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The Journal of Bone and Joint Surgery 79:1452-63 (1997)
© 1997 The Journal of Bone and Joint Surgery, Inc.

The Effect of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) on the Healing of Full-Thickness Defects of Articular Cartilage*

RANI S. SELLERS, D.V.M.{dagger}, DIANE PELUSO, H.T.L.{dagger} and ELISABETH A. MORRIS, D.V.M.{dagger}, CAMBRIDGE, MASSACHUSETTS

Investigation performed at Genetics Institute, Cambridge


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Articular cartilage has a limited capacity for repair. We investigated the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2) on the healing of full-thickness osteochondral defects in adult New Zealand White rabbits. A single defect, three millimeters wide by three millimeters deep, was created in the trochlear groove of the right femur in eighty-nine rabbits. The defect was either left empty, filled with a plain collagen sponge, or filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at four, eight, or twenty-four weeks, and the repair tissue was examined histologically and evaluated with use of a grading scale. The defects also were examined immunohistochemically for the presence of type-II collagen at four and eight weeks. The rate of bone repair was evaluated with fluorescent labeling of bone at two and four weeks and with use of fluorescence microscopy at eight weeks. Treatment with rhBMP-2 greatly accelerated the formation of new subchondral bone and improved the histological appearance of the overlying articular surface. At twenty-four weeks, the thickness of the repair cartilage was 70 per cent that of the normal adjacent cartilage and a new tidemark usually had formed between the repair cartilage and the underlying subchondral bone. The average total scores on the histological grading scale were significantly better (p < 0.01) for the defects treated with rhBMP-2 than for the untreated defects (those left empty or filled with a plain collagen sponge) at all time-points. Immunostaining with an antibody against type-II collagen showed the diffuse presence of this cartilage-specific collagen throughout the repair cartilage in the treated defects. The untreated defects demonstrated minimum staining with this antibody. CLINICAL RELEVANCE: The operative removal of cartilage damaged as a result of trauma or focal osteoarthrosis is of little value because of the limited capability of articular cartilage to repair. If the damaged cartilage were to be removed and the tissue were induced to heal, the self-perpetuating process of osteoarthrosis might be prevented. We describe the capacity of rhBMP-2 to accelerate the healing of full-thickness defects of articular cartilage and to improve the histological appearance and biochemical characteristics of the repair cartilage. These improvements were evident as long as twenty-four weeks postoperatively in adult rabbits. Because of the technical simplicity of delivering a recombinant protein growth factor compared with transplanting cells, and because of the improvement in healing afforded by rhBMP-2, the use of this growth factor and related proteins to influence the healing of defects of articular cartilage should be investigated further.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The limited capacity of articular cartilage to repair after injury has led to the investigation of new therapeutic methods to improve osteochondral regeneration. Because subchondral bone, bone marrow, synovial cells, chondrocytes, and synovial fluid all contribute to the healing of defects of articular cartilage, the use of animal models rather than in vitro models is essential both to understand the process of repair and to assess the value of new therapeutic regimens.

Partial-thickness defects—that is, defects restricted to the substance of articular cartilage—do not heal spontaneously18,26. To elicit repair, a full-thickness defect must be created by penetration into the marrow spaces of subchondral bone2,6,7,11,14,26,36,37. This allows the defect to be populated with undifferentiated, marrow-derived mesenchymal cells that may differentiate into new subchondral bone and overlying cartilage. However, the resulting fibrocartilage does not resemble the original cartilage either biochemically or biomechanically and it ultimately deteriorates1,4,10-13,15,27,37,40. Attempts recently have been made to develop new methods to enhance the repair of full-thickness defects, particularly with use of transplanted mesenchymal stem cells, perichondrium, or chondrocytes implanted either alone or in combination with biodegradable implants1,3-5,10,12,15-17,30,38,39,42-44. A simpler approach would involve the use of a growth factor in a biodegradable matrix; however, this method has received little attention.

