondroprotective agents(such as glucosamine and its derivatives,chondroitin sulfate, and hyaluronic acid) cur-rently are being investigated and theoreticallyhave the potential of reducing cartilage degen-eration or enhancing repair after significant in-jury. Accurate noninvasive markers of carti-lage damage and repair therefore would bevaluable in documenting and validating the ef-fect of various therapeutic approaches.

MATERIALS AND METHODS

Twenty patients were recruited after appropriate In-stitutional Board Review approval. Inclusion crite-ria were age between 18 and 45 years, acute knee in-jury within the previous 6 months, and clinical orMRI evidence of acute joint injury necessitating anarthroscopy. Exclusion criteria were intraarticularbone fracture, preexisting joint lesions, chronicsymptoms, history of previous trauma or surgery, orlocal anatomic deformity. There were 17 men andthree women. The mean age of the patients was 32years (range, 19–43 years) and the mean weight was83 kg (range, 66–125 kg). Sixteen patients sustainedthe knee injury during sporting activities, three re-ported a fall, and one sustained the injury during a

routine activity of daily living. The mean time afterinjury was 2.7 months (range, 1–6 months).

Patients underwent routine diagnostic arthro-scopy as indicated by their clinical examinationand initial clinical MRI findings. Before this diag-nostic arthroscopy, patients underwent an addi-tional MRI examination that included proton den-sity fat saturated fast spin echo and three-dimensionalspoiled gradient echo of the affected knee. Thepresence of any cartilage lesion including the sizeand the grade (ranging from signal heterogeneity tofull-thickness loss of cartilage) was observed. Thepresence and grade of bone marrow edema alsowas documented.

During arthroscopy, the surgeon did a routineexploration of the joint cavity and recorded abnor-mal findings and was unblinded to the results of theresearch MRI. At this time, the surgeon obtained a1.8-mm diameter biopsy specimen at the edge ofany cartilage lesion if present (Figure 1). An 11-gauge Jamshidi bone marrow biopsy needle (Alle-giance Healthcare Corp, McGaw Park, IL) wasused. The surgeon then did any relevant surgicaltherapeutic procedures necessary such as debride-ment and lavage, meniscal excision or repair, andanterior cruciate ligament reconstruction. Cartilagelesions were graded on MRI and arthroscopic find-ings as shown in Table 1.

The biopsy specimen was fixed in 10% bufferedformalin, decalcified and examined histologicallyafter safranin O staining and detection of DNAfragmentation. The safranin O-stained sectionswere graded using the histologic scoring system ofMankin et al,12 which semiquantitatively gradesstructural changes, cellular density, safranin Ostain intensity, and tidemark integrity. Apoptoticcells were detected using the Mebstain kit (MBL,Nagoya, Japan) that uses a fluorescent labeled an-tibody to DNA fragments. Nonapoptotic cells werecounterstained with propidium iodide. The numberof apoptotic cells divided by the total number ofcells present yielded the percent apoptosis in thatsection. Control biopsy specimens were obtainedfrom the femoral condyles of four fresh cadaverdonors and from the intercondylar notch of four pa-tients (before notchplasty) who required anteriorcruciate ligament reconstruction.

RESULTS

All patients successfully underwent the anes-thesia, arthroscopic procedures, and cartilage

biopsy without any adverse events. Twelvepatients required debridement and lavage ofthe joint, eight required meniscal surgery, and11 required anterior cruciate ligament recon-struction. The MRI findings are summarizedin Table 2. Representative images of variousgrades of cartilage lesions are shown in Figure2. There were 13 patients with evidence ofbone marrow edema, 11 in whom the edemawas adjacent to overlying cartilage lesions,and two in whom there were no detectable car-tilage lesions. Figure 2F shows a patient withbone marrow edema without any overlying

cartilage lesion. The arthroscopic findings aresummarized in Table 3. Ten patients had visi-ble cartilage lesions on the lateral femoralcondyle, two in the medial femoral condyle,two in the lateral tibial plateau, one in the me-dial tibial plateau, and one in the patella. Infour of the 20 patients, no significant lesionwas found. Figure 3 shows arthroscopic pho-tographs representative of the different gradesof cartilage lesions.

