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THE CLASSIC: Healing of Articular Cartilage in Intra-Articular Fractures in Rabbits

Mitchell, Nelson; Shepard, Nora

Section Editor(s): Olson, Steven A MD, Guest Editor; Marsh, J Lawrence MD, Guest Editor

Clinical Orthopaedics and Related Research: June 2004 - Volume 423 - Issue - p 3-6
doi: 10.1097/01.blo.0000132632.38338.c2
SECTION I: SYMPOSIUM: Articular Fractures

Abstract: Intra-articular fractures were created in the distal end of the femur in adult rabbits. Fractures were then reduced in three different fashions: incomplete reduction, adequate reduction without compression of the fragments, and ideal reduction, which consisted of reduction with strong compression of the fracture fragments. Animals were killed at intervals ranging from seven weeks to one year, and the cartilage fractures were studied by light, transmission, and scanning electron microscopy, using proteoglycan stains.

Cartilage fractures that were inadequately reduced or were adequately reduced without compression healed by fibrocartilage only. Fractures that were reduced with compression across the fragments healed with a tissue which, by light and electron microscopy, appeared to be hyaline cartilage, which showed the collagen and proteoglycan orientation and relationships of normal hyaline cartilage. The healing of these defects appears to be the result of a proliferation of cells emanating from the region of the tidemark, and perhaps of migration of chondrocytes from the surface into the lesion. We postulate that compression of the cartilage surfaces either creates a physical environment that allows certain chondrocytes to heal the defect with hyaline cartilage, or, by coapting the surfaces, prevents ingrowth of granulation tissue from the subchondral bone that might interfere with repair by hyaline cartilage.

This study suggests a hitherto unappreciated capacity for hyaline cartilage to repair structural defects.

Clinical Relevance: Accurate reduction of intra-articular fractures with interfragmentary compression may create the environment whereby cartilage cells may repair the cartilage fracture—a capacity not previously thought possible in adult cartilage. The repair of cartilage defects provides obvious benefits to the patient.

(Reprinted with permission from Mitchell N, Shepard N: Healing of Articular Cartilage in Intra-Articular Fractures in Rabbits. J Bone Joint Surg 62A: 628–634, 1980.)

Correspondence to: Clinical Orthopaedics and Related Research, 3550 Market Street, Suite 220, Philadelphia, PA 19104. E-mail:

Dr. Mitchell (Fig 1) was born in 1934 in Grandby, Quebec, Canada. He attended the Bishop’s College School in Lennoxville before enrolling in McGill University in Montreal. He received his B.A. degree in 1955 and his medical degree in 1959. He pursued his surgical training at the Royal Victoria Hospital in Montreal and in 1964, he spent a year as an orthopaedic research resident. After a year as an orthopaedic resident at the Shriner’s Hospital in Montreal he was awarded a traveling fellowship which took him to the Nuffield Orthopedic Centre in Oxford. He also received an A.B.C. Exchange Fellowship to Great Britain.

Fig 1.

Fig 1.

After entering practice in Montreal, Mitchell continued to carry on research and was very active in the Orthopaedic Research Society, serving as secretary-treasure for 4 years before being elected president of the society in 1984. In 1979, Mitchell was made Professor of Orthopaedic Surgery at McGill University. From 1992 to 1999 he served as the Associate Dean of the Faculty of Medicine at McGill University.

A Diplomat of the American Board of Surgery and a member of the American Academy of Orthopaedic Surgeons and the American Orthopaedic Association, Mitchell holds membership in many other orthopaedic groups. His many publications reflect a continuing interest in research and clinical problems.

Leonard F. Peltier, MD, PhD

The repair of defects in the structure of articular cartilage depends on the maturity of the animal and the type of defect. Several authors have shown that the healing of incomplete defects does not occur in mature animals and occurs only rarely in immature animals.1–4 We have recently shown that repair of full-thickness defects—those created by drill holes through denuded femoral condyles into the subchondral bone—occurs by ingrowth of granulation tissue from the marrow. Initially this tissue undergoes metaplasia to form hyaline cartilage, but later it becomes fibrocartilage and one year later it appears somewhat degenerated.5 What, then, is the potential for cartilage repair in the full-thickness defects created by intra-articular fractures, when the fracture has been ideally reduced and immobilized? We created intra-articular fractures in mature rabbits and contrasted the results of cartilage repair in those that were ideally reduced and immobilized with those that had less than ideal reductions. The results of this study constitute the substance of this report.