Bone morphogenetic proteins (BMPs) are characterized as members of the TGF-ß (transforming growth factor-ß) superfamily because they have seven highly conserved carboxyl-terminal cysteines. BMP-2 through BMP-7 have been found to be present in extracts of demineralized bone8,33,46. Recombinant human bone morphogenetic protein-2 (rhBMP-2) appears to be intimately involved with the growth and differentiation of mesenchymal cells to chondroblasts and osteoblasts in developing limb-buds25,33. However, increasing evidence has indicated that BMPs are important in many aspects of the differentiation and proliferation of cells in embryogenesis, suggesting a multifunctionality that is influenced by target cells and environment20,21,25. In adults, rhBMP-2 has been shown to induce the expression of cartilage and bone markers in vitro19,41 and the formation of cartilage and bone at ectopic and skeletal sites in vivo45,47. Bone morphogenetic proteins also have been shown to enhance the production of articular cartilage matrix in vitro without inducing the formation of bone24,34,35. These findings prompted us to investigate the ability of rhBMP-2 to modulate the repair of full-thickness defects of articular cartilage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ninety-three eight-month-old female New Zealand White rabbits were anesthetized with an intramuscular injection of ketamine (Fort Dodge, Fort Dodge, Iowa) at a dosage of eighty milligrams per kilogram of body weight and xylazine (Fermenta, Kansas City, Missouri) at a dosage of eight milligrams per kilogram. The right knee joint was approached through a medial parapatellar incision. One full-thickness defect, three millimeters wide by three millimeters deep, was drilled through the articular cartilage into bleeding subchondral bone in the trochlear groove of the femur with use of a low-speed dental drill (Kavo American, East Zurich, Illinois) equipped with a diamond-impregnated stainless-steel burr (Brasseler USA, Savannah, Georgia). In twenty-nine rabbits the defect was left empty, in thirty-two rabbits the defect was filled with a collagen sponge (bovine type-I collagen; Integra Life Sciences, Plainsboro, New Jersey) onto which a buffer (thirty-millimolar L-glutamic acid, 2.5 per cent glycine, 0.5 per cent sucrose, and 0.01 per cent Tween 80, pH 4.5) had been lyophilized, and in thirty-two rabbits the defect was filled with the same type of collagen sponge onto which five micrograms of rhBMP-2 (in the same buffer) had been lyophilized. The collagen sponge is commercially available for use as a hemostatic device. Before each sponge was implanted, it was cut to the approximate size of the defect. The left limb served as the control in each rabbit.

Penicillin G procaine (Fort Dodge), at a dosage of 40,000 international units per kilogram, was administered twenty-four hours preoperatively, at the time of the operation, and at forty-eight hours postoperatively. The rabbits were allowed unrestricted movement in their cages immediately after recovery from anesthesia.

Processing and Evaluation of Tissue
The rabbits were killed with an injection of pentobarbital sodium (Anpro Pharmaceutical, Arcadia, California) at four, eight, or twenty-four weeks, and the defects were photographed and examined grossly. The area of the defect was cut out of the bone, and the defects that were to be examined histologically were fixed in 10 per cent neutral buffered formalin (Fisher Scientific, Pittsburgh, Pennsylvania), decalcified in S/P decalcifying solution (Baxter Healthcare, McGaw, Illinois), and embedded in paraffin. Serial sections were cut transversely, from the proximal aspect to the distal aspect of the trochlear groove. Four-micrometer-thick sections from the center of each defect were stained with toluidine blue and safranin O-fast green22 and were evaluated with use of a histological grading system (to be described). Only sections from the center of the defect were graded in order to ensure unbiased analysis and to allow comparison among specimens studied at different time-points. This area also was chosen because it provides the most stringent test of healing capability, as the least amount of cartilage and bone-healing consistently was found in sections taken from the middle of the defect.

The sections were evaluated blindly by two of us (R. S. S. and E. A. M.) with use of a histological grading scale modified from that of Pineda et al.31 and Wakitani et al.43. The scale was designed to reduce observer bias, to identify subtle changes during repair, and to allow comparisons between standardized studies. Grading was done with use of a section taken from the middle of the defect. The total score on the grading scale ranges from 0 points (normal cartilage) to 31 points (no repair tissue). The modified scale allowed for the evaluation of all relevant aspects of repair of a full-thickness defect of articular cartilage (Table I). Some categories were designed for the evaluation of the entire defect (category 1, filling of the defect relative to the surface of the normal adjacent cartilage; and category 5, architecture within the entire defect, not including the margins). One category (category 7) addressed the repair of subchondral bone, with 100 per cent replacement signifying complete regeneration of subchondral bone to the level of the original tidemark. There also were categories for the evaluation of the repair of the articular cartilage (category 3, staining of the matrix; and category 4, cellular morphology) and for the evaluation of specific aspects of repair (category 2, integration of repair tissue with surrounding cartilage; category 6, architecture of the surface; and category 8, formation of a tidemark).


