Positive Effect of Alendronate on Subchondral Bone Healing and Subsequent Cartilage Repair in a Rabbit Osteochondral Defect Model

Abstract

Background: Cartilage and subchondral bone have recently been considered an osteochondral unit. The treatment of osteo-chondral lesions is still challenging, but better subchondral bone repair may result in higher quality repaired cartilage.

Hypotheses: Alendronate accelerates bone formation in osteochondral defects and affects the quality of the repaired cartilage.

Study Design: Controlled laboratory study.

Methods: Osteochondral defects were made on the left trochleas of 50 rabbits, which were assigned to 1 of 3 groups: control, ALN (weekly subcutaneous injection of 0.14 mg/mL alendronate), and ALN-S (alendronate injection in the first 8 weeks only). They were evaluated at 4, 8, 24, and 52 weeks. Bone repair was evaluated with microcomputed tomography and histologic evaluation. Cartilage repair was evaluated with ultrasound and histologic analyses.

Results: At 4 weeks, the defects were filled, and cartilage-like repair tissue was observed in the ALN group, whereas the defects were incompletely filled in the control group. Alendronate treatment enhanced early bone formation and mineralization in the osteochondral defect for the first 8 weeks. The continuous injection of alendronate for 24 weeks resulted in delayed bone remodeling, but the rabbits in the ALN-S group showed good integrity of the subchondral bone plate, without delayed remodeling. At 52 weeks, the ALN-S group had a columnar arrangement of chondrocytes that had less fibrillation and looked superior to those in the ALN and control groups. Ultrasound analysis showed better quality of repaired cartilage of the ALN and ALN-S group than the control group.

Conclusion: Alendronate accelerated bone formation without inhibiting its mineralization but thereafter inhibited bone remodeling in an osteochondral defect. The withdrawal of alendronate at 8 weeks avoided the delayed remodeling and showed better sub-chondral bone repair. At 52 weeks, better subchondral bone repair resulted in better cartilage quality.

Clinical Relevance: Alendronate administered in the early period accelerates bone formation and improves the quality of the repaired cartilage.

Keywords

cartilage repair subchondral bone repair remodeling osteochondral defect bisphosphonate

The capacity of cartilage defects to heal is limited, and the treatment of chondral or osteochondral defects is still challenging. Autologous osteochondral transplantation,²¹ autologous chondrocyte transplantation,⁴ or marrow stimulation techniques³¹ are used to treat these lesions. These treatments for focal chondral lesions have achieved acceptable results, but the treatment of large, deep osteochondral defects has not shown promising results.

It has been proposed that the failure of subchondral bone maintenance or restoration contributes to the failure of cartilage-forming transplants.²⁴ The increased failure rate of autologous chondrocyte transplantation after marrow stimulation has recently been reported.²² Researchers argue that osteoarthritis is initiated focally and that the quality of the subchondral bone affects the results of autologous chondrocyte transplantation. Although autologous osteochondral transplantation is now a widespread potent procedure, alterations in the subchondral bone sometimes result in a failure to heal.²² Recent studies in which scaffolds were used to treat osteochondral defects have shown that better repair of the subchondral bone improves the mechanical properties of the repaired cartilage.²⁹ The quality of the subchondral bone is critically important in the treatment of cartilage.

In osteoarthritis, the subchondral plate and subchondral cancellous bone contribute greatly to the initiation of osteoarthritis and its progression because increased subchondral bone density results in overloading of and damage to the cartilage.²⁸ Hayami et al¹¹ reported that alendronate suppressed subchondral bone remodeling and had a significant chondroprotective effect in a rat model of secondary osteoarthritis. By contrast, Ding et al⁶ reported that alendronate significantly increased the subchondral bone mass, greatly changed the microarchitecture, increased the bone mineral content and density, and led to accelerated articular cartilage degeneration in a guinea pig model of spontaneous osteoarthritis. Although the effect of alendronate on cartilage in osteoarthritis is still controversial, its effect on the subchondral bone undoubtedly has some sort of indirect effect on the overlying articular cartilage. By improving or maintaining the quality of the subchondral bone, it is possible that the cartilage quality improves during cartilage repair.

