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Meniscal tissue regeneration using a collagenous biomaterial derived from porcine small intestine submucosa
Meniscal Tissue Regeneration Using a Collagenous Biomaterial Derived From Porcine Small Intestine Submucosa Jonathan A. Gastel, M.D., William R. Muirhead, M.D., Joseph T. Lifrak, M.D., Paul D. Fadale, M.D., Michael J. Hulstyn, M.D., and Daniel P. Labrador, B.S.
Purpose: The present investigation is a preliminary study designed to evaluate the use of a collagen-based biomaterial, chemically unaltered porcine small intestine submucosa (SIS), as a scaffold for meniscal tissue regeneration. Type of Study: Basic research. Methods: Surgical defects were created in the lateral menisci of 12 mature New Zealand white rabbits. The defects were repaired with a similarly shaped and sized wedge of a new collagenous biomaterial (SIS) and sutured in place. The opposite knees served as controls by creating a defect in the lateral meniscus without filling with SIS graft. Full cage activity was allowed until the animals were killed at 4, 12, and 24 weeks. Results: At 4 weeks, the graft material retained its physical position and grossly appeared soft and translucent. Histologically, cellular elements had infiltrated between the laminates of the graft. At 12 weeks, the graft grossly appeared more solid and opaque. Histologically, the host meniscal fibrochondrocytes were seen streaming into the peripheral margin of the graft. Early repopulation of the graft with apparently differentiated meniscal tissue was observed. At 24 weeks, the meniscus defect was grossly healed across and looked virtually normal: the normal meniscal shape, contour, consistency, and color had been replicated. Histologically, the healing tissue showed infiltration of what appeared to be meniscal fibrochondrocytes and connective tissue resembling the host meniscal tissue. The graft was nearly totally replaced by host tissue. Conclusions: This pilot animal study demonstrates that the multilaminated collagenous graft is conducive for cellular repopulation with host meniscal elements, and, by 24 weeks, is capable of supporting complete healing of a large meniscal defect. Key Words: Meniscus—Meniscal regeneration—Meniscal repair—Animal study—Collagenous graft—Small intestine submucosa.
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n intact meniscus is clearly vital to the biomechanical and long-term function of the knee joint. The current surgical options for a meniscal tear are repair and debridement. Despite early evidence of the potential for meniscal healing by King1 in the 1930s and Fairbanks’ observations2 of postmeniscectomy osteoarthritic changes in 1948, total
From the Department of Orthopaedics, Brown University, Rhode Island Hospital, Providence, Rhode Island, U.S.A. Supported by an educational grant from DePuy, Inc., Warsaw, Indiana. Address correspondence and reprint requests to Paul D. Fadale, M.D., University Orthopaedics, 2 Dudley Street, Providence, RI 02903, U.S.A. © 2001 by the Arthroscopy Association of North America 0749-8063/01/1702-2038$35.00/0 doi:10.1053/jars.2001.20959
meniscectomy was the standard operative treatment for a torn meniscus until the 1970s. Studies have since proved the functional significance of the menisci to knee stability,3 force transmission,4 and shock absorption.5 Thus, preservation of meniscal tissue became a primary treatment goal. Partial meniscectomy has been shown to preserve knee biomechanics to some degree and to offset articular cartilage changes better than total meniscectomy, but it may still lead to a significant increase in radiographic osteoarthritic changes.6 Repair and preservation of the entire injured meniscus has been shown to be beneficial to the maintenance of normal knee biomechanics7 and important in preventing abnormal articular surface wear. However, the inherent healing capacity of the human meniscus has been shown to be lacking in the central third and
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very limited in the middle third of this poorly vascularized tissue.8 Thus, the applicability of meniscal repair is severely limited by both tear morphology and location. Moreover, the results of attempted repairs are highly variable. These factors have led to a wide variety of techniques aimed at augmenting or inducing the healing of meniscal tissue. Efforts have been directed at augmenting the innate healing response as well as implanting grafts or prostheses to substitute for the irreparably damaged meniscal tissue. Augmentation of healing has been attempted clinically with the insertion of fibrin clot in the repair site9 or by implantation of a vascularized synovial flap.10 The results with meniscal prostheses have been discouraging. A polyurethane-coated Dacron prosthesis (Du Pont, Wilmington, DE) was shown to provide some protection to the articular surface, but did not reproduce the biomechanical functions of the normal meniscus.11 Transplantation techniques have also been used with fresh menisci or menisci preserved by deep freezing, freeze drying (lyophilization), and controlled-rate freezing (cryopreservation). The latter has become the mode of choice based on several factors. This technique has been shown to result in the least amount of cellular destruction, although Arnoczky et al.12 have shown that as little as 10% of the cells remain metabolically active immediately after preservation and only 3% after 12 weeks. Similar to what has been observed in articular cartilage transplantation studies, preserving cell viability should maximize the results of meniscal transplantation.13,14 Arnoczky et al.15 showed this with deep-frozen menisci in a canine model with cellular repopulation thought to be stemming from the synovial tissue. However, good short-term results have also been found with other tissues, such as the anterior cruciate ligament and patellar tendon, that show no signs of donor cellular viability shortly after transplantation.16 Meniscal transplantation has obvious inherent drawbacks. There has been significant shrinkage of the graft with preserved menisci.17 The need for proper preoperative sizing of the transplant requires adequate storage and timely access to a large number of samples. Procurement of adequate samples is limited by the donor supply (e.g., relatively young, disease-free patients with no premorbid meniscal pathology). In addition, the threat of disease transmission is always an issue with human allogeneic tissue transplantation.
