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Early healing processes of free tendon grafts within bone tunnels is bone-specific: a morphological study in a rabbit model
The Knee 9 Ž2002. 21᎐26
Early healing processes of free tendon grafts within bone tunnels is bone-specific: a morphological study in a rabbit model S.R.M. Grassman, D.B. McDonald, G.M. Thornton, N.G. Shrive, C.B. FrankU McCaig Centre for Joint Injury and Arthritis Research, Uni¨ ersity of Calgary, 3330 Hospital Dri¨ e NW, Calgary, Alberta, Canada T2N 4N1 Accepted 28 August 2001
Abstract In order to function as effective ligament replacements, free tendon grafts must become firmly healed into bone tunnels as soon as possible. We hypothesized that graft incorporation would be bone-specific. Free semitendinosus tendon grafts were inserted into drill holes in a lapine medial collateral ligament reconstruction model; thus, creating tibial and femoral bone-specific incorporation sites. Femur-semitendinosus tendon-tibia complexes were harvested from 26 rabbits for histological analysis at various healing times: 0, 6, 12, or 24 weeks post-surgery. Incorporation and remodeling of the graft in the chondral callus was much more extensive at the cancellous-filled femoral insertion than within the marrow-dominated tibial insertion, suggesting that tendon graft healing may depend on the cancellous bone architecture at the graft site. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Free tendon graft; Bone tunnel; Graft healing
1. Introduction Some ligament injuries result in functional instability of a joint and therefore require reconstructive surgery. Unfortunately, even with reconstructive surgery, some graft tissues may never regain all of the properties Žmechanical, structural, histological and biochemical. of the original graft tissue or the native tissue that the graft is replacing. Almost all investigators studying reconstructions with free tendon grafts have described healing within drill holes in a similar manner. In general, they have described osseous ingrowth and incorporation, extending around and then into the tendon substance U
Corresponding author. Tel.: q1-403-220-6881; fax: q1-403283-7742. E-mail address: [email protected] ŽC.B. Frank..
w1᎐4x. Only one study, by Hausman et al. w5x, mentioned a lack of bone growth and ossification of a transplanted tendon, and felt that this demonstrated a reluctance of tendon to heal to metaphyseal bone. This is the only previous speculation that tendon healing to bone may be either environment- or bonespecific. Given the possibility that tendon graft healing may, in fact, be specific to the bone that is receiving the graft, we wanted to examine the morphological appearances of a free tendon reconstruction model in two different osseous environments. We used a rabbit medial collateral ligament ŽMCL. model in which we have considerable background information and have seen some previous evidence of differences in healing between its two ligament insertions w6᎐8x. Our purpose in this study was therefore to test the hypothesis that the healing of a free tendon graft within bone
0968-0160r02r$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 8 - 0 1 6 0 Ž 0 1 . 0 0 1 2 8 - 4
22
S.R.M. Grassman et al. r The Knee 9 (2002) 21᎐26
tunnels in the metaphyseal areas of the rabbit femur and tibia may be bone-specific.
2. Materials and methods 2.1. Surgical procedure Twenty-six skeletally mature Ž1-year-old. female New Zealand White rabbits ŽReimans Fur Ranch, St. Agatha, Ontario. were studied. Six to seven rabbits were used at each of four different time points in this ligament reconstruction study: immediately postsurgery Žtime zero., and at 6 weeks, 12 weeks and 24 weeks post-surgery. Each rabbit underwent the same surgical procedure for medial collateral ligament reconstruction ŽFig. 1.. The right leg of each rabbit was used as the experimental limb while the left leg served as a non-operated contralateral control limb. Both legs of each rabbit were placed in a custom built splint so that the legs were held at a 110⬚ during surgery. This was done to allow comparable placement of all grafts within bones at an angle similar to that with a rabbit sitting. Under general anesthesia, a longitudinal medial incision was made to expose the semitendinosus tendon and medial collateral ligament. Using a scalpel, the medial collateral ligament was incised from its insertional sites. The former medial collateral ligament insertional sites were then scraped, providing a clean surface for drilling bone tunnels. At those insertional sites, the bone tunnels for a subsequent semitendinosus graft tissue were drilled with a Dremel tool ŽDremel Division of Emerson Electric, Racine, WI, USA. and a 1r8-inch drill bit. In attempts to replicate the general fiber orientation at the insertions of the native MCL w9x, the femoral insertion was drilled through the old MCL site at 90⬚ to the metaphyseal surface of the femur while the tibial insertion was drilled at 45⬚ to the long axis of the tibia. Each
Fig. 1. Schematic of model showing rabbit femur and tibia. Note the femoral tendon end is placed within dense cancellous bone while the tibial tendon end is placed within the relatively loose ‘marrow space’.