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  		TABLE I MODIFIED HISTOLOGICAL GRADING SCALE31,43 FOR DEFECTS OF ARTICULAR CARTILAGE

 
When the calculation of a percentage was involved (as for the scores in categories 1, 4, 7, and 8), a reticle was used within the eyepiece of the microscope. In category 1, 100 per cent filling of the defect meant that new tissue filled the entire area of the defect (nine square millimeters) and extended to the level of the original joint surface. In category 4, the percentage of new cartilage that demonstrated organization of chondrocytes into vertical columns in the radial zone was calculated by dividing the width of the portion of tissue that demonstrated such columns by the total width of the repair tissue (three millimeters). In category 7, the percentage of new subchondral bone was calculated by measuring the area beneath the tidemark that was now occupied by new bone. In category 8, the formation of the tidemark was determined by dividing the width of the portion of the defect that had a new tidemark by the original width of the defect (three millimeters).

The architecture within the defect (category 5) was graded by determining if there were any voids within the repair tissue that were not connected to the surface (with the score dependent on the size and number of voids) or if there were large clefts and fissures associated with a collapsed joint surface.

The total scores as well as the scores for each category were compared among the experimental groups. Statistical analysis of the total scores was performed with use of the Student t test.

Preparation of Tissue for Immunohistochemical Analysis
Immunostaining for type-II collagen was done at four weeks (four defects that had been treated with rhBMP-2 and four that had not) and at eight weeks (five defects that had been treated and four that had not). Tissue that was to be evaluated immunohistochemically was fixed in 4 per cent paraformaldehyde in phosphate-buffered saline solution (pH 7.4) for twenty-four hours at 4 degrees Celsius, decalcified in 10 per cent (weight per volume) EDTA in phosphate-buffered saline solution for eighteen days, and embedded in paraffin. The sections then were deparaffinized in xylene, passed through decreasing gradations of ethanol, washed in phosphate-buffered saline solution, and submerged for thirty minutes in methanol with 0.3 per cent hydrogen peroxide to remove endogenous peroxidase activity. Next, the sections were washed again in phosphate-buffered saline solution and incubated for thirty minutes in a blocking buffer (phosphate-buffered saline solution with 10 per cent mouse serum). The sections then were incubated in primary polyclonal goat anti-bovine type-II collagen IgG (Chemicon, Temecula, California), in a 1:10 dilution of blocking buffer, for three hours at room temperature. The sections were washed in phosphate-buffered saline solution, and biotin-conjugated anti-goat IgG (Pierce, Rockford, Illinois) in a 1:500 dilution of phosphate-buffered saline solution was applied for one hour. After another wash in phosphate-buffered saline solution, the sections were incubated for thirty minutes with an avidin-biotin-peroxidase reagent (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, California) and then washed again. Diaminobenzidine substrate (Vector Laboratories) then was applied. Sections that were to serve as controls were treated either with goat serum in blocking buffer or with a secondary antibody alone under the same protocol. The control sections then were washed in water and mounted with Permount (Fisher Scientific).

Labeling of Bone
Bone was labeled at two and four weeks in nine rabbits (six that had an untreated defect and three that had a treated defect) for evaluation of the onset and extent of early repair of subchondral bone. At two weeks, the metabolic bone marker calcein (Sigma Chemical, St. Louis, Missouri) (ten milligrams per kilogram of body weight) in phosphate-buffered saline solution (final pH, 7.4) was injected subcutaneously on two consecutive days. At four weeks, oxytetracycline (Butler, West Falls, Massachusetts) (twenty-five milligrams per kilogram of body weight) was administered subcutaneously on three consecutive days. The rabbits were killed at eight weeks. Tissue was fixed in 4 per cent paraformaldehyde and was embedded undecalcified in methylmethacrylate. Serial sections, ten micrometers thick, were cut through the defect and examined with use of fluorescence microscopy. A 380 to 425-nanometer filter (V2A; Nikon, Melville, New York) was used to examine the incorporation of both oxytetracycline and calcein, and a 450 to 490-nanometer filter (B2E; Nikon) was used to examine the incorporation of calcein alone.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Four rabbits were removed from the protocol because of postoperative complications: two had an empty defect, one had a defect that was filled with a plain collagen sponge, and one had a defect that was treated with rhBMP-2.

Macroscopic Findings
Before tissue was fixed for histological examination, each defect was examined grossly to assess the appearance of the repair cartilage relative to that of the normal adjacent cartilage. The gross appearance of the empty defects did not differ from that of the defects filled with a plain collagen sponge, so both are referred to as untreated defects. At four weeks, the gross appearance of the defects that had been treated with rhBMP-2 was similar to that of the untreated defects: all were primarily white and somewhat irregular. By eight weeks, the treated defects were noticeably smoother than the untreated defects, which generally had a raised and irregular surface. The surface of the treated defects was pink and tan, whereas the surface of the untreated defects was white and tan. At twenty-four weeks, the color and texture of the repair cartilage in the treated defects were similar to those of the normal adjacent cartilage. The color of the untreated defects varied from white to tan to purple, and the texture ranged from irregular to smooth.