The use of a fabricated collagenous graft or prosthesis in meniscal reconstruction and repair has also been studied. Basing their investigation on the use of biologically resorbable scaffolds in the replacement of human skin by Yannas and Burke,18 Stone et al.19 tested the use of a copolymeric collagen scaffold derived from bovine Achilles tendon. After residual blood proteins and watersoluble materials were washed from the tissue, the collagen was purified and cross-linked with glycosaminoglycans. Further cross-linking and lyophilization yielded a collagen scaffold that was shown in vitro to be conducive to cellular migration. The fabricated collagen scaffold was trimmed to size and implanted in canine stifle joints in place of the medial meniscus, which had undergone an 80% subtotal resection. This graft effectively supported meniscal fibrochondrocyte ingrowth and meniscal tissue regeneration. The present investigation is a preliminary study designed to evaluate the use of a collagen-based biomaterial, chemically unaltered porcine small intestine submucosa (SIS), as a scaffold for meniscal tissue regeneration. The SIS biomaterial was selected based on its performance as a ligament graft20 and in vascular surgery studies.21,22 In its use as a vascular graft, it was shown not only to be completely incorporated but, more importantly, the distinct layers of the arterial wall were reconstituted. Thus, the cells that repopulated the graft survived and differentiated in accordance with their physical environment. This characteristic is vital to the success of any biomaterial used in meniscal tissue regeneration. Simply producing a living, meniscus-shaped pad of fibrocartilage is not enough; reconstitution of the intricate collagen fibril networks by living fibrochondrocytes must take place for successful functional meniscal regeneration.
METHODS Bilateral, wedged-shaped, segmental surgical defects were created in the central third of the lateral menisci of 12 male New Zealand White rabbits weighing 3.5 to 4 kg. The defect width was 3 mm, approximately 30% of the meniscus. The SIS biomaterial was supplied by the sponsor (DePuy, Inc.) as sterile, 3-mm thick 1-cm squares. An appropriate triangular graft was fashioned from each sheet and placed in the right limb of each animal and anchored in place with 2 horizontal nonabsorbable sutures. SIS consists of a 0.1-mm thick sheet of tissue composed of the tunica submucosa with attached stratum compactum and muscularis mucosa. Mechanical
MENISCAL TISSUE REGENERATION removal of the other intestinal layers eliminates the need for the potentially damaging use of chemical treatments and preserves the endogenous collagenous and noncollagenous proteins present naturally in the tissue. The harvested tissue is subsequently rinsed in physiologic saline and immersed in an antibiotic solution. A laminate is then prepared by compressing these sheets together mechanically. The left limb of each animal had a partial lateral meniscectomy performed exactly as on the right side but no graft was placed in the defect. The animals were allowed to recover postoperatively for 1 week in standard cages. Afterward, they were kept in freerange pens to allow full range of motion and weight bearing during the survival period. The animals were killed in groups at 4, 12, and 24 weeks, and both lateral menisci were harvested and prepared for histological analysis. Surgical Procedure Approval for this study was obtained from the Rhode Island Hospital Animal Care and Use Committee. Each animal was anesthetized with an intramuscular injection of ketamine and xylazine. The animal was intubated and intravenous access gained. In a supine position, the hind limbs were washed with alcohol and povidone iodine. The animal was then draped to establish a sterile field. A lateral parapatellar approach to the knee was used to gain access to the lateral meniscus. A 3-mm wedge defect was excised from the central third of the meniscus with a No. 11 blade, taking care to protect the articular cartilage. The defect in the right knee was replaced with the wedge-shaped SIS implant, which was sutured to the anterior and posterior horns of the meniscus with 5-0 polypropylene. The defect in the left knee was left untreated. The capsule, deep fascia, and skin were closed in layers in all of the knees. Povidone iodine ointment was placed on the wound and the wound was dressed with collodion. No attempt with dressing or splint material was made to reduce postoperative range of motion. Postoperative antibiotics and analgesics were administered and the animal was allowed to recover. At sacrifice, the animals were anesthetized using a mixture of ketamine and xylazine. Following sedation, peripheral intravenous access was gained using a 22gauge angiocatheter in an ear vein. The animals were
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killed using 3 mL pentobarbital sodium, administered intracardiac. The menisci were excised and prepared for routine histologic analysis. The menisci from 2 animals from each time point were processed for routine histology; the remaining were evaluated grossly. The specimens were fixed in 10% neutral phosphate-buffered saline and formalin for 5 days. The specimens were then embedded in paraffin and parasagittal sections (perpendicular to the implant) were taken. These sections were stained with hematoxylin and eosin, and Masson’s trichrome.
RESULTS 4-Week Group At 4 weeks postoperatively, the SIS implant retained its overall laminar structure and organization. Exuberant cellular infiltration into the layers of the implant was seen under low- and high-power magnification (Figs 1A and 1B, respectively). Interestingly, there was an absence of giant cells or other signs of inflammation or foreign-body reaction. Although the implant remained in contact with the native meniscus, actual incorporation of the graft material was not observed. Some specimens showed a gap between the graft and the meniscus that was filled with a loosely organized fibrous tissue. The controls had a moderately cellular fibrous tissue filling the untreated defect. 12-Week Group At 12 weeks, the implant has started to lose its structure, but some of the implant’s laminar layers were still identifiable amidst the abundant cellular infiltration. The invading host tissue appears slightly less cellular, but more organized and fibrous in nature when compared with the earlier group (Fig 2, low power). Some new tissue was seen growing from the margin of the native meniscus into the defect (Fig 3, high power). The control side showed maturing fibrovascular tissue filling the defect. 24-Week Group At 24 weeks, 2 of the experimental specimens exhibited a gross appearance similar to that of a native uninjured meniscus (Fig 4). The repair appeared to have healed in continuity with new tissue that was opaque and shaped to the contours of the native me-
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FIGURE 1. (A) Low-power magnification view of exuberant cellular infiltration into the layers of SIS on the right, at 4 weeks. (B) High-power magnification of exuberant cellular infiltration into the layers of SIS (blue), at 4 weeks.