When the presence or absence of cartilage le-sions by MRI was correlated with arthroscopy,there was a positive correlation in 50% (10 of

Clinical OrthopaedicsS118 Colwell et al and Related Research

Fig 1. The biopsy technique is shown. An 1.8-mm diameter core of cartilage and subchondral bone washarvested from the edge of the cartilage lesions using an 11-gauge Jamshidi bone marrow biopsy needle.

TABLE 1. Grading of Cartilage Lesions

Grade MRI Arthroscopy

I Signal heterogeneity SofteningII Fraying, fissuring Fraying, fissuringIII Partial loss of Partial loss of

cartilage thickness cartilage thicknessIV Complete loss of Complete loss of

cartilage thickness cartilage thickness

TABLE 2. Magnetic ResonanceImaging Lesions

Grade Number of Patients

I 8II 4III 1IV 1Bone marrow edema 2

without cartilage lesionNo detectable lesion 4

A B

C D

E F

Fig 2A–F. Magnetic resonance images showing different locations and grades of cartilage lesions. (A)Proton density fat saturated fast spin echo image showing signal heterogeneity (arrow) suggestive ofa Grade I lesion in the lateral femoral condylar cartilage; (B) Three-dimensional spoiled gradient echoimage showing partial-thickness (Grade III) cartilage loss (arrow) in the anterior femoral condyle; (C)Proton density fat saturated fast spin echo image of the same section as shown in Figure 2B, high-lighting the area of bone marrow edema (arrow); (D) Proton density fat saturated fast spin echo imageshowing full-thickness (Grade IV) cartilage loss (small arrow) associated with a small area of bone mar-row edema (large arrow) in the coronal plane; (E) The same lesion as shown in Figure 2D, in sagittalsection (small arrow points to cartilage lesion, large arrow points to bone marrow edema); and (F) Largearea of bone marrow edema (arrows) not associated with any obvious overlying cartilage lesion.

Clinical OrthopaedicsS120 Colwell et al and Related Research

20). When the presence of bone marrow edemawithout overlying cartilage lesions was in-cluded, the number of MRI lesions that matchedarthroscopic findings increased to 12 (60%). Allfour cases of partial-thickness or full-thickness

cartilage loss that were seen by arthroscopy alsowere detected by MRI (although the grades didnot always concur).

Control biopsy specimens (from cadaversand femoral intercondylar notch) showed uni-form safranin O stain and a normal Mankinscore. Biopsy specimens from cartilage le-sions showed mild to moderate reduction insafranin O stain intensity especially in the su-perficial third. The Mankin score ranged from3 to 7 with a median of 4. Cells that were pos-itive for DNA fragmentation were signifi-cantly more numerous in biopsy specimensfrom lesions compared with control biopsyspecimens (Fig 4, p � 0.002).

TABLE 3. Arthroscopic Lesions

Grade Number of Patients

I 5II 7III 3IV 1No detectable lesion 4

Fig 3A–D. Arthroscopic images representative of the different grades of cartilage lesions seen areshown. (A) Grade I lesion with intact cartilage surface showing softening on probing; (B) Superficial fi-brillation seen in a Grade II lesion (arrow) in association with an acute meniscal tear that was excised;(C) Partial-thickness Grade III lesion with a flap being raised by the arthroscopic probe; and (D) Full-thickness Grade IV lesion in the femoral condyle (arrow).

A B

C D

DISCUSSION

Mature articular cartilage has poor intrinsic re-pair capability. If repair tissue forms sponta-neously, it is predominantly fibrous in natureand is lacking in biomechanical properties.Various surgical procedures currently are be-ing used for replacement or regeneration ofcartilage.1,6–8,13,14 None has been consistentlysuccessful in restoring the topographic organi-zation of normal hyaline articular cartilage,the biomechanical properties, or in integratingthe edge of the repair tissue with adjacent nor-mal cartilage. Perhaps this may explain thelack of durable long-term results.