semitendinosus tendon was then excised from the right leg and prepared as a graft. Each free end of each semitendinosus tendon was gripped by attaching an absorbable 3.0 Vicryl Bunnell-type suture. This suture at either end was then routed through the respective drill holes into one of the two tunnels and out through two smaller 1r64-inch drill holes through the lateral cortex. The suture ends were then tied over the lateral side of the bone, pulling the semitendinosus tendon into slight tension as these knots were tied. Prior to closing the overlying fascia, the surgical area was sprayed with Gentocin Spray ŽSchering Canada Inc.. and the overlying fascia and skin was then closed with 4.0 Ethilon interrupted sutures. After surgery, the rabbits were left to recover from the anesthesia prior to placing them back in their individual metal cages. Neither leg was immobilized following reconstructive surgery. All rabbits were provided with standardized feed and each rabbit was checked daily for wound sepsis, open wounds, pressure sores, weight loss and knee movement. 2.2. Histological procedure Each rabbit was then euthanized with 1.5 ml of Euthanyl ŽMTC Pharmaceuticals, Cambridge, Ontario.. Each leg was disarticulated from the hip joint and the ankle joint. All surrounding tissue structures, with the exception of the menisci, cruciate ligaments, lateral collateral ligament and medial collateral ligament Žin the case of the contralateral leg. or semitendinosus tendon graft Žin the case of the experimental leg. were removed. Each experimental leg and each contralateral leg were splinted with tongue depressors and rubber bands at roughly 70⬚ of flexion. Both complexes were then placed in paraformaldehyde lysine periodate solution for a minimum of 48 h. The experimental and contralateral legs were subsequently embedded in paraffin according to the methodology of Peterfi w10x or in polymethylmethacrylate according to the method of Matyas et al. w11x. Specimens at each time point were divided equally between these two embedding techniques. The paraffin blocks were cut along the length of the semitendinosus tendon into longitudinal 12-m-thick serial sections using a Wetzlar microtome ŽWalter A. Carveth Ltd., Vancouver, Canada.. The polymethylmethacrylate blocks were cut along the length of the semitendinosus tendon into longitudinal 50-m-thick sections using a Buehler diamond wafer saw with a Proslicer 5 = 12.5-inch diamond wafer blade. The paraffin sections were stained with hematoxylin and eosin to highlight the collagen fibers and the cell nuclei, while the polymethylmethacrylate sections were stained with safranin-O and toluidine blue to highlight proteoglycans and cells, respectively.
S.R.M. Grassman et al. r The Knee 9 (2002) 21᎐26
Morphological observations monitored the appearance of the tendon tissue and the location of the scar tissue over the 24 weeks of healing. In addition, the presence of hypercellular areas and the shapes of cells were documented.
3. Results 3.1. Gross obser¨ ations Each semitendinosus tendon autograft complex at each incorporation period Ž6, 12 and 24 weeks., grossly appeared to be healed into the bone at either end. In its visible extraosseous portion each tendon graft was thicker and wider than it had been at surgery Žtime zero. and its insertional sites were surrounded by new bone. Interestingly, the tibial insertion appeared very quiescent, with no obvious new bone formation, whereas, at the femoral insertion, bone appeared to have grown around the semitendinosus tendon in a conical fashion by 12 and 24 weeks of healing. 3.2. Microscopic obser¨ ations 3.2.1. Time zero Tendons at time zero had a consistent thickness traveling from the femoral drill hole to the tibial drill hole Ža rope-like appearance. ŽTable 1.. Cigar-shaped fibrocytes within the semitendinosus tendon were distributed equally and were oriented along the length of the tendon between its crimped collagen fibers ŽTable 2.. 3.2.2. Six weeks After 6 weeks, the cross-sectional appearance of the semitendinosus tendon at the femoral insertion was very different to that at the tibial insertion. The tendon end within the femur had swelled to take on a bulb-like shape. The tendon in the tibial insertion,
23
however, did not appear to have changed significantly, maintaining its rope-like appearance. Interestingly, the semitendinosus tendon end in the femur was noticeably thicker. The microscopic appearances of the two healing insertions were distinctly different at this interval ŽFig. 2.. At the femoral insertion, the crimped pattern of the fibers had disappeared and large amounts of scar tissue now existed throughout the tendon substance. At 6 weeks, the tendon within the femur was hypercellular with up to 10 times the number of cells in a given area compared with that in the tibial insertion. Most cells within the femoral insertion were round, with chondrocytic-like cells occupying 20᎐50% of the tendon cross-sectional area ŽFig. 3.. Metachromatic toluidine blue and safranin-O staining both indicated that these areas were fibrocartilagenous in makeup, looking similar to typical fracture callus. No such callus was seen around the tibial tendon insertion or within the tibia. Only a small amount of new tissue formed around the graft within the tibia and there were no fibrocartilagenous cells seen within the tendon proper, other than along its perimeter. The tendon at the tibial insertion continued to have fairly typical semitendinosus appearances, with cigar-shaped fibrocytes oriented parallel to crimped collagen fibers. In comparison, the collagen fibers and cells were not organized in an orderly fashion within the femoral insertion. In addition, there was no metachromatic staining of the healing tendon substance within the tibia, and relatively quiescent appearing marrow was still seen surrounding it. Scar tissue within each insertion was also distinctly different. Within the tibial insertion, scar occurred around the perimeter of the tendon and consisted of very loosely arranged and connected non-crimped collagen fibers, small round cells, adipose tissue and elements representing early blood vessels. In contrast, scar tissue within the femoral insertion was distributed throughout the tendon substance and consisted of relatively tightly interwoven collagen fibers.