niscal tissue. Two specimens showed technical failure of the repair, with diastasis between the anterior and posterior portions of the native meniscus. Histologically, at 6 months, the experimental specimens showed more organized tissue resembling that
of the native meniscus (Fig 5). Tissue infiltration into the native meniscus was observed at the interface and fibers were also seen running through the implant. Remnants of the graft were no longer identifiable in the healing tissue, but the original nonabsorbable su-
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FIGURE 2. New tissue on the left abutting host meniscus on the right, at low power. New tissue is still cellular at 12 weeks, but seems to be becoming organized and fibrous.
tures still maintained their positions within the zone of tissue regeneration. The control side meniscus also showed significant signs of healing (Fig 6). On gross examination, this tissue appeared less densely opaque compared with the SIS side (Fig 7).
DISCUSSION This investigation was designed to evaluate the use of a collagen-based biomaterial as a scaffold for meniscal regeneration. The search for the optimum me-
FIGURE 3. High-power view of new tissue (right) growing from the margin of the native meniscus (left) into the defect.
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FIGURE 4.
Native uninjured meniscus of a rabbit.
niscal transplant involves all of the factors addressed above. Human meniscal transplantation has its inherent drawbacks, including sizing, availability, material property changes with preservation techniques, and the risk of disease transmission. Fabricated collagen scaffolds are promising, but the ideal physical construct of the collagen graft has yet to be defined, and the effect of the chemical treatments in the processing of these materials has not been determined. The optimal remodeling of the repopulated graft and regenerated meniscus is likely to be determined, at least in part, by the ultrastructure of the particular implant and its biologic milieu. Just as has been shown with artificial bone grafts or porous ingrowth prostheses, the nature of biologic processes are intimately related to their physical microenvironment. The physical properties of the multilaminated SIS graft and its biologically preserved nature affords a unique look at the regeneration capacity of meniscal tissue in this investigation. Preservation of the functional meniscus has become a primary goal of the knee surgeon. Repair of appropriate meniscal tears and minimizing debridement of irreparable tears has become the standard of care for meniscal injuries as the science regarding the menisci and their vital function has become better understood. Unfortunately, a significant proportion of acute meniscal tears are not amenable to repair and many degenerative tears involve a significant bulk of the meniscal body. Additionally, many patients have been left without any meniscal tissue as a result of total meniscectomy. These situations render the articular surfaces of the knee subject to abnormally high contact stresses leading to premature articular degenera-
tion. Baratz et al.7 have shown that the contact area in the tibiofemoral joint decreases from 6 cm2 with the meniscus intact to 2 cm2 after meniscectomy thus increasing the local contact forces. Similarly, it has been shown that the lateral meniscus bears 70% of the lateral compartment load and the medial meniscus bears 50% of the medial compartment load.23 The accessory functions of the meniscus in joint lubrication,24 shock absorption,3 and joint stability25,26 have also been well documented. The inability to preserve the meniscus in many clinical situations and the need to replace it in others has stimulated the search for a substitute, biologic or otherwise, for the native meniscus. A patient may present to the orthopaedist with vague knee symptoms and a remote history of total meniscectomy or acutely with a symptomatic and irreparable torn meniscus. In the former case, replacement of the meniscus may have a significant role in preventing or slowing the degenerative changes in the knee. In the more acute setting, the detrimental effects of partial meniscectomy may be avoided if the damaged portion can be repaired. These scenarios are examples of the clinical applicability of meniscal preservation that have encouraged investigators to pursue the possibility of using a biologic scaffold or implant to stimulate the regeneration of functional meniscal tissue. The encouraging use of the SIS biomaterial as a scaffold for connective tissue regeneration in ligaments,20 vessels,21,22,27 bladder walls,28 and abdominal walls29 made it a promising candidate for use in meniscal tissue regeneration. The macroscopic and microscopic structure of this material has been shown to be conducive to supporting a collagenous tissue infiltration and subsequent maturation process yielding a well-differentiated, specialized structure with the biologic and physical characteristics vital to the native tissue’s function. This was seen clearly in the vascular graft studies in which the regenerated vessel had the vital constituents of an arterial vessel (i.e., endothelium, muscular media layer).30 For a regenerated connective tissue to have long-lasting value, it must replicate not only the form of the tissue but, more importantly, the structure and function of the tissue. This preliminary study was designed to test the applicability of the SIS graft as a scaffold for meniscal tissue regeneration. Functional considerations of the regenerated tissue will be addressed in future studies using larger animal subjects with a more clinically relevant meniscal injury model. In a previous study, Stone et al.19 outlined the prerequisites of a successful collagen-based scaffold
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FIGURE 5. At 24 weeks, the experimental specimen shows more organized tissue (center of specimen) that resembles a native meniscus (left and right side of specimen) at low power.
for meniscal regeneration. They hypothesized that the resorption rate and the pore size of the resorbable scaffold are critical to successful regeneration of meniscal fibrochondrocytes, and that a critical microstructure will determine the balance between the unwanted prolific vascular ingrowth characteristics of
granulation tissue and meniscal cell precursor ingrowth. They also stressed that the regenerated tissue must contain functioning fibrochondrocytes that produce an appropriate matrix and that the mechanical stresses applied to the tissue during the healing phase will result in orientation of the newly formed collagen
FIGURE 6. Control meniscus at 24 weeks showing significant signs of healing (center of image).
