"Effect of the Intra-Articular Environment on Healing of the Ruptured Anterior Cruciate Ligament"
By Martha Meaney Murray, MD*,
Department of Orthopaedic Surgery,
Brigham and Women's Hospital, Boston, MA
A Perspective on
"Revision Anterior Cruciate Surgery with Use of
Bone-Patellar Tendon-Bone Autogenous Grafts"
by Frank R. Noyes, MD, and Sue D. Barber-Westin, BS
(JBJS, August 2001)
The injury response of the intra-articular anterior cruciate ligament differs from that of the extra-articular medial collateral ligament in several ways, the most important of which is the lack of formation of any bridging tissue in the gap between the ligament remnants1. In extra-articular tissues that heal successfully, a fibrin clot forms that acts as a scaffold which is invaded by fibroblasts and then is gradually replaced by collagen fibers. This sequence of events has been observed in the healing process in both tendon2,3 and the medial collateral ligament4. In the intra-articular milieu, however, it has been demonstrated that a fibrin clot does not form because of the presence of fibrinolytic enzymes5,6. An experimental study of hemarthroses in canine knees (induced by drilling across the joint) demonstrated no clot formation five minutes to four hours after injury, with only bloody fluid found within the joint6. In human knees undergoing surgery in the first ten to twenty-one days after injury, the existing hemarthrosis was noted to be a viscous liquid incapable of holding the remnants of the ruptured ligament together1. The lack of formation or the premature dissolution of the fibrin clot in the intra-articular environment, in combination with the twelve-week latent period preceding revascularization and cell proliferation, suggests that one reason for nonhealing of a ruptured human anterior cruciate ligament is the lack of a bridging scaffold which reapproximates the ligament ends until the proliferative cell response occurs.
A recent study of ruptured human anterior cruciate ligaments retrieved at the time of ligament reconstruction demonstrated four phases in the injury response: an inflammatory phase, an epiligamentous regeneration phase, a proliferative phase, and a remodeling phase. The inflammatory phase, which occurred during the first three weeks after rupture, was characterized by the migration of leukocytes from local vessels (Fig. 2-a), intimal hyperplasia of the vessels (Fig. 2-b), dilatation of blood vessels (Fig. 2-c), and erythrocyte invasion and crimp disruption (Fig. 2-d) in some areas. At no time was there evidence of formation of a fibrin clot or other bridging tissue between the femoral and tibial remnants of the ruptured anterior cruciate ligament. Anterior cruciate ligaments retrieved between three and eight weeks after rupture (the epiligamentous regeneration phase) appeared quiescent, with a loss of the cellular longitudinal alignment (Figs. 3-a and 3-b) and a gradual resynovialization of the ligament remnant (Fig. 3-c). During the proliferative phase, which occurred between sixteen and twenty weeks after injury, increases in cell density were seen (Fig. 4-a) as well as the formation of new capillaries near the rupture site (Fig. 4-b) and of larger vessels more distally (Fig. 4-c). The epiligamentous layer was found to be well vascularized during this phase (Figs. 4-d and 4-e). Between six months and three years after rupture, the ligament remnants had undergone remodeling and retraction. In retrieved specimens, both the cell density (Figs. 1-a and 5-a) and the blood vessel caliber (Figs. 1-b and 5-b) were similar to those seen in the intact ligament.
Systematic histologic analysis of the human anterior cruciate ligament allows us to understand its proclivity to heal insufficiently following primary or secondary reconstruction. Three distinct elements must be analyzed: the intrinsic cells, the epiligamentous tissue, and the extracellular matrix. The cellular distributions in the human anterior cruciate ligament vary7,8, with regions of relatively low cell density and vascularity seen distally (Fig. 1-a) and regions of relatively high cell density and vascularity seen proximally (Fig. 1-b). The surface of the ligament is composed of epiligamentous tissue with a synovial sheath (Fig. 1-c). The collagen of the extracellular matrix forms a crimp (Fig. 1-d)9-13.
Figures 1-a through 1-d: Photomicrographs of the intact human anterior cruciate ligament. (1-a through 1-c: Red designates a positive stain for alpha-smooth muscle actin.)
1-a. The distal region of the ligament, with a low density of rounded cells, some of which stain positively for alpha-smooth muscle actin.
1-b. The more proximal region of the ligament, showing both a higher density of elongated cells and intrafascicular blood vessels (BV).
1-c. The epiligamentous tissue covering the intact anterior cruciate ligament, with a relatively high concentration of blood vessels (BV) and a synovial layer on the surface.