Table 1 Characteristics of tendon tissue appearance and scar tissue location Weeks
0 6 12 24
Tendon tissue appearance
Scar tissue location
Tibia
Femur
Tibia
Femur
Rope-like Organized fibers Rope-like Organized fibers Rope-like Organized fibers Rope-like Organized fibers
Rope-like Organized fibers Bulb-like Fibrocartilagenous Bulb-like Bony protrusions into tendon Substantially replaced with osseous material
None
None
Perimeter
Throughout
Perimeter
Throughout
Perimeter
Throughout
S.R.M. Grassman et al. r The Knee 9 (2002) 21᎐26
24
Table 2 Observation of different cells and hypercellular areas Weeks
0 6 12 24
Cigar-shaped
Hypercellular
Tibia
Femur
⻬ ⻬ ⻬ ⻬
⻬
Tibia
Round-shaped Femur ⻬ ⻬ ⻬
3.2.3. Twel¨ e weeks The femoral insertion appeared to have undergone considerable osteogenesis over the 12-week healing period. The tendon in the femoral insertion in the 12-week complexes still had a bulb shape, but now bone protruded into the tendon substance. Amongst the 12-week complexes, osseous protrusions were randomly located around the area of the femoral insertion only. The tendon traveling over the tibia and into the tibial insertion still had its rope-like shape.
Chondrocytic-like
Tibia
Femur
⻬
⻬ ⻬ ⻬
Tibia
Femur ⻬ ⻬ ⻬
At 12 weeks, the tendon end in the femoral insertion was still hypercellular with round cells and a few chondrocytic-like cells were still visible in each histological section. At this time period, there were many vascular elements within the femoral insertion. In comparison, the tibial insertion still had no chondrocytic-like cells and vessels were seen only along the tendon-bone and tendon-marrow interface. In the femoral insertion, the fingers of new tissue appeared to be associated with fatty deposits, vascular channels,
Fig. 2. Coronal section of a typical rabbit knee at low magnification showing differences between insertional areas. Note the tendon graft within the relatively hollow tibia space invokes a minimal cellular and scar response Žleft. while the other end within the femur Žright. is embedded within cellular callus. Also note that the healing response appears to correlate with the tendon being inserted into the cancellous bone in the femur vs. the ‘marrow space’ in the tibia Žhematoxylin and eosin =25..
S.R.M. Grassman et al. r The Knee 9 (2002) 21᎐26
25
Fig. 3. Typical chondroid cellular response around and within the tendon end in the femur Žhematoxylin and eosin =300.. This is quite different than the minimal response around the tendon end in the tibia Žsee tibial end at top left in Fig. 2..
arterioles, holes, and loose and disorganized collagen fibers. At the tibial insertion, the tendon end had not changed, other than along the tendon-bone and tendon-marrow boundaries, where scar tissue was seen. Collagen fibers remained crimped and fibrocytes were relatively evenly-distributed and aligned with each other and with the collagen fibers. 3.2.4. Twenty-four weeks As in the 6-week and the 12-week autografts, the tendon still had a rope-like appearance along the tibia and into the tibial insertion. Within the femoral insertion, however, there was substantially less tendon material seen as osteogenesis engulfed it. As in the 12-week group, the tendon end protruding from the femur still had a swollen appearance with some adjacent protruding bone growth. Cell shapes within the femoral insertion and within the tibial insertion had not changed from 12 weeks. The only thing that did change from the previous time periods was that the cells in the femoral insertion were now relatively uniformly distributed. The femoral insertion still contained a large number of round-shaped cells and the tibial insertion still contained cigar-shaped fibrocytes with round-shaped cells only in scar tissue located along its tendon-bone and tendon-marrow boundaries.