Bisphosphonate can potentially inhibit bone loss and increase bone mineralization. It is used clinically for the treatment of the hypercalcemia of malignancy, osteoporosis, Paget disease, and bone metastases. Alendronate is a widely used aminobisphosphonate and powerfully inhibits bone resorption without affecting mineralization.² In a fracture healing model, alendronate enhanced fracture healing with large and early callus formation, with no inhibition of bone mineralization.^8,30 Although bisphosphonate promotes callus formation during fracture healing in the long bone, to the best of our knowledge, there is no report that bisphosphonate affects subchondral bone repair in a defect in an articular joint.

The purpose of this study was to show that better subchondral bone repair entails better cartilage repair. We tested the hypotheses that alendronate accelerates subchondral bone repair in a rabbit model of osteochondral defect and that this accelerated subchondral bone repair has a positive effect on the subsequent cartilage repair.

Materials and Methods

Animals

This investigation was approved by the Animal Research Center of our university. Fifty skeletally mature male Japanese White rabbits (age, 6 months; body weight, 3.0–3.5 kg) were used. Five rabbits were randomly assigned to 1 of 10 groups defined by the time point at which the animals were evaluated (4, 8, 24, and 52 weeks after surgery) and by the type of treatment (Table 1). The rabbits were assigned to the control group, the alendronate (ALN) group, or the alendronate-saline (ALN-S) group. The rabbits in the control group received weekly subcutaneous injections of saline. The rabbits in the ALN group were treated with alendronate (0.14 mg/kg per week, given subcutaneously). The rabbits in the ALN-S group, which was derived from the ALN group defined in 24- and 52-week groups, were given ALN injections for the first 8 weeks and saline injections for the remaining periods. The injections were commenced just after the surgical creation of the defect. The animals were acclimated for 2 weeks before the start of the experiments. They were housed, 1 per cage, in a room maintained at 22°C and 50% humidity, with a 14 h/10 h light/dark cycle, and were allowed food and water ad libitum.

Table 1

Number of Each Group^a

	4W	8W	24W	52W
Control	5 (5)	5 (5)	5 (5)	5 (4)
ALN	5 (5)	5 (5)	5 (5)	5 (5)
ALN-S	ND	ND	5 (5)	5 (4)

ALN, alendronate; ALN-S, alendronate-saline; ND, not done. Final evaluated number is indicated in parentheses.

Operation

Intravenous pentobarbital sodium (25 mg/kg body weight) was used to induce and maintain general anesthesia. An intra-articular injection of 3 mL of 1% lidocaine was given. A parapatellar incision was made to expose the left knee joint, and the patella was laterally dislocated. An osteochondral defect was made on the left trochlea of the knee. A full-thickness cylindrical osteochondral plug (6 mm in diameter, 7 mm in depth), which went through the articular surface and into the subchondral bone, was harvested using the Osteochondral Autograft Transfer System (OATS, Arthrex, Naples, Florida). The harvested plug was transplanted to the right knee for use in another study. The joint capsule and skin incision were closed with nylon sutures, and the osteochondral defect was left untreated. The rabbits were allowed full weightbearing postoperatively. The animals were killed with an intravenous injection of pentobarbital sodium (100 mg/kg body weight) and were evaluated macroscopically by 2 orthopaedic surgeons.

Serum Deoxypyridinoline

Peripheral blood was collected before surgery and at 4 and 8 weeks after surgery. The blood remained at room temperature for about 30 minutes and was then centrifuged at 2000g for 15 minutes at 4°C. The serum samples were separated and stored at –80°C until analysis. The quantity of deoxypyridinoline was measured with an enzyme-linked immunosorbent assay (Pyrilinks-D kit, Metra Biosystems Inc, Mountain View, California).