1-d. The regular collagen waveform, or crimp, seen in the intact anterior cruciate ligament, with a periodicity of around 35 micrometers. (Partially polarized light).
Figures 2-a through 2-d: Photomicrographs of the ruptured human anterior cruciate ligament ten days after rupture (during the inflammatory phase). (Red designates a positive stain for alpha-smooth muscle actin.)
2-a. Leukocytes migrating from local vessels into the injured area.
2-b. Intimal hyperplasia of the blood vessel (BV) walls.
2-c. Enlargement of the lumens of the blood vessels (BV) in the epiligamentous tissue, which was not disrupted at the time of injury.
2-d. Loss of the regular crimp waveform with erythrocyte infiltration in the injured tissue. Crimp is maintained in small areas of the injured tissue (seen at center), but lost in many areas (upper left-hand corner).
Figures 3-a through 3-c: Photomicrographs of the ruptured human anterior cruciate ligament at eight weeks after rupture (the epiligamentous regeneration phase). (Red designates a positive stain for alpha-smooth muscle actin.)
3-a. Small areas of a relatively high density of rounded cells with no discernible orientation.
3-b. Loss of the pattern of cellular alignment but with cell density maintained. Note the decrease in intimal hyperplasia in the vessel shown (BV).
3-c. Early reformation of the synovial layer over the injury site. The arrow points to a cell that is positive for alpha-smooth muscle actin in the synovial layer.
Figures 4-a through 4-e: Photomicrographs of the ruptured human anterior cruciate ligament at sixteen to twenty weeks after rupture (the proliferative phase). (Red designates a positive stain for alpha-smooth muscle actin.)
4-a. The increased cell density in the ligament remnant 6 mm from the rupture site. The cells are partially aligned, with crimp beginning to regain an appearance similar to that in the intact ligament.
4-b. New capillary formation in the ligament remnant 2 mm from the rupture site.
4-c. A branching blood vessel (BV) seen 6 mm from the rupture site with erythrocytes in the lumen, suggesting continuing patency and flow.
4-d. Epiligamentous tissue at the rupture site. The synovial layer has reformed, and the epiligamentous tissue has a higher number of blood vessels than that seen at the eight-week time-point.
4-e. Epiligamentous tissue adjacent to the rupture site with more distinct blood vessels (BV) and a more organized synovial layer.
Figures 5-a through 5-c: Photomicrographs of the ruptured human anterior cruciate ligament at fifty-two weeks after rupture (the remodeling phase). (Red designates a positive stain for alpha-smooth muscle actin.)
5-a. The cell density is similar to that in the intact ligaments, and the cell nuclei are ovoid in shape.
5-b. The appearance of blood vessels (BV) in the remnant, 4 mm from the rupture site. Note the lack of vascular hyperplasia.
5-c. The epiligamentous tissue in the ligament, showing fewer blood vessels at this time-point. The synovial layer remains, but is less organized than in the earlier phases.
But if the main problem is lack of reapproximation of the ligament ends, why doesn't primary repair work? In extra-articular tissues, macroscopic reapproximation with use of sutures is supplemented by the formation of blood clot in the injury site, which effectively immobilizes the ends of the ruptured tissue and maintains their microscopic contact. However, this blood clot is not present in the case of intra-articular tissues, and thus sufficient microscopic contact does not occur despite macroscopic reapproximation. The lack of microscopic contact between ligament fascicles in the synovial fluid environment may prevent cell migration and tissue regeneration. A recent in vitro study examining the healing response of the human anterior cruciate ligament demonstrated that anterior cruciate ligament cells would migrate to an adjacent scaffold, but not in the presence of gaps as small as fifty microns (less than the length of an anterior cruciate ligament cell (Fig. 6)14. Moreover, despite macroscopic reapproximation of the tissue fascicles after disruption, no cell or tissue deposition was seen at the transection site unless a bridging scaffold was present. Therefore, it appears likely that one reason for the failure of primary repair of the anterior cruciate ligament is the lack of a biologic scaffold to maintain microscopic contact and facilitate cell migration and scar formation between the reapproximated fascicles. In extra-articular tissues, blood clot bridges the microscopic gap and serves as this preliminary scaffold.
This histologic analysis has led us to hypothesize that one of the reasons for the inability of the anterior cruciate ligament to heal after injury is the lack of formation of a blood clot, or bridge, across the rupture site and that the absence of this clot causes loss of contact between the ligament ends. This hypothesis is one of many proposed to explain the failure of the anterior cruciate ligament to heal, which include lack of vascular supply15,16, deficits in intrinsic cell migration17, an impaired response to growth factors18, and the environmental effect of synovial fluid on cell physiology19,20. A combination of all of these factors may be involved.
