Effects of varying intensities of laser energy on articular cartilage: A preliminary study

Lasers in Surgery and Medicine 5577-588 (1985)

Effects of Varying Intensities of Laser Energy on Articular Cartilage: A Preliminary Study Robert J. Schultz, MD, Shanker Krishnamurthy, MD,William Thelmo, MD, Jose E. Rodriguez, MD, and Gregory Harvey, MD Department of Orthopedic Surgery, New York Medical College, Valhalla (R. J.S., S.K., J.E. R.,G.H.) and Department of Surgical Pathology, Downstate-Kings County Medical Center, Brooklyn (W. T.), New York.

The effects of laser energy on articular cartilage were studied utilizing the neodymium YAG laser. Partial-thickness cartilage defects were surgically attempted in the femoral condyles of knee joints in guinea pigs. The defects were exposed to laser energy of varying intensities [group I, 25 J (5 W X 5 sec); group 11, 75 J (15 W x 5 sec); group 111, 125 J (25 W x 5 sec)]. A fourth group was studied, in which the defect was not lased. Animals were killed at weekly intervals from 1 to 6 weeks and the knee joints were subjected to histological analysis. At 5 weeks, the knees exposed to 25 and 75 J demonstrated a reparative process with chondral proliferation. The knees exposed to 125 J demonstrated fibrotic tissue and tissue necrosis that resulted in fibrosis. In the knees not exposed to laser energy, numerous foci of granulation tissue were present at all stages with the end point of healing being one of fibrosis with disorganized patchy cartilage islands. Key words: Nd:YAG laser, low dosage, articular cartilage, chondral proliferation

INTRODUCTION

Articular cartilage regeneration has been studied by many orthopedic research scientists. Mankin [ 1970, 1982) stated that “articular cartilage is mitotically active only in the immature state of development and with cessation of net synthesis, cartilage cells ‘turn off the switch’ for DNA synthesis (but may ‘turn it on’ again under conditions of stress). ”

Address reprint requests to Robert J. Schultz, MD, Department of Orthopedic Surgery, New York Medical College, Valhalla, NY 10595. Accepted for publication July 23, 1985. This paper was presented at the 5th Annual Meeting of the American Society for Laser Surgery and Medicine, Orlando, Florida, May 28, 1985.

0 1985 Alan R. Liss, Inc.

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Mitchell and Shepard [1976, 19801 demonstrated that early in the course of repair of full-thickness cartilage defects in rabbits, the primary fibrous repair was converted to hyaline chondroid tissue, with high mitotic activity. By 12 months, however, the tissue appeared more fibrous than cartilaginous and the surface layers and cells were more typical of fibrocartilage than hyaline cartilage. Convery et al [ 19721 reported on an experiment in which osteochondral defects of varying sizes were created in the distal end of the femur in horses. They demonstrated that defects less than 3 mm in diameter showed complete repair, whereas those with defects 9 mm or greater showed incomplete repair at the end of 3 months. Salter et al [1980, 19821 noted in rabbit knees that cartilage defects extending through subchondral bone subjected to continuous passive motion immediately after surgery healed more rapidly and completely than those immobilized or subjected to intermittent active motion. The healing tissues also were found to approximate more closely hyaline than fibrocartilage. Fuller and Ghadially [1972] studied the healing of tangential partial thickness cartilage lacerations in rabbits and found no evidence of repair even in young animals. Baker et al [19741 demonstrated accelerated healing of articular cartilage defects in rabbits by the use of electrical fields. Schultz et al [1983] reported attempts on bone-to-bone fusion in guinea pig knees using the Nd-YAG laser. During this research, articular cartilage proliferation in the low-energy-lased joints (less than 75 J) was observed as an incidental finding. In view of the above observation, we were stimulated to investigate this response. This is a preliminary study designed to evaluate the response of articular cartilage exposed to Nd:YAG laser energy of varying intensities. MATERIALS AND METHODS