Ultrasound Evaluation

The ultrasonic measurement system, which has been described previously, provided a quantitative method of assessing the properties of the tissues.^14,16,23,25 In brief, the transducer was about 3 mm in diameter, and the central frequency of the ultrasonic signal was 10 MHz. In the examination of the cartilage, 2 large-amplitude groups of reflected waves were observed (Figure 1). One was from the cartilage surface and the other from the subchondral bone. On the wavelet map, the maximum magnitude of the first wave reflected from the articular cartilage surface was defined as the signal intensity. It reflected the compressive stiffness of the cartilage and was proportional to the aggregate modulus of the cartilage.^9,15 We have previously reported the reproducibility of this system, which was confirmed by the coefficients of variance. Twenty repeated measurements established that the coefficient of variance was 0.9% for signal intensity.¹⁷ Previous studies have shown that articular cartilage degeneration results in reduced signal intensity.^10,15 The repair tissue on the osteochondral defect was evaluated using this system, and each tissue was measured twice.

Bone Morphologic Analysis

Bone morphologic analysis was performed for the 4-, 8-, and 52-week animals. Bone mineral density (BMD) in the area near the osteochondral defect site of the femoral chondyle was measured with a dual-energy X-ray absorptiometer (DXA, model QDR-2000; Hologic Inc, Waltham, Massachusetts). The scan field size was 1.78 × 1.02 cm, and the resolution was 0.0254 × 0.0127 cm². A region of interest (ROI) was determined as the area that was a 0.83 × 1.02 cm rectangle including the defect site (0.6 cm). Bone mineral density was measured with software for high-resolution analysis (Hologic Inc). The CV for the measurement of the BMD of standard samples by this technique was 0.8%.

The microfocused X-ray computed tomography (microCT) system (model NX-CP-C80H-IL) was developed by Nittetsu ELEX Co Ltd (Osaka, Japan). The device has a maximum experimental spatial resolution of 2.5 μm and a minimum slice thickness of 5 μm for measurements of a ceramic standard sample. The fan beam of microCT has a small focal spot size, 3 μm in diameter, a conventional microfocus open vacuum-type X-ray source, rotating sample stage, 1024 linear response image sensors, and image intensifier (image sensors). A cross-sectional tomogram for each specimen was obtained with a slice thickness of 50 μm and reconstructed at 512 × 512 pixels, by use of an acceleration voltage of 55 kV and a current of 0.1 mA. To evaluate the microarchitecture of trabecular bone near the defect site, the center of the slice line was determined as the center of the defect circle area. Bone volume fraction percentage (bone volume [BV]/total volume [TV]) of the osteochondral defect site was quantified.

Histologic Analysis

The specimens were fixed in 10% neutral-buffered formalin, decalcified in Morse solution (10% sodium citrate and 22.5% formic acid), and embedded in paraffin. Sagittal sections (6 μm thick) were cut, stained with safranin O/fast green (safranin O), hematoxylin and eosin (HE), and Masson trichrome, and then examined histologically with a light microscope and a polarized light microscope. The International Cartilage Repair Society (ICRS) Visual Histological Assessment Scale²⁰ (Appendix) was used by 2 evaluators, who were blinded to the treatment groups, to score the repaired cartilage. The number of multinucleate cells was defined as the number of giant cells immediately adjacent to the bone that had more than 3 nuclei. The multinucleate cell number was counted blindly in 5 arbitrary fields at the edge of the osteochondral defect at 100× magnifications.

Statistical Analysis

Data of the histologic score were shown as mean ± SE. Other data were shown as mean ± SD. The histologic scores were analyzed statistically using the nonparametric Kruskal-Wallis test. Other data were analyzed using parametric ANOVA with post hoc comparisons. P < .05 was deemed to indicate statistical significance.