4. Discussion Results of this study show that at all incorporation
times from 6 to 24 weeks, the healing of the free semitendinosis tendon in the femoral bone tunnel of this rabbit model was distinctly different from the healing at the opposite end of the same tendon placed in the tibial bone tunnel. Specifically, the amount and manner of osteogenesis in and around each insertion was notably different. At the femoral insertion, protruding bone apposition that had a cancellous architecture was observed, whereas, at the tibial insertion, modest bone apposition that had a cortical architecture was observed only along the tendon boundary. The reasons for this interesting difference in tendon graft healing are not entirely clear. The most obvious explanation would seem to relate to the normal intrinsic differences between the internal architecture of the femur and tibia in this model, vis-a-vis ` a major difference in cancellous bone quantity, quality and distribution. The entire metaphyseal area of the normal rabbit femur is filled with cancellous bone, providing a relatively firm bed into which a tendon graft used can be fixed. The rabbit tibial metaphysis, however, contains relatively little cancellous bone and is instead filled with what can best be described as loose but very cellular ‘marrow’. Despite the anatomic similarity of the tibia and femur, both being so-called ‘metaphyseal’ regions of these respective bones, each is quite distinct in terms of its internal architecture. This architecture almost certainly reflects major differences in the intrinsic biological responses of each site when injured. Insertion of a tendon graft into each site has clearly invoked a much different cellular response, which likely has quite different functional
26
S.R.M. Grassman et al. r The Knee 9 (2002) 21᎐26
consequences. Based on these results it would seem likely that insertion of a tendon graft into a cancellous bony tunnel would induce a much more rapid and firm attachment than insertion into a similar tunnel in a marrow-filled space. This principle that bone-specific graft incorporation depends on cancellous bone architecture should be tested in other bones to rule out other potential bone-specific factors; for example, loading environment or blood supply. Interestingly, the healing of the tendon in the femoral insertion of the current model appears to be similar to the description given by Forward and Cowan w1x, Whiston and Walmsley w2x and Liu et al. w3x, for the healing of tendon grafts within drill holes in the lapine calcaneus. It is also similar to that shown by Rodeo et al. w4x for healing of an extensor tendon end placed into a drill hole in the proximal tibial metaphysis in a canine model. However, the healing of the tendon in the tibial insertion observed in the current model appears more similar to the healing of flexor carpi radialis tendon in a drill hole in the radial metaphysis in a guinea pig reported by Hausman et al. w5x. These differences support the notion of either bone-, site- or model-specificity of tendon graft healing. In describing the processes of tendinous healing into bone Forward and Cowan w1x and Lui et al. w3x likened it to fracture healing, through formation of a fracture callus. Fractures that occur in cortical bone are known to heal in a different manner w12᎐14x than those in cancellous bone w14x. This may explain all of the differences found in the current study. In summary, the clear clinical implication of these results is that graft healing is different in different bony environments: graft incorporation into a cancellous bone being superior to that in a marrow space. In fact, we speculate that the bony environment is a greater determinant of healing success than even graft type Žfree tendon graft or bone-tendon-bone graft.. In our rabbit MCL reconstruction model, the femur was cancellous-filled and the tibia was marrow-dominated. The human femur and tibia are both more similar to the cancellous-filled rabbit femur. Other locations in the human where free tendon grafts are performed, however, may be more marrow-dominated, like the rabbit tibia Že.g. phalanges or metacarpals, etc... The salient point is that tendon grafts are performed in a variety of locations throughout the human body and successful graft incorporation is likely dependent on the bony environment receiving the graft.
Acknowledgements The authors gratefully acknowledge the administrative assistance of Judy Crawford, as well as the financial support of The Arthritis Society of Canada, the Canadian Institutes of Health Research, the Alberta Heritage Foundation for Medical Research, and the McCaig Professorship Fund. References w1x Forward AD, Cowan RJ. Tendon suture to bone. J Bone Joint Surg ŽAm. 1963;45Ž4.:807᎐823. w2x Whiston TB, Walmsley R. Some observations on the reaction of bone and tendon after tunnelling of bone and insertion of tendon. J Bone Joint Surg ŽBr. 1960;42Ž2.:377᎐386. w3x Liti SH, Panossian V, Al-Shaikh R et al. Morphology and matrix composition during early tendon to bone healing. Clin Orthop 1997;339:253᎐260. w4x Rodeo SA, Arnoczky SP, Torzilli PA, Hidaka C, Warren RF. Tendon-healing in a bone tunnel. J Bone Joint Surg ŽAm. 1993;75Ž12.:1795᎐1803. w5x Hausman M, Bain S, Rubin C. Reluctance of metaphyseal bone to heal to tendon: histologic evidence for poor mechanical strength. Trans Orthop Res Soc 1989;14:277. w6x Frank CB, Loitz BJ, Shrive NG. Injury location affects ligament healing. A morphologic and mechanical study of healing rabbit medial collateral ligament. Acta Orthop 1995;66Ž5.:455᎐462. w7x King GJW, Marchuk LL, Shrive NG, Frank CB. Fresh extraarticular autografts are repopulated by extrinsic cells. Transactions of the 2nd Combined Meeting of the Orthopaedic Research Societies of USA, Japan, Canada and Europe, 1995, p. 96. w8x Matyas JR. The Structure and Function of the Insertions of the Rabbit Medial Collateral Ligament: an Experimental Morphological and Biomechanical Study of the Insertions of the Medial Collateral Ligament in the Skeletally Mature Rabbit. Doctoral Thesis. Calgary, Canada: University of Calgary, Graduate School of Medical Sciences, 1990. w9x Matyas JR, Anton MG, Shrive NG, Frank CB. Stress governs tissue phenotype at the femoral insertion of the rabbit MCL. J Biomech 1995;28Ž2.:147᎐157. w10x Drury RAB, Wallington EA, Cameron SR. Preparation of tissues for microtomy. Carletons Histological Technique. New York: Oxford University Press, 1967:62᎐63. w11x Matyas JR, Anton M, Frank C. Whole joint embedding in polyŽmethymethacrylate .: a method for preparing intact musculoskeletal organs for histology and 3-D reconstruction. Biotech Histochem 1997;72:152᎐157. w12x Chamley J, Baker SL. Compression arthrodesis of the knee. A clinical and histological study. J Bone Joint Surg ŽBr. 1952;34Ž2.:187᎐199. w13x Elkouri ER. Review of cancellous and cortical bone healing after fracture or osteotomy. J Am Podiatry Assoc 1982; 72Ž9.:464᎐466. w14x Frost HM. The biology of fracture healing: an overview for clinicians. Part II. Clin Orthop 1989;248:294᎐309.