The source of laser energy for this experiment was the neodymium YAG laser (Cooper Lasersonics Model 8000). Twenty guinea pigs (Cuviu cobuyu) served as the animal model and were divided into four groups with five animals in each group. In all the animals, partial-thickness cartilage defects were surgically attempted in both knees. The cartilage defects in the first three groups were exposed to varying intensities of laser energy [group I:25 J (5 W/sec); group 11: 75 J (15 W/sec); and group m:125 J (25 W/sec)]. The duration of laser exposure was fixed at 5 seconds in all cases. The spot size of the beam was 3 mm. Energy levels were measured at the laser generator and at the target site with an Apollo Model 101 power density meter (Apollo Instruments, h s Angeles, CA). A power loss of only 0.5% was noted at the target site. This study was conducted over a 6-week period of time. The animals were anesthetized utilizing ketamine hydrochloride (40 mg/kg) and xylazine (6 mg/kg) IM. Under sterile operating-room conditions, both knee joints were exposed through an anterior midline vertical skin incision extending from the distal % of the thigh to the proximal % of the tibia. The knee joint was entered through a medial parapatellar capsular incision. Using loupe magnification ( x 3.5) and a sharp surgical #15 blade, a partial-thickness cartilage core of averaging 4 mm in diameter and 0.14 mm in depth was excised from the center of the intercondylar groove of the femur at a 90" angle. In groups I, 11, and 111, the defect was lased at the intensities assigned to each group. In group IV, the cartilage was not subjected to laser energy. Wound closure was performed in standard fashion.

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The operative time was approximately 15 minutes for each knee. The knees were not immobilized postoperatively. Each animal was placed individually in 24 X 12 inch cages, and their ambulation was restricted to this area. One animal from each group was killed at weekly intervals. The knee joints were analyzed histologically. The specimens were decalcified and serial sagittal sections were made and stained with hematoxylin-eosin. HISTOLOGY DATA RESULTS First Week

Group I (25 J) (Fig. 1A). In this group, the unviolated cartilage base that formed the floor of the partial-thickness surgically created crater demonstrated chondrocytes with viable nuclei and matrix. Overlying the viable chondrocytes was a structureless homogeneous layer in which there was loss of normal basophilia with the presence of increased acidophilia. The zone of calcified cartilage was intact. There was no granulation tissue present and the cartilage bounding the defect was unaffected. Group II (75 J) (Fig. 1B). The findings of the unviolated base were similar to those of group I. In addition, there was evidence of subchondral fibrosis and osteoblastic proliferation. Group 111 (125 J) (Fig. 1C). There was increased acidophilia of the chondrocytes, and the nuclei were pyknotic. The zone of calcified cartilage was intact, as was the subchondral bone. Group IV (nonlased) (Fig. 1D). In this group, the cartilage base of the crater demonstrated loss of architectural integrity with substitution of cartilage by granulation tissue. Also found was disruption of the calcified cartilage zone. Second Week

Group I (25J)(Fig. 2). The chondrocytes demonstrated evidence of acidophiIic nuclei and matrix. The crater depth decreased and the zone of calcified cartilage remained intact. Group 11 (75 J). The specimens from group I1 at the end of 2 weeks demonstrated marked subchondral fibrosis and fibrous tissue replacement of the crater as opposed to hyaline cartilage. The adjacent cartilage demonstrated degeneration with loss of nuclei. Group 111 (125 There was further thinning of the cartilage surface and within each defect were noted areas of cartilage erosion extending to and also through subchondral bone. Group IV (nonlased). The crater base continued to demonstrate loss of architectural integrity with further substitution by granulation tissue. There was fibrosis with myxomatous matrix.

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Third Week

Group I (25 J) (Fig. 3A). The base of the crater demonstrated foci of proliferating cartilage cells that resembled hyaline cartilage. The calcified cartilage zone remained intact and there were islands of cartilage present in the subchondral bone. Group II (75 J).In this group at 3 weeks, there was no evidence of chondral proliferation. There was subchondral disruption. Granulating tissue was present.

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Fig. 1A. Group I (first week). This figure demonstrates the surgically created crater covered by an acidophilic structureless homogeneous material with viable chondrocytes at the base. ( X 145.) B. Group I1 (first week). Section taken through the crater shows a pale (acidophilic) tissue involving almost the entire thickness of the cartilage layers. The calcified cartilage zone is intact. There is also adjacent fibrosis extending up to the subchondral level. Normal cartilage is seen on the left side. ( X 145.)

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Fig. 1C. Group 111 (first week). On the left corner of this section there is necrosis and sloughing of superficial layer of the chondral surface. Also noted was increased acidophilia of chondrocytes with pyknotic degeneration of nuclei, which cannot be appreciated on a black and white print. ( X 145.) D. Group IV (first week). Note the loss of architectural integrity with substitution of cartilage by granulation tissue at the base of the crater. ( x 145.)