Histological Findings

Healing of Subchondral Bone
At four and eight weeks, in both the empty defects and the defects that had been filled with a plain collagen sponge, a large portion below the original tidemark had been replaced with cartilage rather than with new subchondral bone (Figs. 1-A and 2-A). The percentage of new subchondral bone was significantly greater (p < 0.01) at all time-points in the defects treated with rhBMP-2 (Table II). At four and eight weeks, there was an average of 88 and 100 per cent new subchondral bone in the defects treated with rhBMP-2, an average of 59 and 62 per cent in the defects filled with a plain collagen sponge, and an average of 58 and 65 per cent in the empty defects. By twenty-four weeks, there was an average of 130 per cent new subchondral bone in the defects treated with rhBMP-2, an average of 88 per cent in the defects filled with a plain collagen sponge, and an average of 100 per cent in the empty defects. The thickness of the repair cartilage in the treated defects was an average of 70 per cent that of the normal cartilage.



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  		Figs. 1-A and 1-B: Photomicrographs of full-thickness defects of articular cartilage, showing repair at four weeks (toluidine blue, x 40). r = repair cartilage and b = new subchondral bone. The arrows denote the margins of the defect. Fig. 1-A: Section of a defect filled with a plain collagen sponge, demonstrating fibrillation of the surface, fissuring through the repair cartilage, and incomplete replacement of subchondral bone.

 


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  		Figs. 2-A and 2-B: Photomicrographs of full-thickness defects of articular cartilage, showing repair at eight weeks (toluidine blue, x 40). r = repair tissue, b = new subchondral bone, and c = normal adjacent cartilage. The arrows denote the margins of the defect. Fig. 2-A: Section of a defect filled with a plain collagen sponge, demonstrating incomplete replacement of subchondral bone as well as disruption of the surface of the fibrous or fibrocartilaginous repair tissue.

 

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  		TABLE II REPAIR OF SUBCHONDRAL BONE IN FULL-THICKNESS DEFECTS OF ARTICULAR CARTILAGE

 

Repair of Articular Cartilage in Untreated Defects
At four weeks, the histological appearance of the empty defects was identical to that of the defects filled with a plain collagen sponge (Fig. 1-A). Gaps or discontinuities between the repair tissue and the adjacent cartilage were rare. However, the tissue at the edges of the defect was hypocellular, with areas of cloning in the normal cartilage adjacent to the defect. Safranin O-fast green staining was evident predominantly in the area of hypertrophic chondrocytes at the base of the defect. The repair tissue consisted of a mixture of hypercellular fibrous tissue and fibrocartilage. Ten of the twenty defects had deep clefts extending from the surface into the center of the repair tissue, and twelve of the twenty defects had substantial disruption of the surface (a score of 3 points in category 6). No tidemark was evident in any specimen.

The histological features of the defects studied at eight weeks were similar to those of the defects studied at four weeks (Fig. 2-A). Eleven of the eighteen defects had a gap or lack of continuity between the repair tissue and the adjacent articular cartilage on one or both sides. This finding indicates a decrease in the integration of repair tissue with the surrounding articular cartilage compared with that seen at four weeks. Nine of the eighteen defects demonstrated clefts and fissures in the fibrous and fibrocartilaginous repair cartilage as well as severe fibrillation of the surface. Safranin O-fast green staining of the matrix was always evident in the region of hypertrophic cartilage; staining within the defect was variable depending on the type of repair tissue. Initiation of a tidemark was not observed in any defect.

At twenty-four weeks, the empty defects could not be differentiated histologically from the defects filled with a plain collagen sponge and both types of defect demonstrated extreme variability in the extent of repair (Figs. 3-A, 3-B, 4-A, 4-B, and 4-C). Eleven of the twenty defects had a gap or lack of continuity between the repair tissue and the adjacent articular cartilage on one or both sides. Safranin O-fast green staining was similar to that seen at four and eight weeks. Ten of the twenty defects demonstrated repair cartilage consisting predominantly of fibrous tissue, and the other ten were filled with disorganized fibrocartilage. Twelve defects had clefts and fissures extending into the repair cartilage, and fourteen had severe disruption of the surface. Nine of the twenty demonstrated formation of a tidemark over more than 75 per cent of the defect.