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J. A. GASTEL ET AL. 2. It is not destroyed early in the regeneration process by highly vascular granulation tissue. 3. It is not rejected, as in a giant-cell–mediated foreign-body reaction. 4. It has a microstructure and macrostructure conducive to cellular infiltration and adherence. 5. It possesses adequate structural support to withstand the biomechanical stresses applied to the meniscus during the prolonged regeneration phase.
FIGURE 7. Control meniscus with reparative tissue that appears less dense (opposite suture).
bundles along the lines of those stresses. Lastly, they outlined specific requirements for a successful collagenous graft in order to modify the resistance of the implant to the degradative processes within the synovial joint. These include: survival of the implant long enough for essential vascular, cellular, and matrix elements to be established; to not evoke a potentially destructive foreign-body reaction; and to reapproximate the stability of the native meniscus during the regeneration phase. In this preliminary study, the SIS implant successfully provided a reasonable scaffold for the complete regeneration of a large, complete radial defect in the adult rabbit meniscus. From a gross anatomic perspective, the specimen had the appearance of a normal meniscus with respect to contour, color, and consistency. Histologically, there was early infiltration of the laminated implant with host cellular elements without evidence of rejection or giant-cell reaction. At 6 months, there was maturation of a moderately cellular connective tissue uniformly coapted to the margin of the native meniscal tissue. Incorporation of the new tissue was shown by the streaming of tissue seen between the native and regenerate tissues (Fig 3). These findings strongly support the following theories regarding the SIS biomaterial: 1. It is resorbed at an appropriate rate that allows for gradual population with connective tissue cellular elements.
These properties are critical for the success of a collagenous scaffold and indicate that further work in assessing the applicability of this biomaterial in meniscal regeneration is warranted. Further studies would need to address some of the shortcomings encountered in this pilot study. A large animal model would likely result in fewer technical failures related to the implantation procedure. In this study, we needed to use a full-thickness, segmental, radial defect, which is highly destructive and not analogous to defects observed clinically. A larger meniscus would allow for more secure suturing of the graft into a larger and more clinically relevant meniscal defect. Future studies should also include a longer postoperative period to allow for better characterization of the regenerated tissue. This would also allow for biomechanical testing of the regenerated tissue, both in comparison with the control healing tissue as well as with normal meniscal tissue. Such functional evaluation is critical to the final assessment of any regenerated tissue. In addition, the applicability of SIS in complete versus partial meniscal regeneration should be assessed. This study evaluated the use of the SIS scaffold for regeneration of a segmental defect with the native meniscus in direct contact with the implant.
REFERENCES 1. King D. The healing of semilunar cartilages. J Bone Joint Surg 1936;18:333-338. 2. Fairbanks TJ. Knee joint changes after meniscectomy. J Bone Joint Surg Br 1948;30:664-670. 3. Krause WR, Pope M, Johnson RJ, et al. Mechanical changes in the knee after meniscectomy. J Bone Joint Surg Am 1976;58: 599. 4. Walker P, Erkman M. The role of the menisci in force transmission across the knee. Clin Orthop Rel Res 1975;109:184192. 5. Shrive NG, Phil D, O’Connor JJ, et al. Load-bearing in the knee joint. Clin Orthop Rel Res 1978;131:279-287. 6. McGinty JB, Geuss LF, Marvin RA. Partial or total menisectomy. J Bone Joint Surg Am 1977;59:763-766. 7. Baratz ME, Fu FH, Mengato R. Meniscal tears: The effect of
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8. 9. 10. 11. 12.
13. 14.
15. 16.
17. 18. 19.
menisectomy and repair on intraarticular contact areas and stress in the human knee. Am J Sports Med 1986;14:270-275. Cannon D, Vittori J. The incidence of healing in arthroscopic meniscal repairs in anterior cruciate ligament-reconstructed knees versus stable knees. Am J Sports Med 1992;20:176-181. Arnoczky S, Warrren RF, Spivak JM. Meniscal repair using exogenous fibrin clot. An experimental study in dogs. J Bone Joint Surg Am 1988;70:1209-1217. Cisa J, Basora J, Madarnas P, et al. Meniscal repair by synovial flap transfer. Acta Orthop Scand 1995;66:38-40. Sommerlath K, Gillquist J. The effect of a meniscal prosthesis on knee biomechanics and cartilage: An experimental study in rabbits. Am J Sports Med 1992;20:73-81. Arnoczky SP, McDevitt CA, Schmidt MB, et al. The effect of cryopreservation on canine menisci: A biochemical, morphological, and biomechanical evaluation. J Orthop Res 1988;6: 1-12. Czitrom AA, Keating S, Gross AE. The viability of articular cartilage in fresh osteochondral allografts after clinical transplantation. J Bone Joint Surg Am 1990;72:574-581. Arnoczky SP, Milachowski KA. Meniscal allografts: Where do we stand? In: Ewing JW, ed. Articular cartilage and knee joint function: Basic science and arthroscopy. New York: Raven, 1990. Arnoczky SP, O’Brien SJ, Dicarl, EF, et al. Cellular repopulation of deep-frozen meniscal autografts. An experimental study in the dog. Orthop Trans 1988;12:431-433. Jackson DW, Simon TM, Kurzweil PR, et al. Survival of cells after intra-articular transplantation of fresh allografts of the patellar and anterior cruciate ligaments, DNA-probe analysis in a goat model. J Bone Joint Surg Am 1992;74:112-118. Milachowski KA, Weismeier K, Wirth CJ. Homologous meniscus transplantation. Int Orthop 1989;13:1-11. Yannas IV, Burke JF. Design of an artificial skin. Basic design principles. J Biomed Mater Res 1980;14:65-81. Stone KR, Rodkey WG, Webber R, et al. Meniscal regeneration with copolymeric collagen scaffolds: In vitro and in vivo
20. 21. 22. 23. 24.