Results

One rabbit from the 52-week control group and 1 rabbit from the 52-week ALN-S group were excluded because of femoral fracture. The final numbers evaluated are shown in Table 1. No other rabbits died or showed significant loss of body weight during the evaluation periods.

Confirmation of Effects of Alendronate

Serum deoxypyridinoline levels were significantly reduced by treatment with alendronate. The serum deoxypyridinoline of the control group at 8 weeks was 15.9 ± 3.0 nM, and that of the ALN group at 8 weeks was 10.5 ± 0.8 nM (P = .032). The BMD of the femoral chondyle was significantly increased in the ALN group at 8 weeks. At 52 weeks, the ALN group had significantly higher BMD than that of the control group, but the ALN-S group had almost similar BMD to that of the control group (Table 2).

Table 2

Effects of Alendronate on Bone Mineral Density of Femoral Condyle in the Rabbit Full-Thickness Defect Model^a

BMD (g/cm²)	4 Weeks	8 Weeks	52 Weeks
Control	0.0309 ± 0.0074	0.0308 ± 0.0071	0.0490 ± 0.0048
ALN	0.0262 ± 0.0030	0.0433 ± 0.0072^b	0.0621 ± 0.0031^b
ALN-S	ND	ND	0.0515 ± 0.0063

BMD, bone mineral density; ALN, alendronate; ALN-S, alendronate-saline; ND, not done.

P < .01 to control group.

Macroscopic Appearances

The macroscopic appearances of the defects differed dramatically after 4 weeks (Figure 2). At 4 weeks, hemorrhage or red tissue was observed in the defect in all rabbits in the control group. In the ALN group, white opaque tissue was observed in most parts of the defect in all rabbits, with no hemorrhage. Although some red tissue remained at the edges of the defect in the 4-week ALN group, the macroscopic appearance was very different from that of the control group.

Histologic Study for Bone Repair

Histologic study for subchondral bone showed early bone repair in the ALN group (Figure 3). At 4 weeks, the defects in the control group were filled with connective tissue and were still below the level of the subchondral plate. By contrast, the defects in the ALN group showed new bone formation and were filled with repair tissue up to the level of the subchondral plate. At 8 weeks, the defects in the controls were filled with patchy tissue mixed with connective tissue and newly formed bone. In the ALN group, the newly formed bone containing thick subchondral plate, with small bone lacunae and less trabeculae, reached the level of native subchondral plate. At 24 weeks, the continuity of the subchondral bone was still imperfect, and the amount of newly formed bone was still small in the control group. In the ALN group, the thick subchondral plate lacking trabecular structure and remaining thick cartilage tissue indicated delayed remodeling. In the ALN-S group, the repaired subchondral plate was continuous with the native subchondral plate, and remodeling was seen. Under the subchondral plate, a bone mass was observed that was just being remodeled and included connective tissue and multinucleate cells in the bone lacunae. At 52 weeks, the newly formed bone was remodeled in all 3 groups; they seemed no different from each other.

We counted the multinucleate cells at the borders of the bone regeneration areas at 4 and 8 weeks. There were significantly fewer multinucleate cells in the ALN group at 4 weeks (5.5 ± 1.0) than in the control group (12.8 ± 1.7) (P = .033). At 8 weeks, there were also fewer multinucleate cells in the ALN group (5.8 61.4) than in the control group (10.2 ± 2.6), but the difference was not significant (P = .108).

MicroCT

The mineralization of the newly formed bone at the defect site was delayed in the control group (Figure 4). At 4 weeks, soft tissue was seen in the defect, but only a little bone formation was observed, and the trabecular network was absent in both groups. In the 8-week control group, newly formed bone was only seen at the edges of the defects. Conversely, in the ALN group, mineralized bone repair was observed extending from the bottoms and sides of the defects. The newly formed bone appeared as an immature thick bone mass and had no trabecular network. At 52 weeks, the newly formed bone in the defect had a trabecular network, and remodeling was observed in all 3 groups. The BV/TV of the 8-week ALN group (27.1 ± 7.7) was greater than that of the control group (18.1 ± 3.3) (P = .029). There was no significant difference in BV/TV in other periods (data not shown).