Figure 6: Photomicrograph of human anterior cruciate ligament cells migrating from a tissue explant in culture, demonstrating cell lengths in excess of 50 micrometers.
How do we overcome this problem? One potential solution is the surgical implantation of an engineered substitute for blood clot in the rupture site. This tissue-engineering approach requires design of a non-toxic surgical implant that maintains microscopic contact, resists degradation by synovial fluid, and stimulates cell invasion and the regeneration of the extracellular matrix in the rupture site. Research on collagen-based scaffolds has demonstrated the ingrowth potential of cells from the human anterior cruciate ligament in vitro14 as well as collagen production by the cells that invade the scaffold. Additional studies, on tissue engineering of menisci, have also focused on the interaction of cells and scaffolds21,22 and the use of fibrin clot as a scaffold for the enhancement of healing of meniscal tears23-25. Extensive research characterizing the interaction between cells and matrix in articular cartilage before and after injury26-29 has led to the investigation of polymer scaffolds30, periosteal flaps31-33, collagen scaffolds34 and gels35,36, and autologous chondrocyte transplantation37-39 as tools and techniques for articular cartilage repair. Further optimization of bioengineered substitutes for blood clot can be accomplished with the adsorption of growth factors40-43 or gene delivery by viral vectors44,45, seeding with autogenous ligament cells46-48, mesenchymal stem cells49-51, or genetically altered cells45,52.
Further study is needed to better define the spectrum of the normal histology of the anterior cruciate ligament. For example, variations between species, the elucidation of which could be helpful in selecting appropriate animal models for in vivo studies, as well as the effects of aging and gender on the histology of the human ligament have yet to be defined. An understanding of the effects of histologic changes on the mechanical properties and function of the ligament is important in assessing new treatment techniques. For example, recent concerns about early ligament rupture following the use of radiofrequency treatment for the lax anterior cruciate ligament53 raise questions about the effect of histologic changes, including cell necrosis and collagen denaturation, on the ability of the ligament to maintain its structural integrity over time.
In summary, the histologic study of human intra-articular tissues after injury provides insight into factors that contribute to their failure to heal. In the case of the anterior cruciate ligament, the lack of blood clot formation and the failure to re-establish both macroscopic and microscopic contact between the ligament ends contribute to the inability of the ligament to heal. The other phases of healing seen in extra-articular tissues (revascularization and proliferation) do occur in this intra-articular ligament. The development of implantable engineered substitutes for blood clot will hopefully emerge from our ongoing analysis of the failure of these ligaments to heal and will result in new surgical treatments for intra-articular pathology.
*In support of her research or preparation of this manuscript, the author received grants or outside funding from NIAMS (Grant No. R03 AR46356-01), the Center for the Integration of Medicine and Innovative Technology (CIMIT), and Partner's Department of Orthopaedic Surgery. The author did not receive payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the author is affiliated or associated.
References
1. Murray MM, Martin SD, Martin TL, Spector M. Histological changes in the human anterior cruciate ligament after rupture. J Bone Joint Surg Am. 2000;82:1387-97.
2. Buck RC. Regeneration of tendon. J Pathol Bacteriol. 1953;66:1-18.
3. Peach R, Williams G, Chapman JA. A light and electron optical study of regenerating tendon. Am J Pathol. 1961;38:495-513.
4. Frank C, Amiel D, Akeson WH. Healing of the medial collateral ligament of the knee. A morphological and biochemical assessment in rabbits. Acta Orthop Scand. 1983;54:917-23.
5. Andersen RB, Gormsen J. Fibrin dissolution in synovial fluid. Acta Rheum Scand. 1970;16:319-33.
6. Harrold AJ. The defect of blood coagulation in joints. J Clin Path. 1961;14:305-8.
7. Murray MM, Spector M. Fibroblast distribution in the anteromedial bundle of the human anterior cruciate ligament: the presence of alpha-smooth muscle actin-positive cells. J Orthop Res. 1999;17:18-27.
8. Petersen W, Tillmann B. Structure and vascularization of the cruciate ligaments of the human knee joint. Anat Embryol (Berl). 1999;200:325-34.
9. Clark JM, Sidles JA. The interrelation of fiber bundles in the anterior cruciate ligament. J Orthop Res. 1990;8:180-8.
10. Danylchuk KD, Finlay JB, Krcek JP. Microstructural organization of human and bovine cruciate ligaments. Clin Orthop. 1978;131:294-8.