Magnetic resonance imaging has potential asa safe and noninvasive means of detecting carti-lage lesions that may progress to degenerationand significant disability. Magnetic resonanceimaging sequences have been developed to op-timize cartilage imaging. Fast spin echo2,15,22

and fat-suppressed three-dimensional spoiled

gradient echo5,18 are established MRI tech-niques that allow excellent assessment of car-tilage lesions with a high sensitivity and speci-ficity. Between 60% and 80% of patients withMRI or arthroscopically documented cartilageinjury had cartilage degeneration develop bythe 5-year followup.9 The correlation ofarthroscopic evaluation, histologic features,and sequential MRI examination should pro-vide valuable information in defining the nat-ural history of posttraumatic cartilage degen-eration. The current authors will continue tomonitor these patients with sequential MRIscans as many as 2 years after the initialarthroscopy. In the current study, the overallcorrelation between MRI and arthroscopicfindings was positive in 50% of the patients.However, all four cases of full-thickness orpartial-thickness cartilage loss as seen onarthroscopy also were detected by MRI. Thissuggests that MRI is relatively more accuratein detecting Grade III and IV lesions. Two pa-

Number 391SOctober, 2001 In Vivo Changes After Mechanical Injury S121

Fig 4. The mean chondrocyte apoptosis in cartilage biopsy specimens is shown. Cells positive for DNAfragmentation were counted and apoptosis was quantitated as a percentage of all cells. Mean (� stan-dard deviation) percent apoptosis in biopsy specimens from cartilage lesions was significantly higherthan control biopsy specimens (p � 0.002).

tients with only bone marrow edema on MRIhad Grade I or Grade II lesions on arthro-scopy. When these patients were included inthe correlation, the overall MRI accuracy in-creased to 60%.

A high incidence of bone marrow edema onMRI has been seen in association with anteriorcruciate ligament injuries.19 Bone marrowedema seems to be an important marker ofacute injury because there was disappearanceof this signal on sequential MRI scans (at 6months and 1 year after arthroscopy, data notshown). This may serve to differentiate acutefrom chronic cartilage injury. In addition, thetwo patients who had bone marrow edemaalone on MRI were found to have Grade I orGrade II cartilage lesions on arthroscopy, sug-gesting that bone marrow edema is supportiveevidence of significant joint trauma.

The histologic findings of decreasedsafranin O stain and increased Mankin scoreprovide evidence of cartilage matrix damage.In addition, the high percentage of cells posi-tive for DNA fragmentation suggests thatchondrocytes undergo apoptosis after joint in-jury. Recent studies have reported that chon-drocytes undergo apoptosis in response towounding and injurious compression.4,10,21

The current study provides additional supportby showing apoptosis with acute joint trauma.

Cartilage cellularity varies among speciesand changes during skeletal development andaging. Only 19 cells/mm3 are present in thecartilage of young adults.17 This cell numberdecreases by approximately 75% during theaging process. A study on human cartilage ag-ing indicated that individuals older than 90years with intact knee articular cartilagesurfaces are distinguished by a significantlyincreased number of chondrocytes per tissuevolume.16 The level of cartilage cellularity de-termines the tissue volume that is being main-tained by one chondrocyte and may have pro-found implications for cartilage repair. Ittherefore may be possible to limit cartilage de-generation and promote repair if cell viabilityis maintained. This opens potential therapeutic

avenues for the treatment of posttraumatic car-tilage lesions through chondroprotection.

References1. Brittberg M, Lindahl A, Nilsson A, et al: Treatment of

deep cartilage defects in the knee with autologous chon-drocyte transplantation. N Engl J Med 331:889–895,1994.

2. Broderick LS, Turner DA, Renfrew DL, et al: Severityof articular cartilage abnormality in patients with os-teoarthritis: Evaluation with fast spin-echo MR vsarthroscopy. AJR Am J Roentgenol 162:99–103, 1994.

3. Calandruccio RA, Gilmer WS: Proliferation and re-pair of articular cartilage of immature animals. JBone Joint Surg 44A:431–455, 1962.