Early healing processes of free tendon grafts within bone tunnels is bone-specific: a morphological study in a rabbit model S.R.M. Grassman, D.B. McDonald, G.M. Thornton, N.G. Shrive, C.B. FrankU McCaig Centre for Joint Injury and Arthritis Research, Uni¨ ersity of Calgary, 3330 Hospital Dri¨ e NW, Calgary, Alberta, Canada T2N 4N1 Accepted 28 August 2001
Abstract In order to function as effective ligament replacements, free tendon grafts must become firmly healed into bone tunnels as soon as possible. We hypothesized that graft incorporation would be bone-specific. Free semitendinosus tendon grafts were inserted into drill holes in a lapine medial collateral ligament reconstruction model; thus, creating tibial and femoral bone-specific incorporation sites. Femur-semitendinosus tendon-tibia complexes were harvested from 26 rabbits for histological analysis at various healing times: 0, 6, 12, or 24 weeks post-surgery. Incorporation and remodeling of the graft in the chondral callus was much more extensive at the cancellous-filled femoral insertion than within the marrow-dominated tibial insertion, suggesting that tendon graft healing may depend on the cancellous bone architecture at the graft site. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Free tendon graft; Bone tunnel; Graft healing
1. Introduction Some ligament injuries result in functional instability of a joint and therefore require reconstructive surgery. Unfortunately, even with reconstructive surgery, some graft tissues may never regain all of the properties Žmechanical, structural, histological and biochemical. of the original graft tissue or the native tissue that the graft is replacing. Almost all investigators studying reconstructions with free tendon grafts have described healing within drill holes in a similar manner. In general, they have described osseous ingrowth and incorporation, extending around and then into the tendon substance U
Corresponding author. Tel.: q1-403-220-6881; fax: q1-403283-7742. E-mail address: [email protected] ŽC.B. Frank..
w1᎐4x. Only one study, by Hausman et al. w5x, mentioned a lack of bone growth and ossification of a transplanted tendon, and felt that this demonstrated a reluctance of tendon to heal to metaphyseal bone. This is the only previous speculation that tendon healing to bone may be either environment- or bonespecific. Given the possibility that tendon graft healing may, in fact, be specific to the bone that is receiving the graft, we wanted to examine the morphological appearances of a free tendon reconstruction model in two different osseous environments. We used a rabbit medial collateral ligament ŽMCL. model in which we have considerable background information and have seen some previous evidence of differences in healing between its two ligament insertions w6᎐8x. Our purpose in this study was therefore to test the hypothesis that the healing of a free tendon graft within bone
0968-0160r02r$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 8 - 0 1 6 0 Ž 0 1 . 0 0 1 2 8 - 4
22
S.R.M. Grassman et al. r The Knee 9 (2002) 21᎐26
tunnels in the metaphyseal areas of the rabbit femur and tibia may be bone-specific.
2. Materials and methods 2.1. Surgical procedure Twenty-six skeletally mature Ž1-year-old. female New Zealand White rabbits ŽReimans Fur Ranch, St. Agatha, Ontario. were studied. Six to seven rabbits were used at each of four different time points in this ligament reconstruction study: immediately postsurgery Žtime zero., and at 6 weeks, 12 weeks and 24 weeks post-surgery. Each rabbit underwent the same surgical procedure for medial collateral ligament reconstruction ŽFig. 1.. The right leg of each rabbit was used as the experimental limb while the left leg served as a non-operated contralateral control limb. Both legs of each rabbit were placed in a custom built splint so that the legs were held at a 110⬚ during surgery. This was done to allow comparable placement of all grafts within bones at an angle similar to that with a rabbit sitting. Under general anesthesia, a longitudinal medial incision was made to expose the semitendinosus tendon and medial collateral ligament. Using a scalpel, the medial collateral ligament was incised from its insertional sites. The former medial collateral ligament insertional sites were then scraped, providing a clean surface for drilling bone tunnels. At those insertional sites, the bone tunnels for a subsequent semitendinosus graft tissue were drilled with a Dremel tool ŽDremel Division of Emerson Electric, Racine, WI, USA. and a 1r8-inch drill bit. In attempts to replicate the general fiber orientation at the insertions of the native MCL w9x, the femoral insertion was drilled through the old MCL site at 90⬚ to the metaphyseal surface of the femur while the tibial insertion was drilled at 45⬚ to the long axis of the tibia. Each
Fig. 1. Schematic of model showing rabbit femur and tibia. Note the femoral tendon end is placed within dense cancellous bone while the tibial tendon end is placed within the relatively loose ‘marrow space’.