11. Harner CD, Livesay GA, Kashiwaguchi S, Fujie H, Choi NY, Woo SL. Comparative study of the size and shape of human anterior and posterior cruciate ligaments. J Orthop Res. 1995;13:429-34.
12. Mommersteeg TJ, Blankevoort L, Kooloos JG, Hendriks JC, Kauer JM, Huiskes R. Nonuniform distribution of collagen density in human knee ligaments. J Orthop Res. 1994;12:238-45.
13. Mommersteeg TJ, Kooloos JG, Blankevoort L, Kauer JM, Huiskes R, Roeling FQ. The fibre bundle anatomy of human cruciate ligaments. J Anat. 1995;187:461-71.
14. Murray MM, Martin SD, Spector M. Migration of cells from human anterior cruciate ligament explants into collagen-glycosaminoglycan scaffolds. J Orthop Res. 2000;18:557-64.
15. Ghadially FN, Wedge JH, Lalonde JM. Experimental methods of repairing injured menisci. J Bone Joint Surg Br. 1986;68:106-10.
16. Heatley FW. The meniscus--can it be repaired? An experimental investigation in rabbits. J Bone Joint Surg Br. 1980;62:397-402.
17. Geiger MH, Green MH, Monosov A, Akeson WH, Amiel D. An in vitro assay of anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cell migration. Connect Tissue Res. 1994;30:215-24.
18. Spindler KP, Imro AK, Mayes CE, Davidson JM. Patellar tendon and anterior cruciate ligament have different mitogenic responses to platelet-derived growth factor and transforming growth factor beta. J Orthop Res. 1996;14:542-6.
19. Andrish J, Holmes R. Effects of synovial fluid on fibroblasts in tissue culture. Clin Orthop. 1979;138:279-83.
20. Nickerson DA, Joshi R, Williams S, Ross SM, Frank C. Synovial fluid stimulates the proliferation of rabbit ligament. Fibroblasts in vitro. Clin Orthop. 1992;274:294-9.
21. Arnoczky SP. Building a meniscus. Biologic considerations. Clin Orthop. 1999;367 Suppl:S244-53.
22. Ibarra C, Koski JA, Warren RF. Tissue engineering meniscus: cells and matrix. Orthop Clin North Am. 2000;31:411-8.
23. Ishimura M, Ohgushi H, Habata T, Tamai S, Fujisawa Y. Arthroscopic meniscal repair using fibrin glue. Part I: Experimental study. Arthroscopy. 1997;13:551-7.
24. McAndrews PT, Arnoczky SP. Meniscal repair enhancement techniques. Clin Sports Med. 1996;15:499-510.
25. van Trommel MF, Simonian PT, Potter HG, Wickiewicz TL. Arthroscopic meniscal repair with fibrin clot of complete radial tears of the lateral meniscus in the avascular zone. Arthroscopy. 1998;14:360-5.
26. Buckwalter JA, Mankin HJ. Articular cartilage: tissue design and chondrocyte-matrix interactions. Instr Course Lect. 1998;47:477-86.
27. Mankin HJ. The response of articular cartilage to mechanical injury. J Bone Joint Surg Am. 1982;64:460-6.
28. Reimann I, Mankin HJ, Trahan C. Quantitative histologic analyses of articular cartilage and subchondral bone from osteoarthritic and normal human hips. Acta Orthop Scand. 1977;48:63-73.
29. Treadwell BV, Mankin HJ. The synthetic processes of articular cartilage. Clin Orthop. 1986;213:50-61.
30. Perka C, Sittinger M, Schultz O, Spitzer RS, Schlenzka D, Burmester GR. Tissue engineered cartilage repair using cryopreserved and noncryopreserved chondrocytes. Clin Orthop. 2000;378:245-54.
31. Carranza-Bencano A, Garcia-Paino L, Armas Padron JR, Cayuela Dominguez A. Neochondrogenesis in repair of full-thickness articular cartilage defects using free autogenous periosteal grafts in the rabbit. A follow-up in six months. Osteoarthritis Cartilage. 2000;8:351-8.
32. Katsube K, Ochi M, Uchio Y, Maniwa S, Matsusaki M, Tobita M, Iwasa J. Repair of articular cartilage defects with cultured chondrocytes in Atelocollagen gel. Comparison with cultured chondrocytes in suspension. Arch Orthop Trauma Surg. 2000;120:121-7.
33. Wang CJ, Chen CY, Tsung SM, Chen WJ, Huang HY. Cartilage repair by free periosteal grafts in the knees of pigs: a histologic study. J Formos Med Assoc. 2000;99:324-9.