Ultrasound Analysis

Ultrasound analysis indicated better mechanical properties in the ALN and ALN-S groups (Figure 5). Signal intensity continued to increase in the first 8 weeks in both the control and ALN groups. The signal intensity of the ALN group also continued to increase up to 24 and 52 weeks. The signal intensity of the ALN-S group had declined slightly at 24 weeks but was restored almost to the level of the ALN group at 52 weeks. The signal intensity of the control group did not increase after 8 weeks. The signal intensity of the ALN and ALN-S groups at 4, 24, and 52 weeks was significantly higher than that of the control group.

Histologic Study for Cartilage Repair

Histologic study of the cartilage showed early cartilage repair in the ALN group and hyaline cartilage–like repair in the ALN-S group (Figure 6). In the 4-week control group, the defect was not completely filled, and little cartilage-like tissue was seen. The ALN group had cartilage-like tissue on the repaired bone tissue without subchondral bone plate. There was a layer of hypertrophic chondrocytes and dense safranin O staining in the deep layer of the cartilage-like tissue.

In the 8-week control group, mixed tissue containing hypercellular cartilage–like mass with dappled safranin O staining and hypocellular fibrous tissue was observed. In the ALN group, the major type of tissue was fibrocartilage-like tissue, but it contained a slight columnar arrangement, and the cartilage looked more mature. The border between the cartilage-like tissue and the subchondral bone became obscure.

At 52 weeks, thin cartilage tissue was observed, and its thickness did not appear different in the 3 groups, but the good repaired tidemark was seen in the ALN and ALN-S group. The repair tissue of the control group consisted totally of fibrocartilage-like tissue. The repair tissue of the ALN group consisted partially of hyaline-like cartilage, which had a columnar arrangement of chondrocytes, but mostly of fibrocartilage-like tissue with fibrillated surface. The ALN-S group had a hyaline cartilage–like columnar arrangement of chondrocytes. Safranin O staining in the ALN-S group looked slightly denser than in the other groups but was less dense than in normal cartilage.

ICRS Visual Histological Assessment Scale

We evaluated the repaired osteochondral unit with the ICRS Visual Histological Assessment Scale (Table 3). Criteria II indicated that the matrix of the ALN group was superior to that of the control group at 4 and 8 weeks. At 24 weeks, the ALN-S group had a significantly better score than the control group. As for cell distribution, the 4-week ALN group had a better score than the control group. As indicated in the histology, the cartilage of the ALN-S group had columnar distribution, and the score of the ALN-S group was better at 24 and 52 weeks, but there was a statistical difference only at 24 weeks. The early subchondral bone repair in the ALN group was confirmed by the statistical significance of criteria V at 4 weeks. The scores of criteria V and VI were gradually increased, indicating the maturation of subchondral bone and calcified cartilage.

Table 3

International Cartilage Repair Society (ICRS) Visual Histological Assessment Scale^a