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  		Figs. 3-A through 3-D: Photomicrographs of full-thickness defects of articular cartilage, showing repair at twenty-four weeks (safranin O-fast green, x 40 [Figs. 3-A and 3-C] and x 200 [Figs. 3-B and 3-D]). r = repair cartilage, b = new subchondral bone, and c = normal adjacent cartilage. The arrows denote the margins of the defect. Figs. 3-A and 3-B: Section of a defect filled with a plain collagen sponge.

 


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  		Figs. 3-A and 3-B: Section of a defect filled with a plain collagen sponge.

 


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  		Figs. 4-A through 4-F: Photomicrographs of full-thickness defects of articular cartilage, demonstrating the animal-to-animal variation in repair at twenty-four weeks (toluidine blue, x 40). Figs. 4-A, 4-B, and 4-C: Sections of defects filled with a plain collagen sponge.

 


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  		Figs. 4-A, 4-B, and 4-C: Sections of defects filled with a plain collagen sponge.

 


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  		Figs. 4-A, 4-B, and 4-C: Sections of defects filled with a plain collagen sponge.

 

Repair of Articular Cartilage in Treated Defects
At four weeks, the defects that had been treated with rhBMP-2 were completely filled with cells and matrix. Gaps or discontinuities at the margin were rare (Fig. 1-B). The margins of the repair tissue were hypocellular, with regions of clusters of chondrocytes. Such clusters also were seen in the normal cartilage adjacent to the repair tissue. Safranin O-fast green staining of the matrix was evident primarily in the region of hypertrophic chondrocytes at the base of the defect. The repair tissue consisted primarily of hypercellular fibrocartilage filled with round cells. No defect had clefts or fissures in the repair cartilage or evidence of a tidemark.



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  		Fig. 1-B Section of a defect treated with rhBMP-2, demonstrating uninterrupted repair cartilage and accelerated replacement of subchondral bone.

 
The repair tissue in the defects studied at eight weeks appeared similar to that in the defects studied at four weeks (Fig. 2-B). Three of the twelve defects demonstrated a gap or lack of continuity at one interface. Safranin O-fast green staining was similar to that seen at four weeks. The repair cartilage was predominantly fibrocartilage, with 25 per cent of the cartilage appearing hyaline-like. No defect had severe fibrillation of the surface, although slight disruptions were evident.



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  		Fig. 2-B Section of a defect treated with rhBMP-2, demonstrating complete replacement of subchondral bone below undisrupted repair cartilage.

 
At twenty-four weeks, the defects were completely healed and the over-all histological appearance of the repair tissue was similar to that of normal cartilage and bone (Figs. 3-C, 3-D, 4-D, 4-E, and 4-F). Four of the ten defects demonstrated a gap or lack of continuity between the repair tissue and the surrounding cartilage, whereas the other six demonstrated integration of the margins with the normal surrounding cartilage. Safranin O-fast green staining was evident throughout the repair cartilage. The intensity of the staining in the repair tissue was slightly or moderately reduced in seven of the defects and was substantially reduced in three compared with that in the normal adjacent cartilage. Most of the repair cartilage appeared hyaline-like, with five defects demonstrating vertical columns of chondrocytes in the radial zone (a typical feature of normal articular cartilage). The architecture within the defect was normal in seven of the defects. Eight of the defects had only mild fibrillation of the surface, usually at the margins. The thickness of the repair cartilage was 70 per cent that of the normal adjacent cartilage, and a complete tidemark was evident in all but one defect.



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  		Figs. 3-C and 3-D: Section of a defect treated with rhBMP, demonstrating a complete cartilage layer and healing of the subchondral bone. Note the formation of the tidemark (Fig. 3-D).

 


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  		Figs. 3-C and 3-D: Section of a defect treated with rhBMP, demonstrating a complete cartilage layer and healing of the subchondral bone. Note the formation of the tidemark (Fig. 3-D).

 


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  		Figs. 4-D, 4-E, and 4-F: Sections of defects treated with rhBMP-2. The histological appearance was consistently improved by treatment with rhBMP-2.

 


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  		Figs. 4-D, 4-E, and 4-F: Sections of defects treated with rhBMP-2. The histological appearance was consistently improved by treatment with rhBMP-2.

 


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  		Figs. 4-D, 4-E, and 4-F: Sections of defects treated with rhBMP-2. The histological appearance was consistently improved by treatment with rhBMP-2.