34. Sellers RS, Zhang R, Glasson SS, Kim HD, Peluso D, D'Augusta DA, Beckwith K, Morris EA. Repair of articular cartilage defects one year after treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2). J Bone Joint Surg Am. 2000;82:151-60.
35. Kawamura S, Wakitani S, Kimura T, Maeda A, Caplan AI, Shino K, Ochi T. Articular cartilage repair. Rabbit experiments with a collagen gel-biomatrix and chondrocytes cultured in it. Acta Orthop Scand. 1998;69:56-62.
36. Wakitani S, Goto T, Young RG, Mansour JM, Goldberg VM, Caplan AI. Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel. Tissue Eng. 1998;4:429-44.
37. Minas T, Nehrer S. Current concepts in the treatment of articular cartilage defects. Orthopedics. 1997;20:525-38.
38. Nehrer S, Spector M, Minas T. Histologic analysis of tissue after failed cartilage repair procedures. Clin Orthop. 1999;365:149-62.
39. Richardson JB, Caterson B, Evans EH, Ashton BA, Roberts S. Repair of human articular cartilage after implantation of autologous chondrocytes. J Bone Joint Surg Br. 1999;81:1064-8.
40. Frank C, Shrive N, Hiraoka H, Nakamura N, Kaneda Y, Hart D. Optimisation of the biology of soft tissue repair. J Sci Med Sport. 1999;2:190-210.
41. Hildebrand KA, Woo SL, Smith DW, Allen CR, Deie M, Taylor BJ, Schmidt CC. The effects of platelet-derived growth factor-BB on healing of the rabbit medial collateral ligament. An in vivo study. Am J Sports Med. 1998;26:549-54.
42. Spindler KP, Mayes CE, Miller RR, Imro AK, Davidson JM. Regional mitogenic response of the meniscus to platelet-derived growth factor (PDGF-AB). J Orthop Res. 1995;13:201-7.
43. Stanislawski L, De Nechaud B, Christel P. Plasma protein adsorption to artificial ligament fibers. J Biomed Mater Res. 1995;29:315-23.
44. Hildebrand KA, Deie M, Allen CR, Smith DW, Georgescu HI, Evans CH, Robbins PD, Woo SL. Early expression of marker genes in the rabbit medial collateral and anterior cruciate ligaments: the use of different viral vectors and the effects of injury. J Orthop Res. 1999;17:37-42.
45. Menetrey J, Kasemkijwattana C, Day CS, Bosch P, Fu FH, Moreland MS, Huard J. Direct-, fibroblast- and myoblast-mediated gene transfer to the anterior cruciate ligament. Tissue Eng. 1999;5:435-42.
46. Bellincampi LD, Closkey RF, Prasad R, Zawadsky JP, Dunn MG. Viability of fibroblast-seeded ligament analogs after autogenous implantation. J Orthop Res. 1998;16:414-20.
47. Dunn MG, Liesch JB, Tiku ML, Zawadsky JP. Development of fibroblast-seeded ligament analogs for ACL reconstruction. J Biomed Mater Res. 1995;29:1363-71.
48. Lin VS, Lee MC, O'Neal S, McKean J, Sun KL. Ligament tissue engineering using synthetic biodegradable fiber scaffolds. Tissue Eng. 1999;5:443-52.
49. Johnstone B, Yoo JU. Autologous mesenchymal progenitor cells in articular cartilage repair. Clin Orthop. 1999;367 Suppl:S156-62.
50. Mosca JD, Hendricks JK, Buyaner D, Davis-Sproul J, Chuang LC, Majumdar MK, Chopra R, Barry F, Murph M, Thiede MA, Junker U, Rigg RJ, Forestell SP, Bohnlein E, Storb R, Sandmaier BM. Mesenchymal stem cells as vehicles for gene delivery. Clin Orthop. 2000;379 Suppl;S71-90.
51. Solchaga LA, Dennis JE, Goldberg VM, Caplan AI. Hyaluronic acid-based polymers as cell carriers for tissue-engineered repair of bone and cartilage. J Orthop Res. 1999;17:205-13.
52. Gerich TG, Kang R, Fu FH, Robbins PD, Evans CH. Gene transfer to the patellar tendon. Knee Surg Sports Traumatol Arthrosc. 1997;5:118-23.
53. Perry JJ, Higgins LD. Anterior and posterior cruciate ligament rupture after thermal treatment. Arthroscopy. 2000;16:732-6.
|