25. 26. 27. 28. 29. 30.
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studies evaluated clinically, histologically, and biochemically. Am J Sports Med 1992;20:104-111. Aiken SW, Badylak SF, Toombs JP, et al. Small intestinal submucosa as an intra-articular graft material: A pilot study in dogs. VCOT 1994;37:124-128. Badylak S.F, Lantz GC, Coffey AC, et al. Small intestinal submucosa as a large diameter vascular graft in the dog. J Surg Res 1989;47:74-80. Lantz GC, Badylak SF, Coffey AC, et al. Small intestine submucosa as a superior vena cava graft in the dog. J Surg Res 1992;53:175-181. Seedhom BB, Wright V. Functions of the menisci. J Bone Joint Surg Br 1974;56:381-382. Arnoczky SP, Adams M, DeHaven K, et al. Meniscus. In: Woo S, Buckwalter JA, eds. Injury and repair of the musculoskeletal soft tissues. Park Ridge, IL: American Academy of Orthopaedic Surgeons, 1988;487. Levy IM, Torzilli PA, Warren RF. The effect of medial meniscectomy on the anterior-posterior motion of the knee. J Bone Joint Surg Am 1982;64:883-888. Shoemaker SC, Markolf KL. The role of the meniscus in the anterior-posterior stability of the loaded anterior cruciate-deficient knee. J Bone Joint Surg Am 1986;68:71-79. Lantz GC, Badylak SF, Coffoy AC, et al. Small intestinal submucosa as a small-diameter arterial graft in the dog. J Invest Surg 1990;3:217-227. Kropp BP, Epley BL, Prevel CD, et al. Experimental assessment of small intestinal intestinal submucosa as a bladder wall substitute. Urology 1995;46:396-400. Prevel DC, Eppley BL, Summerlin DJ, et al. Small intestinal submucosa: Utilization for repair of rodent abdominal wall defects. Ann Plast Surg 1995;35:374-380. Sandusky GE Jr, Badylak SF, Morff RJ, et al. Histologic findings after in vivo placement of small intestine submucosal vascular grafts and saphenous vein grafts in the carotid artery in dogs. Am J Pathol 1992;140:317-324.
Purpose: The present investigation is a preliminary study designed to evaluate the use of a collagen-based biomaterial, chemically unaltered porcine small intestine submucosa (SIS), as a scaffold for meniscal tissue regeneration. Type of Study: Basic research. Methods: Surgical defects were created in the lateral menisci of 12 mature New Zealand white rabbits. The defects were repaired with a similarly shaped and sized wedge of a new collagenous biomaterial (SIS) and sutured in place. The opposite knees served as controls by creating a defect in the lateral meniscus without filling with SIS graft. Full cage activity was allowed until the animals were killed at 4, 12, and 24 weeks. Results: At 4 weeks, the graft material retained its physical position and grossly appeared soft and translucent. Histologically, cellular elements had infiltrated between the laminates of the graft. At 12 weeks, the graft grossly appeared more solid and opaque. Histologically, the host meniscal fibrochondrocytes were seen streaming into the peripheral margin of the graft. Early repopulation of the graft with apparently differentiated meniscal tissue was observed. At 24 weeks, the meniscus defect was grossly healed across and looked virtually normal: the normal meniscal shape, contour, consistency, and color had been replicated. Histologically, the healing tissue showed infiltration of what appeared to be meniscal fibrochondrocytes and connective tissue resembling the host meniscal tissue. The graft was nearly totally replaced by host tissue. Conclusions: This pilot animal study demonstrates that the multilaminated collagenous graft is conducive for cellular repopulation with host meniscal elements, and, by 24 weeks, is capable of supporting complete healing of a large meniscal defect. Key Words: Meniscus—Meniscal regeneration—Meniscal repair—Animal study—Collagenous graft—Small intestine submucosa.