 

Grading
The average total scores on the histological grading scale were significantly better (p < 0.01) for the defects treated with rhBMP-2 than for the untreated defects (Table III): the scores at four, eight, and twenty-four weeks were 12.5, 12.4, and 8.6 points for the treated defects compared with 18.4, 18.7, and 18.1 points for the defects filled with a plain collagen sponge and 19.4, 18.6, and 17.1 points for the empty defects. With the numbers available, we could not detect a significant difference between the total scores for the defects filled with a plain collagen sponge and those for the empty defects. At all time-points, the treated defects had better scores for cellular morphology (category 4), architecture within the defect (category 5), and architecture of the surface (category 6). Additionally, at twenty-four weeks, the scores for safranin O-fast green staining of the matrix (category 3) and formation of the tidemark (category 8) were better for the defects treated with rhBMP-2.


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  		TABLE III SCORES ON THE MODIFIED HISTOLOGICAL GRADING SCALE31,43

 

Results of Immunohistochemical Analysis
At four weeks, the untreated defects demonstrated variable levels of immunoreactivity to type-II collagen, ranging from completely negative to partially immunoreactive; the levels were much lower than those in the normal adjacent cartilage. At eight weeks, the untreated defects showed improvement but variable staining throughout, depending on the type of repair tissue (Fig. 5-A). For example, no immunoreactivity to type-II collagen was evident in tissue that was primarily fibrous. In contrast, immunoreactivity to type-II collagen was demonstrated throughout the defects treated with rhBMP-2, at levels approximately the same as those in the normal adjacent cartilage. At eight weeks, the defects treated with rhBMP-2 demonstrated immunoreactivity to type-II collagen at levels greater than those seen at four weeks (Fig. 5-B); in some defects, the staining of the repair cartilage could not be differentiated from that of the normal adjacent cartilage. The intensity and distribution of immunostaining was consistently greatest in the defects in which the healing of subchondral bone was more advanced.



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  		Figs. 5-A and 5-B: Photomicrographs of defects that were immunostained for type-II collagen at eight weeks (x 200). r = repair tissue and c = normal adjacent cartilage. Fig. 5-A: Section of a defect filled with a plain collagen sponge, showing variable areas of type-II collagen in the repair tissue.

 


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  		Fig. 5-B Section of a defect treated with rhBMP-2, demonstrating diffuse type-II collagen throughout the repair tissue.

 

Labeling of Bone
There was no difference between the empty defects and the defects filled with a plain collagen sponge with regard to the incorporation of calcein or oxytetracycline, so both are referred to as the untreated defects. The incorporation of calcein was evident over a larger area in the defects treated with rhBMP-2 than in the untreated defects (Figs. 6-A and 6-C). The incorporation of oxytetracycline was evident throughout most of the new subchondral bone, indicating that most bone repair had taken place between two and four weeks (Figs. 6-B and 6-D). However, the highest point of incorporation of oxytetracycline in the defects treated with rhBMP-2 was consistently closer to the joint surface, reflecting the accelerated replacement of subchondral bone.



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  		Figs. 6-A through 6-D: Fluorescent photomicrographs of full-thickness defects of articular cartilage at eight weeks. Filters to demonstrate incorporation of calcein (which had been injected at two weeks) were used for Figs. 6-A and 6-C, and filters to demonstrate incorporation of oxytetracycline (which had been injected at four weeks) were used for Figs. 6-B and 6-D. Incorporation of calcein is hardly evident in the defect filled with a plain collagen sponge (Fig. 6-A) and is evident over one-eighth of the base of the defect treated with rhBMP-2 (Fig. 6-C). Incorporation of oxytetracycline is considerably increased in the treated defect (Fig. 6-D) compared with the defect filled with a plain collagen sponge (Fig. 6-B), indicating accelerated repair of subchondral bone between two and four weeks.

 


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  		Figs. 6-A through 6-D: Fluorescent photomicrographs of full-thickness defects of articular cartilage at eight weeks. Filters to demonstrate incorporation of calcein (which had been injected at two weeks) were used for Figs. 6-A and 6-C, and filters to demonstrate incorporation of oxytetracycline (which had been injected at four weeks) were used for Figs. 6-B and 6-D. Incorporation of calcein is hardly evident in the defect filled with a plain collagen sponge (Fig. 6-A) and is evident over one-eighth of the base of the defect treated with rhBMP-2 (Fig. 6-C). Incorporation of oxytetracycline is considerably increased in the treated defect (Fig. 6-D) compared with the defect filled with a plain collagen sponge (Fig. 6-B), indicating accelerated repair of subchondral bone between two and four weeks.