4. D’Lima DD, Hashimoto S, Chen PC, et al: Impact ofmechanical trauma on matrix and cells. Clin Orthop391 (Suppl):S90–S99, 2001.

5. Disler DG: Fat-suppressed three-dimensional spoiledgradient-recalled MR imaging: assessment of articu-lar and physeal hyaline cartilage. AJR Am J Roentgenol169:1117–1123, 1997.

6. Ghazavi MT, Prizker KP, Davis AM, et al: Fresh os-teochondral allografts for post-traumatic osteochon-dral defects of the knee. J Bone Joint Surg79B:1008–1013, 1997.

7. Hangody L, Kish G, Karpati Z, et al: Mosaicplasty forthe treatment of articular cartilage defects: Applica-tion in clinical practice. Orthopedics 21:751–756,1998.

8. Homminga GN, Bulstra SK, Bouwmeester PS, et al:Perichondral grafting for cartilage lesions of theknee. J Bone Joint Surg 72B:1003–1007, 1990.

9. Johnson DL, Urban WPJ, Caborn DN, et al: Articu-lar cartilage changes seen with magnetic resonanceimaging-detected bone bruises associated with acuteanterior cruciate ligament rupture. Am J Sports Med26:409–414, 1998.

10. Loening AM, James IE, Levenston ME, et al: Injuri-ous mechanical compression of bovine articular car-tilage induces chondrocyte apoptosis. Arch BiochemBiophys 381:205–212, 2000.

11. Mankin HJ: Localisation of tritiated thymidine incartilage: I. Growth in immature cartilage. J BoneJoint Surg 44 A:682–688, 1962.

12. Mankin HJ, Dorfman H, Lippiello L, et al: Bio-chemical and metabolic abnormalities in articularcartilage from osteo-arthritic human hips: II. Corre-lation of morphology with biochemical and meta-bolic data. J Bone Joint Surg 53A:523–537, 1971.

13. Meyers MH, Akeson W, Convery FR: Resurfacingof the knee with fresh osteochondral allograft. JBone Joint Surg 71A:704–713, 1989.

14. Peterson L, Minas T, Brittberg M, et al: Two- to 9-year outcome after autologous chondrocyte transplan-tation of the knee. Clin Orthop 374:212–234, 2000.

15. Potter HG, Linklater JM, Allen AA, et al: Magneticresonance imaging of articular cartilage in the knee:An evaluation with use of fast-spin-echo imaging. JBone Joint Surg 80A:1276–1284, 1998.

16. Quintero M, Mitrovic DR, Stankovic A, et al: Cellular

Clinical OrthopaedicsS122 Colwell et al and Related Research

Number 391SOctober, 2001 In Vivo Changes After Mechanical Injury S123

aspects of the aging of articular cartilage: I. Condylarcartilage with a normal surface sampled from normalknees. Rev Rhum Mal Osteoartic 51:375–379, 1984.

17. Quintero M, Mitrovic DR, Stankovic MA, et al: Cel-lular aspects of the aging of the articular cartilage: II.Condylar cartilage with fissured surface taken fromnormal and arthritic knees. Rev Rhum Mal Osteoar-tic 51:445–449, 1984.

18. Recht MP, Resnick D: MR imaging of articular car-tilage: Current status and future directions. AJR AmJ Roentgenol 163:283–290, 1994.

19. Rosen MA, Jackson DW, Berger PE: Occult osseous

lesions documented by magnetic resonance imagingassociated with anterior cruciate ligament ruptures.Arthroscopy 7:45–51, 1991.

20. Rudel T: Caspase inhibitors in prevention of apopto-sis. Herz 24:236–241, 1999.

21. Tew SR, Kwan AP, Hann A, et al: The reactions ofarticular cartilage to experimental wounding: Role ofapoptosis. Arthritis Rheum 43:215–225, 2000.

22. Yao L, Gentili A, Thomas A: Incidental magneti-zation transfer contrast in fast spin-echo imagingof cartilage. J Magn Reson Imaging 6:180–184,1996.