	I. Surface	II. Matrix	III. Cell Distribution	IV. Cell Population Viability	V. Subchondral Bone	VI. Cartilage Mineralization
4W
Control	0.3 ± 0.3	0.5 ± 0.3	0.5 ± 0.2	2.7 ± 0.3	0.5 ± 0.2	0.6 ± 0.4
ALN	1.2 ± 0.6	1.6 ± 0.1^b	1.5 ± 0.0^b	3.0 ± 0.0	1.2 ± 0.1^b	0.9 ± 0.4
8W
Control	0.6 ± 0.6	0.6 ± 0.2	1.0 ± 0.4	2.4 ± 0.6	0.8 ± 0.4	0.6 ± 0.4
ALN	0.9 ± 0.6	1.5 ± 0.2^b	1.7 ± 0.1	2.9 ± 0.1	1.4 ± 0.3	0.9 ± 0.6
24W
Control	1.5 ± 0.7	0.5 ± 0.2	0.4 ± 0.2	2.2 ± 0.2	2.1 ± 0.2	2.1 ± 0.6
ALN	1.8 ± 0.7	1.4 ± 0.2	1.5 ± 0.3	2.6 ± 0.4	2.0 ± 0.3	2.1 ± 0.4
ALN-S	2.1 ± 0.4	1.8 ± 0.1^b	1.7 ± 0.1^b	3.0 ± 0.0	2.4 ± 0.1	3.0 ± 0.0
52W
Control	1.5 ± 0.9	1.1 ± 0.4	0.8 ± 0.3	2.1 ± 0.3	2.3 ± 0.3	2.3 ± 0.8
ALN	2.1 ± 0.6	1.5 ± 0.4	1.5 ± 0.4	2.7 ± 0.3	2.5 ± 0.3	2.7 ± 0.3
ALN-S	2.3 ± 0.8	2.4 ± 0.4	2.3 ± 0.3	3.0 ± 0.0	2.9 ± 0.1	3.0 ± 0.0

Mean ± standard error. ALN, alendronate; ALN-S, alendronate-saline.

P < .05 to control group.

Discussion

We tested the hypotheses that alendronate accelerates subchondral bone repair in a rabbit model of osteochondral defect and that the accelerated subchondral bone repair positively affects the subsequent cartilage repair. In this study, we have shown that alendronate accelerated the subchondral bone repair in the early stage of the process, but continuous administration of alendronate inhibited subchondral bone remodeling. We have also shown that the early subchondral bone repair and its maintenance in appropriate condition resulted in better cartilage quality after 52 weeks.

This study has shown that new bone formation was accelerated with alendronate. When we considered the macroscopic appearances of the tissues at 4 weeks, the ALN group showed dramatically early repair compared with that in the control group, which still displayed hemorrhage. At 4 weeks, new bone formation was initiated in the ALN group, whereas it was observed only at the bottoms and edges of the defects in the control group. The newly formed bone could not be detected with microCT, indicating that the repaired subchondral bone lacked mineralization and was at the stage of woven bone. At 8 weeks, the mineralization of the newly formed subchondral bone was initiated. MicroCT showed the mineralization of the newly formed bone in the ALN group, but it was observed only at the edges of the defects in the control group. Thus, ALN accelerated bone formation without inhibiting its mineralization. At this time point, ALN had a significant positive effect on the subchondral repair of an osteochondral defect.

At 24 weeks, remodeling of the newly formed subchondral bone was seen in the ALN-S group, which had ceased alendronate treatment at 8 weeks, and the subchondral plate was continuous with the surrounding native subchondral bone and looked completely healed. However, remodeling was delayed in the ALN group, which underwent continuous treatment for 24 weeks. Cartilage tissue that had not changed to bone tissue remained, and a thick subchondral bone mass without trabecular structure was present. In the control group, no delayed remodeling was seen, but the subchondral plate was thin, and the trabeculae looked loose compared with the surrounding native trabeculae. The main effect of alendronate acts through the inhibition of osteoclast and macrophage activity.³ In our study, multinucleate cell numbers were significantly reduced in the ALN group relative to those in the control group, and we consider that alendronate inhibited the remodeling of the cartilage tissue and the newly formed bone by inhibiting osteoclast and macrophage activity, so that the bone mass remained below the subchondral bone plate in the ALN group. In the period from 8 weeks to 24 weeks, continuous injections of alendronate had a negative effect on subchondral bone repair by inhibiting bone remodeling. However, at 52 weeks, the subchondral bone repair was complete in all 3 groups.