 


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  		Figs. 6-A through 6-D: Fluorescent photomicrographs of full-thickness defects of articular cartilage at eight weeks. Filters to demonstrate incorporation of calcein (which had been injected at two weeks) were used for Figs. 6-A and 6-C, and filters to demonstrate incorporation of oxytetracycline (which had been injected at four weeks) were used for Figs. 6-B and 6-D. Incorporation of calcein is hardly evident in the defect filled with a plain collagen sponge (Fig. 6-A) and is evident over one-eighth of the base of the defect treated with rhBMP-2 (Fig. 6-C). Incorporation of oxytetracycline is considerably increased in the treated defect (Fig. 6-D) compared with the defect filled with a plain collagen sponge (Fig. 6-B), indicating accelerated repair of subchondral bone between two and four weeks.

 


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  		Figs. 6-A through 6-D: Fluorescent photomicrographs of full-thickness defects of articular cartilage at eight weeks. Filters to demonstrate incorporation of calcein (which had been injected at two weeks) were used for Figs. 6-A and 6-C, and filters to demonstrate incorporation of oxytetracycline (which had been injected at four weeks) were used for Figs. 6-B and 6-D. Incorporation of calcein is hardly evident in the defect filled with a plain collagen sponge (Fig. 6-A) and is evident over one-eighth of the base of the defect treated with rhBMP-2 (Fig. 6-C). Incorporation of oxytetracycline is considerably increased in the treated defect (Fig. 6-D) compared with the defect filled with a plain collagen sponge (Fig. 6-B), indicating accelerated repair of subchondral bone between two and four weeks.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings of the present study demonstrate the capacity of five micrograms of rhBMP-2 to accelerate and improve the repair of full-thickness defects of articular cartilage. A commercially available type-I collagen sponge was used as a delivery matrix for rhBMP-2. There was no substantial difference at any time-point between the empty defects and the defects filled with a plain collagen sponge with regard to the histological appearance or the score on the histological grading scale. As early as four weeks after the defect had been created, the repair cartilage in the treated defects was morphologically and biochemically superior to that in the untreated defects. The repair cartilage in the treated defects lacked fissures and consisted of chondrocytes embedded in a matrix containing type-II collagen; these characteristics were not found in the untreated defects. The presence of type-II collagen at four weeks indicated that the new chondrocytes were synthesizing appropriate matrix components, and the repair cartilage therefore could be classified as hyaline-like. In addition, the treated defects were almost completely filled with new woven bone and lamellar bone at four weeks. The untreated defects had much less new subchondral bone at that time. At eight weeks, the levels of immunoreactivity to type-II collagen were nearly normal in the treated defects and there was almost complete replacement of the subchondral bone. The cartilage and subchondral bone in the untreated defects began to degenerate at twenty-four weeks, but they continued to improve in the defects treated with rhBMP-2. At twenty-four weeks, the defects that had been treated with rhBMP-2 were filled with minimally disrupted hyaline and hyaline-like cartilage. All but one of the treated defects had a continuous tidemark without hypertrophic chondrocytes, indicating cessation of endochondral ossification. To our knowledge, we are the first to report consistent success for as long as six months after the use of a recombinant protein to repair an osteochondral defect.

Although the morphological features of the repair cartilage and the composition of the matrix in the treated defects were superior to those in the untreated defects, the integration of the repair tissue with the normal adjacent cartilage was not better in the treated compared with the untreated defects. Other investigators have commented that the lack of integration of repair tissue with adjacent cartilage is unrelated to the method of treatment or the size of the defect4,10,12,13,29,30,32,39,43,44. Integration at the margins is a difficult problem to overcome because chondrocytes in normal cartilage are not involved in repair and cannot migrate14,37. The ultimate success of repair cartilage may require its integration with the surrounding articular cartilage in order to maintain biomechanical integrity. Some investigators have reported that better integration at the interface is associated with enzymatic treatment of the defect at the time of the operation30,37. Treatment of the edges of the exposed cartilage may enhance cellular adhesion by removing matrix factors that inhibit such adhesion as well as by enhancing integration of the matrix18,28. Enzymatic treatment of the full-thickness defects before the insertion of the collagen sponge (with or without rhBMP-2) did not yield an advantage in our study (unpublished results).