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Intra-articular fractures were created in the distal medial femoral condyle in twenty-eight mature rabbits. Following the creation of this consistently reproducible lesion, each fracture was reduced and immobilized with an AO navicular lag screw. In some of the fractures the screw was tightened only sufficiently to approximate the fracture fragments. In others strong compression was applied to the fragments, as the dense cortical nature of rabbit femoral bone makes it possible to achieve rather large compression forces in these fractures. Postoperatively, the rabbits were returned to normal cage activity and quickly regained full use of their limbs.

Groups of four to six animals were killed at intervals from seven weeks to twelve months following fracture. When they were killed, the joints were photographed and specimens were prepared as follows. The fractured distal end of the femur was removed and fixed un 2 per cent buffered glutaraldehyde containing either 0.2 per cent ruthenium red or 0.1 per cent toluidine blue 0, as described previously.6,7 The fractured femur was then decalcified in 4.13 per cent EDTA, pH 7.4;8 trimmed into small blocks containing the fracture; rinsed in buffer; and fixed in 2 per cent osmium tetroxide containing either 0.05 per cent ruthenium red or 0.025 per cent toluidine blue 0. The ruthenium red-fixed cartilage was additionally stained with p-phenylenediamine during the dehydration phase, as we have reported previously.7 During embedding in Spurr resin, the blocks were oriented so that full-thickness sections of the fracture could be cut for visualization by either light or electron microscopy. The sections to be studied by transmission electron microscopy were also stained with uranyl acetate and lead citrate and were examined with a Phillips 400 transmission electron microscope.

Several joints were removed at six and twelve months, fixed, critically point-dried, coated with gold-palladium, and studied with a Phillips scanning electron microscope.

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Seven weeks after reduction without compression, a broad area of whitish tissue could be seen filling the defect between the fracture fragments. By light microscopy, it was noted that the subchondral bone had united but the defect between the cartilaginous surfaces was filled by highly cellular tissue. Although many of these cells had the appearance of chondrocytes by light microscopy, their electron microscopic appearance was that of fibrocytes. The cells were stellate in appearance, the organization of collagen was irregular, proteoglycan was deficient about them, and there were no cytoplasmic footlets. Seven weeks after reduction of fractures with maximum compression, the cartilage fracture was barely visible on inspection of the joint. Histologic examination revealed that the hyaline cartilage defect had been united by a cluster of cells whose maximum activity arose from the tidemark and by migration of cells at the surface of the cartilage into the fracture. The electron microscopic examination of the cells at this time, however, suggested that fibrochondrocytes rather than hyaline chondrocytes were filling the defect.

On inspection of the joint three months after reduction without compression, a similar whitish material could be seen occupying the site of the old cartilage fracture. By both light and electron microscopy, this material had the appearance of fibrocartilage. However, three months after reduction and strong compression, no sign of the old fracture line could be seen on the cartilaginous surface. Histologically, the subchondral bone was healed and the cartilage matrix defect appeared to have repaired, with some residual cellular activity noted just above the tidemark. At the surface of the defect, elongated cells similar to those of normal hyaline cartilage were found. Deeper in the matrix, at the junction of the old cartilage matrix with the new reparative tissue, only a thin seam could be seen separating the new and the old material. Higher magnification of the area showed that the collagen pattern on both sides of this seam appeared similar. The ruthenium-red staining of the proteoglycan showed similar proteoglycan and collagen relationships in both new and old matrix.

Six months after reduction with maximum compression, an examination of the joint surface again showed new evidence of the old fracture line. By scanning electron microscopy, the surface of the defect showed a small depression, the floor of which was filled with collagenous bundles running tangentially in the same direction as the normal cartilage surface. Transmission electron microscopy revealed the surface to be intact, and confirmed that the orientation of the collagen was the same as that of normal cartilage. An examination of the deeper zones of the defect revealed that the collagen bundles, although they were somewhat less compact in the repair cartilage, were oriented in a similar direction toward the surface. The ruthenium and proteoglycan stain demonstrated fine proteoglycan precipitates attached to the collagen fibers6,7 in both the repair matrix and the normal cartilage. Six-month specimens from fractures that had been reduced without compression were similar to the three-month specimens.

Of the four animals that were killed at twelve months, three showed an excellent repair of the cartilage. Scanning electron microscopy showed the defect as either a small heaped-up area or a small depressed area, with continuity between the new and the old matrix. Transmission electron microscopy demonstrated a healed cartilage defect with a small seam of reparative tissue extending from the area of the tidemark to the surface. Within this seam and adjacent to it were healthy-appearing chondrocytes. In the region of calcified cartilage, below the tidemark, the impression was that cells had divided and migrated upward in the defect, carrying small traces of the tidemark with them. Higher magnification of the area of cartilage repair demonstrated a normal-appearing relationship between collagen and proteoglycan.