Several studies have shown that early callus formation during healing of fractures is induced by bisphosphonate. Using a murine model, Gerstenfeld et al⁸ showed that alendronate increases the callus volume and bone mineral content without inhibiting mineralization and enhances the stiffness and strength of the fractured bone. Similar results have been reported in canine, sheep, and rat models treated with alendronate or other bisphosphonates.^5,18, ²⁷ Consistent with these studies, our study showed the acceleration of new bone formation in an osteochondral defect of an articular joint. By contrast, another study showed that alendronate inhibits osteoblast activity. Bisphosphonate delays new bone formation by inhibiting osteoblast activity in vivo¹² and dose-dependently inhibits osteoblast function in vitro.²⁶ In our study, weekly subcutaneous injection of rabbits with 0.14 mg/kg alendronate had no such effect. A number of previous reports have also suggested that continuous treatment with bisphosphonate delays the remodeling of the large callus or cartilaginous tissue at the fracture site. To examine this problem, we prepared an ALN-S group in which treatment with alendronate was discontinued at 8 weeks. Because a previous report indicates alendronate inhibits bone remodeling at 16 weeks in a dog fracture model,²⁷ we decided to test the cessation of alendronate earlier, at 8 weeks. At 24 weeks, the remodeling of the newly formed bone was delayed in the ALN group, whereas the ALN-S group exhibited remodeled subchondral bone. Thus, the effects of alendronate differed depending on the time of its administration. Alendronate accelerated new bone formation in the first 8 weeks but impaired remodeling in the following 16 weeks. Therefore, alendronate has both positive and negative effects on the repair of an osteochondral defect. To the best of our knowledge, this is the first study to describe the effects of bisphosphonate on enchondral ossification in an osteochondral defect in an articular joint or to show that alendronate has a beneficial effect in the early stages of such repair.

In cartilage repair, the early repair of the subchondral bone seems to have a positive effect on the repaired cartilage tissue. Together with the accelerated subchondral bone repair, cartilage-like tissue repair was seen at 4 weeks in the ALN group, although it was very immature cartilage, with randomly arranged hypercellular tissue, including partially hypertrophic chondrocytes. At 8 weeks, the cartilage-like tissue of the ALN group was more matured. The major type of tissue was fibrocartilage-like tissue, but it contained a slight columnar arrangement, like that of native cartilage. The cartilage-like tissue of the 8-week control group was very thick and patchy, with hypercellular tissue and hypocellular fibrous tissue. At 52 weeks, 2 of the 4 rabbits in the ALN-S group had a columnar arrangement of chondrocytes. There was continuous regeneration of the tidemark and less fibrillation at the surface. In the ALN group, there was mixed tissue, with mostly fibrocartilage-like tissue and with partially hyaline cartilage–like tissue. The surface of the repaired cartilage had fibrillation, and there were few chondrocytes in the surface layer. The cartilage of the control group was a fibrocartilage-like tissue, and there was no columnar arrangement of the chondrocytes.

At 52 weeks, the cartilage of the ALN group looked inferior to that of the ALN-S group, which we attributed, at least partly, to the delayed subchondral bone remodeling. Some reports have indicated that the thickening of the subchondral bone increases the internal cartilage stress, leading to increased hardening of the subchondral bone (sclerosis) and resulting in the degeneration of the articular cartilage.^6,28 With delayed remodeling, the newly formed bone remained as a bone mass, resulting in thick subchondral bone at 24 weeks in the ALN group. By contrast, the subchondral bone looked similar to the surrounding native bone in the ALN-S group. The BMD of the 52-week ALN group was significantly higher than that of the other groups. Ding et al⁶ recently reported that alendronate increases the subchondral bone density and promotes the progression of osteoarthritis. The degenerative changes in the osteochondral unit are possibly the effects of the thick subchondral bone mass and increased BMD. Although the cartilage of the ALN-S group looked superior to that of the other 2 groups, the cartilage was thin, with low safranin O staining compared with that of native cartilage. The subchondral bone was protuberant, and the tidemark had advanced, which may correspond to the thinning of the articular cartilage. The thinning cartilage and the advanced tidemark make the cartilage more susceptible to damage and further degeneration.¹