semitendinosus tendon was then excised from the right leg and prepared as a graft. Each free end of each semitendinosus tendon was gripped by attaching an absorbable 3.0 Vicryl Bunnell-type suture. This suture at either end was then routed through the respective drill holes into one of the two tunnels and out through two smaller 1r64-inch drill holes through the lateral cortex. The suture ends were then tied over the lateral side of the bone, pulling the semitendinosus tendon into slight tension as these knots were tied. Prior to closing the overlying fascia, the surgical area was sprayed with Gentocin Spray ŽSchering Canada Inc.. and the overlying fascia and skin was then closed with 4.0 Ethilon interrupted sutures. After surgery, the rabbits were left to recover from the anesthesia prior to placing them back in their individual metal cages. Neither leg was immobilized following reconstructive surgery. All rabbits were provided with standardized feed and each rabbit was checked daily for wound sepsis, open wounds, pressure sores, weight loss and knee movement. 2.2. Histological procedure Each rabbit was then euthanized with 1.5 ml of Euthanyl ŽMTC Pharmaceuticals, Cambridge, Ontario.. Each leg was disarticulated from the hip joint and the ankle joint. All surrounding tissue structures, with the exception of the menisci, cruciate ligaments, lateral collateral ligament and medial collateral ligament Žin the case of the contralateral leg. or semitendinosus tendon graft Žin the case of the experimental leg. were removed. Each experimental leg and each contralateral leg were splinted with tongue depressors and rubber bands at roughly 70⬚ of flexion. Both complexes were then placed in paraformaldehyde lysine periodate solution for a minimum of 48 h. The experimental and contralateral legs were subsequently embedded in paraffin according to the methodology of Peterfi w10x or in polymethylmethacrylate according to the method of Matyas et al. w11x. Specimens at each time point were divided equally between these two embedding techniques. The paraffin blocks were cut along the length of the semitendinosus tendon into longitudinal 12-m-thick serial sections using a Wetzlar microtome ŽWalter A. Carveth Ltd., Vancouver, Canada.. The polymethylmethacrylate blocks were cut along the length of the semitendinosus tendon into longitudinal 50-m-thick sections using a Buehler diamond wafer saw with a Proslicer 5 = 12.5-inch diamond wafer blade. The paraffin sections were stained with hematoxylin and eosin to highlight the collagen fibers and the cell nuclei, while the polymethylmethacrylate sections were stained with safranin-O and toluidine blue to highlight proteoglycans and cells, respectively.
S.R.M. Grassman et al. r The Knee 9 (2002) 21᎐26
Morphological observations monitored the appearance of the tendon tissue and the location of the scar tissue over the 24 weeks of healing. In addition, the presence of hypercellular areas and the shapes of cells were documented.
3. Results 3.1. Gross obser¨ ations Each semitendinosus tendon autograft complex at each incorporation period Ž6, 12 and 24 weeks., grossly appeared to be healed into the bone at either end. In its visible extraosseous portion each tendon graft was thicker and wider than it had been at surgery Žtime zero. and its insertional sites were surrounded by new bone. Interestingly, the tibial insertion appeared very quiescent, with no obvious new bone formation, whereas, at the femoral insertion, bone appeared to have grown around the semitendinosus tendon in a conical fashion by 12 and 24 weeks of healing. 3.2. Microscopic obser¨ ations 3.2.1. Time zero Tendons at time zero had a consistent thickness traveling from the femoral drill hole to the tibial drill hole Ža rope-like appearance. ŽTable 1.. Cigar-shaped fibrocytes within the semitendinosus tendon were distributed equally and were oriented along the length of the tendon between its crimped collagen fibers ŽTable 2.. 3.2.2. Six weeks After 6 weeks, the cross-sectional appearance of the semitendinosus tendon at the femoral insertion was very different to that at the tibial insertion. The tendon end within the femur had swelled to take on a bulb-like shape. The tendon in the tibial insertion,
23
however, did not appear to have changed significantly, maintaining its rope-like appearance. Interestingly, the semitendinosus tendon end in the femur was noticeably thicker. The microscopic appearances of the two healing insertions were distinctly different at this interval ŽFig. 2.. At the femoral insertion, the crimped pattern of the fibers had disappeared and large amounts of scar tissue now existed throughout the tendon substance. At 6 weeks, the tendon within the femur was hypercellular with up to 10 times the number of cells in a given area compared with that in the tibial insertion. Most cells within the femoral insertion were round, with chondrocytic-like cells occupying 20᎐50% of the tendon cross-sectional area ŽFig. 3.. Metachromatic toluidine blue and safranin-O staining both indicated that these areas were fibrocartilagenous in makeup, looking similar to typical fracture callus. No such callus was seen around the tibial tendon insertion or within the tibia. Only a small amount of new tissue formed around the graft within the tibia and there were no fibrocartilagenous cells seen within the tendon proper, other than along its perimeter. The tendon at the tibial insertion continued to have fairly typical semitendinosus appearances, with cigar-shaped fibrocytes oriented parallel to crimped collagen fibers. In comparison, the collagen fibers and cells were not organized in an orderly fashion within the femoral insertion. In addition, there was no metachromatic staining of the healing tendon substance within the tibia, and relatively quiescent appearing marrow was still seen surrounding it. Scar tissue within each insertion was also distinctly different. Within the tibial insertion, scar occurred around the perimeter of the tendon and consisted of very loosely arranged and connected non-crimped collagen fibers, small round cells, adipose tissue and elements representing early blood vessels. In contrast, scar tissue within the femoral insertion was distributed throughout the tendon substance and consisted of relatively tightly interwoven collagen fibers.