A
n intact meniscus is clearly vital to the biomechanical and long-term function of the knee joint. The current surgical options for a meniscal tear are repair and debridement. Despite early evidence of the potential for meniscal healing by King1 in the 1930s and Fairbanks’ observations2 of postmeniscectomy osteoarthritic changes in 1948, total
From the Department of Orthopaedics, Brown University, Rhode Island Hospital, Providence, Rhode Island, U.S.A. Supported by an educational grant from DePuy, Inc., Warsaw, Indiana. Address correspondence and reprint requests to Paul D. Fadale, M.D., University Orthopaedics, 2 Dudley Street, Providence, RI 02903, U.S.A. © 2001 by the Arthroscopy Association of North America 0749-8063/01/1702-2038$35.00/0 doi:10.1053/jars.2001.20959
meniscectomy was the standard operative treatment for a torn meniscus until the 1970s. Studies have since proved the functional significance of the menisci to knee stability,3 force transmission,4 and shock absorption.5 Thus, preservation of meniscal tissue became a primary treatment goal. Partial meniscectomy has been shown to preserve knee biomechanics to some degree and to offset articular cartilage changes better than total meniscectomy, but it may still lead to a significant increase in radiographic osteoarthritic changes.6 Repair and preservation of the entire injured meniscus has been shown to be beneficial to the maintenance of normal knee biomechanics7 and important in preventing abnormal articular surface wear. However, the inherent healing capacity of the human meniscus has been shown to be lacking in the central third and
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very limited in the middle third of this poorly vascularized tissue.8 Thus, the applicability of meniscal repair is severely limited by both tear morphology and location. Moreover, the results of attempted repairs are highly variable. These factors have led to a wide variety of techniques aimed at augmenting or inducing the healing of meniscal tissue. Efforts have been directed at augmenting the innate healing response as well as implanting grafts or prostheses to substitute for the irreparably damaged meniscal tissue. Augmentation of healing has been attempted clinically with the insertion of fibrin clot in the repair site9 or by implantation of a vascularized synovial flap.10 The results with meniscal prostheses have been discouraging. A polyurethane-coated Dacron prosthesis (Du Pont, Wilmington, DE) was shown to provide some protection to the articular surface, but did not reproduce the biomechanical functions of the normal meniscus.11 Transplantation techniques have also been used with fresh menisci or menisci preserved by deep freezing, freeze drying (lyophilization), and controlled-rate freezing (cryopreservation). The latter has become the mode of choice based on several factors. This technique has been shown to result in the least amount of cellular destruction, although Arnoczky et al.12 have shown that as little as 10% of the cells remain metabolically active immediately after preservation and only 3% after 12 weeks. Similar to what has been observed in articular cartilage transplantation studies, preserving cell viability should maximize the results of meniscal transplantation.13,14 Arnoczky et al.15 showed this with deep-frozen menisci in a canine model with cellular repopulation thought to be stemming from the synovial tissue. However, good short-term results have also been found with other tissues, such as the anterior cruciate ligament and patellar tendon, that show no signs of donor cellular viability shortly after transplantation.16 Meniscal transplantation has obvious inherent drawbacks. There has been significant shrinkage of the graft with preserved menisci.17 The need for proper preoperative sizing of the transplant requires adequate storage and timely access to a large number of samples. Procurement of adequate samples is limited by the donor supply (e.g., relatively young, disease-free patients with no premorbid meniscal pathology). In addition, the threat of disease transmission is always an issue with human allogeneic tissue transplantation.
The use of a fabricated collagenous graft or prosthesis in meniscal reconstruction and repair has also been studied. Basing their investigation on the use of biologically resorbable scaffolds in the replacement of human skin by Yannas and Burke,18 Stone et al.19 tested the use of a copolymeric collagen scaffold derived from bovine Achilles tendon. After residual blood proteins and watersoluble materials were washed from the tissue, the collagen was purified and cross-linked with glycosaminoglycans. Further cross-linking and lyophilization yielded a collagen scaffold that was shown in vitro to be conducive to cellular migration. The fabricated collagen scaffold was trimmed to size and implanted in canine stifle joints in place of the medial meniscus, which had undergone an 80% subtotal resection. This graft effectively supported meniscal fibrochondrocyte ingrowth and meniscal tissue regeneration. The present investigation is a preliminary study designed to evaluate the use of a collagen-based biomaterial, chemically unaltered porcine small intestine submucosa (SIS), as a scaffold for meniscal tissue regeneration. The SIS biomaterial was selected based on its performance as a ligament graft20 and in vascular surgery studies.21,22 In its use as a vascular graft, it was shown not only to be completely incorporated but, more importantly, the distinct layers of the arterial wall were reconstituted. Thus, the cells that repopulated the graft survived and differentiated in accordance with their physical environment. This characteristic is vital to the success of any biomaterial used in meniscal tissue regeneration. Simply producing a living, meniscus-shaped pad of fibrocartilage is not enough; reconstitution of the intricate collagen fibril networks by living fibrochondrocytes must take place for successful functional meniscal regeneration.