The production of matrix by chondrocytes and cartilage explants has been shown to be increased in vitro after treatment with BMPs23,34,35. This activity may have played a role in the improved characteristics of the matrix that were seen in the repair cartilage of the treated defects in the present study. In addition, the biomechanical stability afforded by the acceleration of subchondral-bone formation in the defects treated with rhBMP-2 may be a determining factor for improved repair. It has been found that rhBMP-2 is a potent inducer of endochondral ossification44, and subchondral bone was rapidly replaced in the treated defects. Bone repair was initiated earlier (as indicated by the amount of calcein incorporated at two weeks) and continued more rapidly (as indicated by the amount of oxytetracycline incorporated at four weeks) in the treated compared with the untreated defects. It is interesting to note that most of the new-bone formation took place between two and four weeks after the defect had been created. Histologically, there was a significantly greater (p < 0.01) percentage of new subchondral bone at four and eight weeks in the treated compared with the untreated defects. Although the new bone tended to be somewhat sclerotic initially, remodeling of the subchondral bone over twenty-four weeks resulted in lamellar bone with an appearance similar to that of normal subchondral bone. Histological and immunohistochemical analysis demonstrated a consistent positive relationship between an accelerated rate of repair of subchondral bone and an improvement in the morphological features of cartilage with increased production of type-II collagen. This finding suggests that the accelerated repair of subchondral bone provided support to the overlying cartilage, allowing normal production of matrix and preventing fissuring of the cartilage as a result of biomechanical instability.

The ability of rhBMP-2 to induce the formation of bone was an initial concern with regard to its use for the repair of articular cartilage. We wondered if treatment with rhBMP-2 would result in the formation of new subchondral bone extending to the joint surface without replacement of the articular cartilage. Although the repair cartilage in the treated defects was 30 per cent thinner than normal cartilage, the tidemark was replaced and endochondral bone formation had ceased. This thinning of the repair cartilage cannot be attributed to the activity of rhBMP-2. Shapiro et al.37 studied the repair of untreated osteochondral defects and demonstrated that the thickness of repair cartilage was half that of the normal adjacent cartilage at twenty-four weeks. Hence, the thinning of cartilage in osteochondral defects in rabbits is a normal finding and was not altered by the presence of rhBMP-2. The ability of the joint to maintain cartilage at its surface even after exposure to rhBMP-2 may be the result of many things, including biomechanical forces as well as factors related to synovial tissue or cartilage, such as the recently discovered cartilage-derived morphogenetic proteins9,23.

Several investigators have noted the considerable animal-to-animal variation in the repair of articular cartilage10,13,15,16,30,37. Dahlberg and Kreicbergs13 concluded that it was difficult to evaluate the regeneration of cartilage because healing varied within and between animals despite identical treatment. This variability necessitates a detailed, objective method of histological analysis that will obviate both subjective bias and the presentation of only selected examples of anticipated results. A detailed histological grading system with consistent parameters of measurement allows for the comparison of results within and between studies. Although many investigators have evaluated the repair of full-thickness defects of articular cartilage, only a few have used a detailed grading system4,43,44. The grading scale used in the present study allowed for an objective analysis of the healing of the defects. It also enabled the comparison of experimental groups by individual categories, thereby allowing identification of the component of tissue repair that was most affected by the treatment. Ultimately, the use of a computerized imaging system to analyze defects could provide the most unbiased presentation of data.

Precise identification of the location of sections examined within the defect is also essential for critical evaluation. Because defects repair centripetally, the center of the defect is always the last area to heal and is most often the site of fissures and collapse of bone and cartilage repair tissue. This area of the defect is therefore most appropriate for comparative evaluation. In the present study, scores were determined only for histological sections taken from the middle of the defect, thereby increasing the stringency of the analysis.

A cytokine-based therapy for damaged cartilage would be more clinically useful and efficient than cell-based therapies, which involve removal of autologous cells derived from marrow43 or from cartilage3-5,10,15,17,35,42 followed by expansion in culture and then by a second operation for implantation into the defect. A single operation in which a cytokine is used to elicit repair of cartilage would substantially expedite the treatment process as well as reduce costs and decrease the morbidity associated with the operation and rehabilitation. The ability of rhBMP-2 to consistently accelerate and improve cartilage repair in full-thickness osteochondral defects emphasizes its promise as a practical and important candidate for cartilage repair.

NOTE: The authors are grateful for the operative assistance of T. Haire and L. Grosser, the histological expertise of G. Jolly, and the contribution of A. Gaskin.


   Footnotes
 
*One or more of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding source was Genetics Institute.

{dagger}Genetics Institute, 87 Cambridge Park Drive, Cambridge, Massachusetts 02140. E-mail address for Dr. Morris: emorris@genetics.com.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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