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Fig. 2. Group I (second week). Discloses a more shallow crater with no granulation tissue. ( x 145.)

Group III (125 (Fig. 3B). The cartilage base of the crater demonstrated total loss of architectural definition of all tissue elements. Extensive fibrosis was noted. Fibrous tissue was found replacing the entire thickness of the cartilage defect with extension into subchondral bone. Group IV (nonlased). The depth of injury extended to subchondral bone with extensive granulation tissue being present. Fourth Week

Group I (25 J) (Fig. 4). The specimens in this group were found to show continued increase in chondrocytic proliferation. In addition, normal basophilia had returned and the zone of calcified cartilage was still intact. Group 11 (75 J). There was some chondrocytic proliferation but significantly less than that observed in group I. Acidophilia remained present. There was also evidence of granulation tissue extending into the subchondral bone. Group III (125 3.In this group, there was no evidence of chondral proliferation. The defect was filled with granulation tissue that extended into the subchondral bone. Group IV (nonlased). By the fourth week, there was evidence of fibroblastic proliferation with scattered disorganized foci of cartilage cells. The nuclei and matrix demonstrated marked acidophilia. Sixth Week

Group I (25 J) (Fig. 5A,B). By the sixth week, the specimens demonstrated chondrocytic proliferation having the characteristics of hyaline cartilage. The cells were organized in a horizontal pattern and the defect had been reconstituted to the level of the surface of normal cartilage. There was no evidence of cartilage overgrowth beyond the limits of the original defect. The cells and the matrix demonstrated normal basophilic staining.

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Fig. 3A. Group I (third week). Chondrocytic proliferation similar to hyaline cartilage is present. Also detectable is a focal area of cartilage in the subchondral zone. ( X 145.) B. Group III (third week). A segment of degenerating chondral surface is shown with fibrous tissue replacing the entire thickness of chondral defect. Normal cartilage is present on the left. ( X 145.)

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Fig. 4. Group I (fourth week).The lased area shows an area of chondrocytic proliferation. A broad zone of fibrolastic proliferation is also noted extending to the subchondral bone. ( X 145.)

Group II (75 J) (Fig. 5C). At the end of 6 weeks, these specimens demonstrated evidence of patchy, disorganized chondrocytic proliferation. In addition, the cells and the matrix demonstrated persistent acidophilia. There was evidence of degenerative surface cartilage with marked fibrosis. Group III (125 J) (Fig. 5Jl). This group demonstrated extensive chondral necrosis with marked subchondral fibrosis. Group IV (nonlased) (Fig. a). In the crater there was absence of all chondral elements. Islands of cartilage were found located in the subchondral bone. There was replacement of the surface cartilage and the subchondral bone in the area of the crater by fibrous tissue. DISCUSSION

This experiment was instituted following our observation of articular cartilage proliferation in response to laser energy during another study [Schultz et al, 19831. The possibility of articular cartilage possessing the capability to regenerate and the repair of partial thickness defects are important issues. Mankin [ 1970, 19821 is of the opinion that cartilage cells have the potential to regenerate when the appropriate stimulus is provided. Although cartilage regeneration in full-thickness defects has been shown to occur, cartilage regeneration in partial-thickness articular cartilage defects has not been conclusively demonstrated. In this study, partial-thickness articular cartilage defects were surgically attempted and subjected to varying intensities of laser energy in groups I, 11, and III. In group IV, the defects were not exposed to laser energy. In this manner we have attempted to determine (1) whether laser energy can provide the correct stimulus for

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Fig. 5A. Group I (sixth week). The original crater has been completely replaced by hyaline cartilage with reconstitution up to the level of the normal joint surface. (The stained sections demonstrated return of normal basophilic staining, which is characteristic of hyaline cartilage.) ( x 145.) B. Group I (sixth week). At higher power, the chondrocytes are seen within lacunae. Note the increased cellularity present. ( X 160.)

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Fig. 5C. Group I1 (sixth week). Extensive fibrosis with patchy, disorganized cartilage islands in the basal layers is noted. The superficial layers are acellular. ( X 145.) D. Group 111 (sixth week). Section shows necrotic chondral surface that is being sloughed off and extensive subchondral fibrosis. ( X 145.)

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Fig. 5E.Group IV (sixth week). The crater shows absence of chondral elements with islands of cartilage cells located in the subchondral bone and fibrous tissue replacement. ( X 145.)