Table 1 Characteristics of tendon tissue appearance and scar tissue location Weeks
0 6 12 24
Tendon tissue appearance
Scar tissue location
Tibia
Femur
Tibia
Femur
Rope-like Organized fibers Rope-like Organized fibers Rope-like Organized fibers Rope-like Organized fibers
Rope-like Organized fibers Bulb-like Fibrocartilagenous Bulb-like Bony protrusions into tendon Substantially replaced with osseous material
None
None
Perimeter
Throughout
Perimeter
Throughout
Perimeter
Throughout
S.R.M. Grassman et al. r The Knee 9 (2002) 21᎐26
24
Table 2 Observation of different cells and hypercellular areas Weeks
0 6 12 24
Cigar-shaped
Hypercellular
Tibia
Femur
⻬ ⻬ ⻬ ⻬
⻬
Tibia
Round-shaped Femur ⻬ ⻬ ⻬
3.2.3. Twel¨ e weeks The femoral insertion appeared to have undergone considerable osteogenesis over the 12-week healing period. The tendon in the femoral insertion in the 12-week complexes still had a bulb shape, but now bone protruded into the tendon substance. Amongst the 12-week complexes, osseous protrusions were randomly located around the area of the femoral insertion only. The tendon traveling over the tibia and into the tibial insertion still had its rope-like shape.
Chondrocytic-like
Tibia
Femur
⻬
⻬ ⻬ ⻬
Tibia
Femur ⻬ ⻬ ⻬
At 12 weeks, the tendon end in the femoral insertion was still hypercellular with round cells and a few chondrocytic-like cells were still visible in each histological section. At this time period, there were many vascular elements within the femoral insertion. In comparison, the tibial insertion still had no chondrocytic-like cells and vessels were seen only along the tendon-bone and tendon-marrow interface. In the femoral insertion, the fingers of new tissue appeared to be associated with fatty deposits, vascular channels,
Fig. 2. Coronal section of a typical rabbit knee at low magnification showing differences between insertional areas. Note the tendon graft within the relatively hollow tibia space invokes a minimal cellular and scar response Žleft. while the other end within the femur Žright. is embedded within cellular callus. Also note that the healing response appears to correlate with the tendon being inserted into the cancellous bone in the femur vs. the ‘marrow space’ in the tibia Žhematoxylin and eosin =25..
S.R.M. Grassman et al. r The Knee 9 (2002) 21᎐26
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Fig. 3. Typical chondroid cellular response around and within the tendon end in the femur Žhematoxylin and eosin =300.. This is quite different than the minimal response around the tendon end in the tibia Žsee tibial end at top left in Fig. 2..
arterioles, holes, and loose and disorganized collagen fibers. At the tibial insertion, the tendon end had not changed, other than along the tendon-bone and tendon-marrow boundaries, where scar tissue was seen. Collagen fibers remained crimped and fibrocytes were relatively evenly-distributed and aligned with each other and with the collagen fibers. 3.2.4. Twenty-four weeks As in the 6-week and the 12-week autografts, the tendon still had a rope-like appearance along the tibia and into the tibial insertion. Within the femoral insertion, however, there was substantially less tendon material seen as osteogenesis engulfed it. As in the 12-week group, the tendon end protruding from the femur still had a swollen appearance with some adjacent protruding bone growth. Cell shapes within the femoral insertion and within the tibial insertion had not changed from 12 weeks. The only thing that did change from the previous time periods was that the cells in the femoral insertion were now relatively uniformly distributed. The femoral insertion still contained a large number of round-shaped cells and the tibial insertion still contained cigar-shaped fibrocytes with round-shaped cells only in scar tissue located along its tendon-bone and tendon-marrow boundaries.