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At all stages of this study of mature animals, compression of the fracture fragments appeared to consistently produce a better repair of the cartilage articular surface than did reduction and immobilization without compression. In the latter fractures a fibrocartilaginous material appeared to fill the gap and there was little evidence that this had converted to hyaline cartilage. In the specimens that had had strong compression, a considerable amount of repair appeared to be taking place as early as seven weeks. The reparative tissue appeared to arise from activity in cells at the region of the tidemark. By three, six, and twelve months following compression of the fragments, both the matrix in the cartilage fracture and the old matrix outside it exhibited similar morphologic patterns of collagen orientation and collagen-proteoglycan relationships. In addition, cells in the defect had the morphologic appearance of hyaline chondrocytes. Although on occasion a small depression was seen in the surface by both light and scanning electron microscopy, in all successful instances of cartilage healing there was continuity of tangential orientation of matrix at the surface of the defect.

In our previous study, in which holes were drilled in the subchondral bone, a tissue grew into the drill-holes and initially repaired the defect in a fashion similar to that seen in the fractures in this study that were not rigidly compressed.5 In that study, hyaline cartilage appeared initially, which then became fibrocartilage and eventually fibrillated.

It must be assumed that the compression of the fracture surfaces in this study is only temporary, and that these surfaces are not under continuous load for any significant period of time following the tightening of the screw. How, then, can compression mediate the changes described, and induce apparent hyaline cartilage repair? Two possibilities arise: first, forceful compression may so coapt the surfaces as to seal the subchondral bone and prevent the ingrowth of granulation tissue that was seen in the experiments in which drill-holes were created.5 The absence of this granulation tissue and the initial force exerted by compression may produce a physical environment that induces chondrocytes at the tidemark to divide and migrate into the defect. Evidence for such migration perhaps is scanty, although there were suggestions that the cells as they migrated up into the defect were carrying along with them wisps of the electron-dense, amorphous material seen at the tidemark. The second possibility is that the physical environment created by compression may stimulate chondrocytes in the normal cartilage, close to the area of the fracture, to also participate in the repair. However, evidence for the migration of these cells into the defect was not seen.

It was previously thought that structural defects could not be repaired in mature cartilage. This report suggests that under certain conditions mature articular cartilage may undergo satisfactory repair. The only variable in this experiment was the degree of compression across the fracture surfaces. In what way this compression produces the proper biologic requirements for repair is unknown. However, the possible relationship between the degree of compression of the surfaces of a cartilage defect and the quality of the repair of that defect may have important implications, not only for intra-articular fractures but also for other disease states involving articular cartilage.

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1. Bennett GA: Bauer, Walter; and Maddock, S.J.: A Study of the Repair of Articular Cartilage and the Reaction of Normal Joints of Adult Dogs to Surgically Created Defects of Articular Cartilage, “Joint Mice” and Patellar Displacement. Am. J. Pathol. 8:499–524, 1932.
2. Calandruccio, R.A., and Gilmer, W.S., Jr.: Proliferation, Regeneration, and Repair of Articular Cartilage of Immature Animals. J. Bone and Joint Surg., 44-A:431–455, April 1962.
3. Campbell CJ: The Healing of Cartilage Defects. Clin. Orthop. 64:45–63, 1969.
4. Mankin H.J.: Localization of Tritiated Thymidine in Articular Cartilage of Rabbits. II. Repair in Immature Cartilage. J. Bone and Joint Surg., 44-A:688–698, June 1962.
5. Mitchell, Nelson, and Shepard, Nora: The Resurfacing of Adult Rabbit Articular Cartilage by Multiple Perforations through the Subchondral Bone. J. Bone and Joint Surg., 58-A:230–233, March 1976.
6. Shepard, Nora, and Mitchell, Nelson: Simultaneous Localization of Proteoglycan by Light and Electron Microscopy using Toluidine Blue 0. A Study of Epiphyseal Cartilage. J. Histochem. and Cytochem., 24:621–629, 1976.
7. Shepard, Nora, and Mitchell, Nelson: The Use of Ruthenium Red and p-Phenylenediamine to Stain Cartilage Simultaneously for Light and Electron Microscopy. J. Histochem. and Cytochem., 25:1163–1168, 1977.
8. Warshawsky, H, and Moore, G.A.: Technique for the Fixation and Decalcification of Rat Incisors for Electron Microscopy. J. Histochem. and Cytochem., 15:542–549, 1967.
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