(a) cartilage cells to regenerate and (b) repair to occur in partial-thickness cartilage defects and (2) the appropriate dosage required to produce this effect. By histologic sections, we were able to follow the cartilage tissue response to laser energy over a 6-week period of time. In addition, we studied the course adopted by a partial-thickness defect in cartilage when not exposed to the laser or any other external stimulus. In group I, the knees exposed to 25 J of laser energy clearly demonstrated, initially, preservation of the cartilage cells bordering the surface of the crater with some change in staining characteristics and maintenance of the zone of calcified cartilage. Evidence of chondral proliferation was found to occur by the fourth week after lasing. By the sixth week, the defect had been reconstituted to the level of the surface cartilage. The staining characteristics of the cells had reverted back to that of normal basophilia. There was no evidence of cartilage overgrowth beyond the boundaries of the original defect, although some hypercellularity was noted. Therefore, we feel that this level of energy is compatible with the tissue tolerance of the adjacent tissues. In group II, in which the crater was exposed to 75 J, although there was evidence of cartilage regeneration, it was patchy, disorganized, and the defect was not reconstituted. Furthermore, by 6 weeks, the staining characteristics of the tissues had still not reverted to normal. This has led us to infer that this level of laser energy can result in isolated areas of cell reconstitution, but it is probably too high to allow uniform regeneration. In group IIl (125 J), we noted progressive tissue destruction with ultimate replacement of the crater by fibrous tissue. This level of laser energy far exceeds the tissue tolerance level, resulting in destruction of cartilage and subchondral bone damage.

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In group IV, we found no evidence of restoration of the crater by cartilage. Nests of cartilage cells were found in the subchondral bone. We have no good explanation for this particular finding. Thus we can conclude from this preliminary study that Nd:YAG laser energy of low dosage provides an appropriate stimulus for the regeneration of partial-thickness cartilage defects. The mechanism by which this regeneration occurs is not clear at the present time. It is possible that laser energy of appropriate dosage is capable of reactivating the mechanism controlling cartilage cell replication. We can also conclude that higher doses of laser energy do not provide a greater response of cartilage regeneration. In fact, higher levels of energy produce cell death and destruction with healing by formation of fibrous tissue. Whether this low-dose relationship of 25 J can be linearly or longitudinally applied to other animal models will be the focus of further studies so that we can eventually find the correct dose required for cartilage stimulation in humans. In addition, histochemical and immunochemical studies are planned to analyze better the composition of the “regenerated cartilage.” ACKNOWLEDGMENTS

This study was supported by grants from Cooper Lasersonics and the Department of Orthopedics of New York Medical College. REFERENCES 1. Baker B, Becker RO, Spadaro J: A study of electrochemical enhancement of articular cartilage repair. Clin Orthop 102:251-267, 1974. 2. Convery FR, Akeson WH, Keown GH: The repair of large osteochondral defects, an experimental study in horses. Clin Orthop 82:253-262, 1972. 3. Fuller JA, Ghadially FN: Ultrastructural observations on surgically produced partial thickness defects in articular cartilage. Clin Orthop 86:192-205, 1972. 4. Mankin HJ: “The Articular Cartilage: A Review.” AAOS Instructional Course Lectures. Mosby CO, VOI 19, pp 204-224, 1970. 5. Mankin HJ: The response of articular cartilage to mechanical injury. J Bone Joint Surg 64A:460466, 1982. 6. Mitchell N, Shepard N: The resurfacing of rabbit articular cartilage by multiple perforations through the subchondral bone. J Bone Joint Surg 58A:230-233, 1976. 7. Mitchell N, Shepard N: Healing of articular cartilage in intra-articular fractures in rabbits. J Bone Joint Surg 62A:628-634, 1980. 8. Salter RB, Minster RR, Bell RS, Wong DA, Bogoeh ER: Continuous passive motion and the repair of full thickness articular cartilage defects: A one year follow up. Trans Orthop Res SOC7:167, 1982. 9. Salter RB, Simmonds DF, Malcolm BW, Rumble DJ, McMichael D, Clements ND: The biological effects of continuous passive motion on the healing of full thickness defects in articular cartilage: An experimental investigation in the rabbit. J Bone Joint Surg 62A: 1232-1251, 1980. 10. Schultz, RJ, Krishnamurthy S , Thelmo W, Rodriguez JE, Harvey GP: Laser arthrodesis of guinea pig knee joints. Presented at the Fifth Annual Meeting of the International Laser Society in Detroit, Michigan, 1983.