4. Discussion Results of this study show that at all incorporation
times from 6 to 24 weeks, the healing of the free semitendinosis tendon in the femoral bone tunnel of this rabbit model was distinctly different from the healing at the opposite end of the same tendon placed in the tibial bone tunnel. Specifically, the amount and manner of osteogenesis in and around each insertion was notably different. At the femoral insertion, protruding bone apposition that had a cancellous architecture was observed, whereas, at the tibial insertion, modest bone apposition that had a cortical architecture was observed only along the tendon boundary. The reasons for this interesting difference in tendon graft healing are not entirely clear. The most obvious explanation would seem to relate to the normal intrinsic differences between the internal architecture of the femur and tibia in this model, vis-a-vis ` a major difference in cancellous bone quantity, quality and distribution. The entire metaphyseal area of the normal rabbit femur is filled with cancellous bone, providing a relatively firm bed into which a tendon graft used can be fixed. The rabbit tibial metaphysis, however, contains relatively little cancellous bone and is instead filled with what can best be described as loose but very cellular ‘marrow’. Despite the anatomic similarity of the tibia and femur, both being so-called ‘metaphyseal’ regions of these respective bones, each is quite distinct in terms of its internal architecture. This architecture almost certainly reflects major differences in the intrinsic biological responses of each site when injured. Insertion of a tendon graft into each site has clearly invoked a much different cellular response, which likely has quite different functional
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consequences. Based on these results it would seem likely that insertion of a tendon graft into a cancellous bony tunnel would induce a much more rapid and firm attachment than insertion into a similar tunnel in a marrow-filled space. This principle that bone-specific graft incorporation depends on cancellous bone architecture should be tested in other bones to rule out other potential bone-specific factors; for example, loading environment or blood supply. Interestingly, the healing of the tendon in the femoral insertion of the current model appears to be similar to the description given by Forward and Cowan w1x, Whiston and Walmsley w2x and Liu et al. w3x, for the healing of tendon grafts within drill holes in the lapine calcaneus. It is also similar to that shown by Rodeo et al. w4x for healing of an extensor tendon end placed into a drill hole in the proximal tibial metaphysis in a canine model. However, the healing of the tendon in the tibial insertion observed in the current model appears more similar to the healing of flexor carpi radialis tendon in a drill hole in the radial metaphysis in a guinea pig reported by Hausman et al. w5x. These differences support the notion of either bone-, site- or model-specificity of tendon graft healing. In describing the processes of tendinous healing into bone Forward and Cowan w1x and Lui et al. w3x likened it to fracture healing, through formation of a fracture callus. Fractures that occur in cortical bone are known to heal in a different manner w12᎐14x than those in cancellous bone w14x. This may explain all of the differences found in the current study. In summary, the clear clinical implication of these results is that graft healing is different in different bony environments: graft incorporation into a cancellous bone being superior to that in a marrow space. In fact, we speculate that the bony environment is a greater determinant of healing success than even graft type Žfree tendon graft or bone-tendon-bone graft.. In our rabbit MCL reconstruction model, the femur was cancellous-filled and the tibia was marrow-dominated. The human femur and tibia are both more similar to the cancellous-filled rabbit femur. Other locations in the human where free tendon grafts are performed, however, may be more marrow-dominated, like the rabbit tibia Že.g. phalanges or metacarpals, etc... The salient point is that tendon grafts are performed in a variety of locations throughout the human body and successful graft incorporation is likely dependent on the bony environment receiving the graft.
Acknowledgements The authors gratefully acknowledge the administrative assistance of Judy Crawford, as well as the financial support of The Arthritis Society of Canada, the Canadian Institutes of Health Research, the Alberta Heritage Foundation for Medical Research, and the McCaig Professorship Fund. References w1x Forward AD, Cowan RJ. Tendon suture to bone. J Bone Joint Surg ŽAm. 1963;45Ž4.:807᎐823. w2x Whiston TB, Walmsley R. Some observations on the reaction of bone and tendon after tunnelling of bone and insertion of tendon. J Bone Joint Surg ŽBr. 1960;42Ž2.:377᎐386. w3x Liti SH, Panossian V, Al-Shaikh R et al. Morphology and matrix composition during early tendon to bone healing. Clin Orthop 1997;339:253᎐260. w4x Rodeo SA, Arnoczky SP, Torzilli PA, Hidaka C, Warren RF. Tendon-healing in a bone tunnel. J Bone Joint Surg ŽAm. 1993;75Ž12.:1795᎐1803. w5x Hausman M, Bain S, Rubin C. Reluctance of metaphyseal bone to heal to tendon: histologic evidence for poor mechanical strength. Trans Orthop Res Soc 1989;14:277. w6x Frank CB, Loitz BJ, Shrive NG. Injury location affects ligament healing. A morphologic and mechanical study of healing rabbit medial collateral ligament. Acta Orthop 1995;66Ž5.:455᎐462. w7x King GJW, Marchuk LL, Shrive NG, Frank CB. Fresh extraarticular autografts are repopulated by extrinsic cells. Transactions of the 2nd Combined Meeting of the Orthopaedic Research Societies of USA, Japan, Canada and Europe, 1995, p. 96. w8x Matyas JR. The Structure and Function of the Insertions of the Rabbit Medial Collateral Ligament: an Experimental Morphological and Biomechanical Study of the Insertions of the Medial Collateral Ligament in the Skeletally Mature Rabbit. Doctoral Thesis. Calgary, Canada: University of Calgary, Graduate School of Medical Sciences, 1990. w9x Matyas JR, Anton MG, Shrive NG, Frank CB. Stress governs tissue phenotype at the femoral insertion of the rabbit MCL. J Biomech 1995;28Ž2.:147᎐157. w10x Drury RAB, Wallington EA, Cameron SR. Preparation of tissues for microtomy. Carletons Histological Technique. New York: Oxford University Press, 1967:62᎐63. w11x Matyas JR, Anton M, Frank C. Whole joint embedding in polyŽmethymethacrylate .: a method for preparing intact musculoskeletal organs for histology and 3-D reconstruction. Biotech Histochem 1997;72:152᎐157. w12x Chamley J, Baker SL. Compression arthrodesis of the knee. A clinical and histological study. J Bone Joint Surg ŽBr. 1952;34Ž2.:187᎐199. w13x Elkouri ER. Review of cancellous and cortical bone healing after fracture or osteotomy. J Am Podiatry Assoc 1982; 72Ž9.:464᎐466. w14x Frost HM. The biology of fracture healing: an overview for clinicians. Part II. Clin Orthop 1989;248:294᎐309.