The ALN and ALN-S groups showed higher signal intensity than did the control group. Previous studies have shown that degenerated cartilage displays lower signal intensity,^10,15 so high signal intensity indicates less degenerated cartilage. The signal intensity reflects the compressive stiffness of the cartilage and is proportional to the aggregate modulus of the cartilage.^9,15 The signal intensity of the cartilage gradually increased. The cartilage in the early periods had more cells and less matrix, and we consider that the increase in signal intensity reflected the maturation of the cartilage matrix. At 52 weeks, the signal intensities of the ALN-S and ALN groups were higher than those of the control group. In this study, the differences in the signal intensities of the 3 experimental groups at 52 weeks indicated a better quality of matrix in the repaired cartilage of the ALN-S and ALN groups.

In this study, the direct effect of alendronate on chondrocyte was not considered. Alendronate acts by inhibiting osteoclast and macrophage activity, and it is not thought to act directly on cartilage. We showed that the use of alendronate in the early stages of repair resulted in superior cartilage quality, and we propose that this result was a secondary effect of the early and improved subchondral bone repair. However, we did not investigate whether alendronate has a direct effect on cartilage. Recently, several studies have shown a chondroprotective effect of bisphosphonate, with antioxidant potential,⁷ and its prevention of dexamethasone-induced growth retardation and apoptosis.³² There is a little possibility that alendronate directly affects chondrocytes and influences the cartilage matrix.

This study has provided some new insights but also has some limitations. First, the number of animals in each group was limited, although we obtained statistically significant results with 5 rabbits in each group. Most unfortunately, the numbers of rabbits in the control and ALN-S groups at 52 weeks were further reduced because of femoral fracture. The histologic appearance and the ICRS Visual Histological Assessment Scale scores of these groups differed at 52 weeks, but statistical significance was not obtained possibly because of the further limited number. Second, we created 6-mm osteochondral defects, but we did not confirm that this defect size was ideal. Mature articular cartilage does not have an intrinsic capacity to heal itself,³³ and in the rabbit model, 1- to 4-mm osteochondral defects are reported to heal after 128 days. However, the quality reflects partial fibrocartilage-based healing.¹⁹ In gout, 6-mm osteochondral defects do not heal, and the cavitary lesion remains unfilled with fibrocartilage after 28 weeks.¹³ In our groups, the 6-mm osteochondral defects did not heal, although subchondral bone repair was complete after 52 weeks (unpublished data). Therefore, we defined a 6-mm osteochondral defect as an “irreparable” model and investigated the ameliorating capacity of alendronate.

Third, the defects were located on the trochleas of the rabbits to create the critical defect size of 6 mm. The patellofemoral joints of rabbits suffer from some degree of compressive force because the knee joints of rabbits are always in the flexed position. However, there is no direct weightbearing force on the patellofemoral joint. To determine the effects of alendronate on subchondral bone repair and cartilage repair in a weightbearing area, a larger animal study is required.

Fourth, the ideal timing of the cessation of alendronate is still unclear. Ideally, we should perform the experiments with cessation of alendronate at different time points, such as 4, 8, and 24 weeks. However, if so, the experimental system would become complicated, and far more rabbits would be required. So, we decided to cease the alendronate injection at 8 weeks in the ALN-S group.

In conclusion, alendronate promoted new bone formation and cartilage-like tissue formation in an osteochondral defect in the first 8 weeks. However, at 24 weeks, it inhibited bone remodeling. The withdrawal of alendronate at 8 weeks did not inhibit remodeling, and good continued subchondral bone formation was seen. At 52 weeks, there was partial hyaline cartilage–like repair in the ALN-S group. Alendronate accelerated new bone formation in an osteochondral defect. With its withdrawal, good subchondral bone formation was achieved and maintained, leading to the formation of better-quality cartilage.

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