METHODS Bilateral, wedged-shaped, segmental surgical defects were created in the central third of the lateral menisci of 12 male New Zealand White rabbits weighing 3.5 to 4 kg. The defect width was 3 mm, approximately 30% of the meniscus. The SIS biomaterial was supplied by the sponsor (DePuy, Inc.) as sterile, 3-mm thick 1-cm squares. An appropriate triangular graft was fashioned from each sheet and placed in the right limb of each animal and anchored in place with 2 horizontal nonabsorbable sutures. SIS consists of a 0.1-mm thick sheet of tissue composed of the tunica submucosa with attached stratum compactum and muscularis mucosa. Mechanical
MENISCAL TISSUE REGENERATION removal of the other intestinal layers eliminates the need for the potentially damaging use of chemical treatments and preserves the endogenous collagenous and noncollagenous proteins present naturally in the tissue. The harvested tissue is subsequently rinsed in physiologic saline and immersed in an antibiotic solution. A laminate is then prepared by compressing these sheets together mechanically. The left limb of each animal had a partial lateral meniscectomy performed exactly as on the right side but no graft was placed in the defect. The animals were allowed to recover postoperatively for 1 week in standard cages. Afterward, they were kept in freerange pens to allow full range of motion and weight bearing during the survival period. The animals were killed in groups at 4, 12, and 24 weeks, and both lateral menisci were harvested and prepared for histological analysis. Surgical Procedure Approval for this study was obtained from the Rhode Island Hospital Animal Care and Use Committee. Each animal was anesthetized with an intramuscular injection of ketamine and xylazine. The animal was intubated and intravenous access gained. In a supine position, the hind limbs were washed with alcohol and povidone iodine. The animal was then draped to establish a sterile field. A lateral parapatellar approach to the knee was used to gain access to the lateral meniscus. A 3-mm wedge defect was excised from the central third of the meniscus with a No. 11 blade, taking care to protect the articular cartilage. The defect in the right knee was replaced with the wedge-shaped SIS implant, which was sutured to the anterior and posterior horns of the meniscus with 5-0 polypropylene. The defect in the left knee was left untreated. The capsule, deep fascia, and skin were closed in layers in all of the knees. Povidone iodine ointment was placed on the wound and the wound was dressed with collodion. No attempt with dressing or splint material was made to reduce postoperative range of motion. Postoperative antibiotics and analgesics were administered and the animal was allowed to recover. At sacrifice, the animals were anesthetized using a mixture of ketamine and xylazine. Following sedation, peripheral intravenous access was gained using a 22gauge angiocatheter in an ear vein. The animals were
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killed using 3 mL pentobarbital sodium, administered intracardiac. The menisci were excised and prepared for routine histologic analysis. The menisci from 2 animals from each time point were processed for routine histology; the remaining were evaluated grossly. The specimens were fixed in 10% neutral phosphate-buffered saline and formalin for 5 days. The specimens were then embedded in paraffin and parasagittal sections (perpendicular to the implant) were taken. These sections were stained with hematoxylin and eosin, and Masson’s trichrome.
RESULTS 4-Week Group At 4 weeks postoperatively, the SIS implant retained its overall laminar structure and organization. Exuberant cellular infiltration into the layers of the implant was seen under low- and high-power magnification (Figs 1A and 1B, respectively). Interestingly, there was an absence of giant cells or other signs of inflammation or foreign-body reaction. Although the implant remained in contact with the native meniscus, actual incorporation of the graft material was not observed. Some specimens showed a gap between the graft and the meniscus that was filled with a loosely organized fibrous tissue. The controls had a moderately cellular fibrous tissue filling the untreated defect. 12-Week Group At 12 weeks, the implant has started to lose its structure, but some of the implant’s laminar layers were still identifiable amidst the abundant cellular infiltration. The invading host tissue appears slightly less cellular, but more organized and fibrous in nature when compared with the earlier group (Fig 2, low power). Some new tissue was seen growing from the margin of the native meniscus into the defect (Fig 3, high power). The control side showed maturing fibrovascular tissue filling the defect. 24-Week Group At 24 weeks, 2 of the experimental specimens exhibited a gross appearance similar to that of a native uninjured meniscus (Fig 4). The repair appeared to have healed in continuity with new tissue that was opaque and shaped to the contours of the native me-
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FIGURE 1. (A) Low-power magnification view of exuberant cellular infiltration into the layers of SIS on the right, at 4 weeks. (B) High-power magnification of exuberant cellular infiltration into the layers of SIS (blue), at 4 weeks.
niscal tissue. Two specimens showed technical failure of the repair, with diastasis between the anterior and posterior portions of the native meniscus. Histologically, at 6 months, the experimental specimens showed more organized tissue resembling that
of the native meniscus (Fig 5). Tissue infiltration into the native meniscus was observed at the interface and fibers were also seen running through the implant. Remnants of the graft were no longer identifiable in the healing tissue, but the original nonabsorbable su-
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FIGURE 2. New tissue on the left abutting host meniscus on the right, at low power. New tissue is still cellular at 12 weeks, but seems to be becoming organized and fibrous.
tures still maintained their positions within the zone of tissue regeneration. The control side meniscus also showed significant signs of healing (Fig 6). On gross examination, this tissue appeared less densely opaque compared with the SIS side (Fig 7).
DISCUSSION This investigation was designed to evaluate the use of a collagen-based biomaterial as a scaffold for meniscal regeneration. The search for the optimum me-
FIGURE 3. High-power view of new tissue (right) growing from the margin of the native meniscus (left) into the defect.
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FIGURE 4.
Native uninjured meniscus of a rabbit.
niscal transplant involves all of the factors addressed above. Human meniscal transplantation has its inherent drawbacks, including sizing, availability, material property changes with preservation techniques, and the risk of disease transmission. Fabricated collagen scaffolds are promising, but the ideal physical construct of the collagen graft has yet to be defined, and the effect of the chemical treatments in the processing of these materials has not been determined. The optimal remodeling of the repopulated graft and regenerated meniscus is likely to be determined, at least in part, by the ultrastructure of the particular implant and its biologic milieu. Just as has been shown with artificial bone grafts or porous ingrowth prostheses, the nature of biologic processes are intimately related to their physical microenvironment. The physical properties of the multilaminated SIS graft and its biologically preserved nature affords a unique look at the regeneration capacity of meniscal tissue in this investigation. Preservation of the functional meniscus has become a primary goal of the knee surgeon. Repair of appropriate meniscal tears and minimizing debridement of irreparable tears has become the standard of care for meniscal injuries as the science regarding the menisci and their vital function has become better understood. Unfortunately, a significant proportion of acute meniscal tears are not amenable to repair and many degenerative tears involve a significant bulk of the meniscal body. Additionally, many patients have been left without any meniscal tissue as a result of total meniscectomy. These situations render the articular surfaces of the knee subject to abnormally high contact stresses leading to premature articular degenera-
tion. Baratz et al.7 have shown that the contact area in the tibiofemoral joint decreases from 6 cm2 with the meniscus intact to 2 cm2 after meniscectomy thus increasing the local contact forces. Similarly, it has been shown that the lateral meniscus bears 70% of the lateral compartment load and the medial meniscus bears 50% of the medial compartment load.23 The accessory functions of the meniscus in joint lubrication,24 shock absorption,3 and joint stability25,26 have also been well documented. The inability to preserve the meniscus in many clinical situations and the need to replace it in others has stimulated the search for a substitute, biologic or otherwise, for the native meniscus. A patient may present to the orthopaedist with vague knee symptoms and a remote history of total meniscectomy or acutely with a symptomatic and irreparable torn meniscus. In the former case, replacement of the meniscus may have a significant role in preventing or slowing the degenerative changes in the knee. In the more acute setting, the detrimental effects of partial meniscectomy may be avoided if the damaged portion can be repaired. These scenarios are examples of the clinical applicability of meniscal preservation that have encouraged investigators to pursue the possibility of using a biologic scaffold or implant to stimulate the regeneration of functional meniscal tissue. The encouraging use of the SIS biomaterial as a scaffold for connective tissue regeneration in ligaments,20 vessels,21,22,27 bladder walls,28 and abdominal walls29 made it a promising candidate for use in meniscal tissue regeneration. The macroscopic and microscopic structure of this material has been shown to be conducive to supporting a collagenous tissue infiltration and subsequent maturation process yielding a well-differentiated, specialized structure with the biologic and physical characteristics vital to the native tissue’s function. This was seen clearly in the vascular graft studies in which the regenerated vessel had the vital constituents of an arterial vessel (i.e., endothelium, muscular media layer).30 For a regenerated connective tissue to have long-lasting value, it must replicate not only the form of the tissue but, more importantly, the structure and function of the tissue. This preliminary study was designed to test the applicability of the SIS graft as a scaffold for meniscal tissue regeneration. Functional considerations of the regenerated tissue will be addressed in future studies using larger animal subjects with a more clinically relevant meniscal injury model. In a previous study, Stone et al.19 outlined the prerequisites of a successful collagen-based scaffold
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FIGURE 5. At 24 weeks, the experimental specimen shows more organized tissue (center of specimen) that resembles a native meniscus (left and right side of specimen) at low power.
for meniscal regeneration. They hypothesized that the resorption rate and the pore size of the resorbable scaffold are critical to successful regeneration of meniscal fibrochondrocytes, and that a critical microstructure will determine the balance between the unwanted prolific vascular ingrowth characteristics of
granulation tissue and meniscal cell precursor ingrowth. They also stressed that the regenerated tissue must contain functioning fibrochondrocytes that produce an appropriate matrix and that the mechanical stresses applied to the tissue during the healing phase will result in orientation of the newly formed collagen
FIGURE 6. Control meniscus at 24 weeks showing significant signs of healing (center of image).
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J. A. GASTEL ET AL. 2. It is not destroyed early in the regeneration process by highly vascular granulation tissue. 3. It is not rejected, as in a giant-cell–mediated foreign-body reaction. 4. It has a microstructure and macrostructure conducive to cellular infiltration and adherence. 5. It possesses adequate structural support to withstand the biomechanical stresses applied to the meniscus during the prolonged regeneration phase.
FIGURE 7. Control meniscus with reparative tissue that appears less dense (opposite suture).
bundles along the lines of those stresses. Lastly, they outlined specific requirements for a successful collagenous graft in order to modify the resistance of the implant to the degradative processes within the synovial joint. These include: survival of the implant long enough for essential vascular, cellular, and matrix elements to be established; to not evoke a potentially destructive foreign-body reaction; and to reapproximate the stability of the native meniscus during the regeneration phase. In this preliminary study, the SIS implant successfully provided a reasonable scaffold for the complete regeneration of a large, complete radial defect in the adult rabbit meniscus. From a gross anatomic perspective, the specimen had the appearance of a normal meniscus with respect to contour, color, and consistency. Histologically, there was early infiltration of the laminated implant with host cellular elements without evidence of rejection or giant-cell reaction. At 6 months, there was maturation of a moderately cellular connective tissue uniformly coapted to the margin of the native meniscal tissue. Incorporation of the new tissue was shown by the streaming of tissue seen between the native and regenerate tissues (Fig 3). These findings strongly support the following theories regarding the SIS biomaterial: 1. It is resorbed at an appropriate rate that allows for gradual population with connective tissue cellular elements.
These properties are critical for the success of a collagenous scaffold and indicate that further work in assessing the applicability of this biomaterial in meniscal regeneration is warranted. Further studies would need to address some of the shortcomings encountered in this pilot study. A large animal model would likely result in fewer technical failures related to the implantation procedure. In this study, we needed to use a full-thickness, segmental, radial defect, which is highly destructive and not analogous to defects observed clinically. A larger meniscus would allow for more secure suturing of the graft into a larger and more clinically relevant meniscal defect. Future studies should also include a longer postoperative period to allow for better characterization of the regenerated tissue. This would also allow for biomechanical testing of the regenerated tissue, both in comparison with the control healing tissue as well as with normal meniscal tissue. Such functional evaluation is critical to the final assessment of any regenerated tissue. In addition, the applicability of SIS in complete versus partial meniscal regeneration should be assessed. This study evaluated the use of the SIS scaffold for regeneration of a segmental defect with the native meniscus in direct contact with the implant.
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