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Marrow Stimulation Improves Meniscal Healing at Early Endpoints in a Rabbit Meniscal Injury Model
Marrow Stimulation Improves Meniscal Healing at Early Endpoints in a Rabbit Meniscal Injury Model Matthew D. Driscoll, M.D., Brett N. Robin, M.D., Masafumi Horie, M.D., Ph.D., Zachary T. Hubert, B.S., H. Wayne Sampson, Ph.D., Daniel C. Jupiter, Ph.D., Binu Tharakan, Ph.D., and Robert E. Reeve, M.D.
Purpose: To critically evaluate the effect of marrow stimulation (MS) on the extent of healing and the local biological environment after meniscal injury in ligamentously stable knees in a rabbit model. Methods: A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in 18 New Zealand White rabbits (36 knees). In right knees (MS knees), a 2.4-mm Steinman pin was drilled into the apex of the femoral intercondylar notch and marrow contents were observed spilling into the joint. Left knees served as controls. Rabbits were killed in 3 groups (n ¼ 6 rabbits each) at 1, 4, and 12 weeks with meniscal harvest and blinded histomorphometric and histologic evaluation using an established 3-component tissue quality score (range, 0 to 6). One-week specimens were also evaluated for the presence of proregenerative cytokines using immunohistochemistry. Results: The mean proportion of the avascular zone defect bridged by reparative tissue was greater in MS knees than in controls at each endpoint (1 week, 55% v 30%, P ¼ .02; 4 weeks, 71% v 53%, P ¼ .047; 12 weeks, 96% v 77%, P ¼ .16). Similarly, there was a consistent trend toward superior tissue quality scores in knees treated with MS compared with controls (1 week, 1.8 v 0.3, P ¼ .03; 4 weeks, 4.3 v 2.8, P ¼ .08; 12 weeks, 5.9 v 4.5, P ¼ .21). No statistically significant differences, however, were observed at the 12-week endpoint. Increased staining for insulin-like growth factor I, transforming growth factor-b, and platelet-derived growth factor was observed in regenerated tissue, compared with native meniscal tissue, in all specimens at 1 week. Staining density for all growth factors was similar, however, in reparative tissue of MS and control knees. Conclusions: The results of this study suggest that marrow stimulation leads to modest improvements in quality and quantity of reparative tissue bridging a meniscal defect, particularly during the early recovery period. Clinical Relevance: Clinical evaluation of marrow stimulation techniques designed to enhance healing in isolated meniscus repair surgery may be indicated.
eniscal fibrocartilage is a largely avascular tissue with limited inherent healing capacity.1 Conventional meniscal suture repair techniques approximate torn tissue edges but do little to enhance the local biological environment, often leading to failure. For
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From the Department of Orthopaedic Surgery, Scott and White Memorial Hospital (M.D.D., B.N.R., Z.T.H., R.E.R.); Texas A&M Institute for Regenerative Medicine (M.H.); Department of Systems Biology and Translational Medicine, Scott & White Memorial Hospital (H.W.S.); Department of Surgery, Texas A&M University Health Science Center (D.C.J., B.T.), Temple, Texas, U.S.A.; and Tokyo Medical and Dental University (M.H.), Tokyo, Japan. The authors report that they have no conflicts of interest in the authorship and publication of this article. Received November 29, 2011; accepted June 25, 2012. Address correspondence to Matthew D. Driscoll, M.D., Department of Orthopaedic Surgery, Scott and White Memorial Hospital, Temple, TX, 76502, U.S.A. E-mail: [email protected] Ó 2013 by the Arthroscopy Association of North America 0749-8063/11790/$36.00 http://dx.doi.org/10.1016/j.arthro.2012.06.023
example, reported rates of clinical failure for isolated meniscus tears repaired in ligamentously stable knees range from 24% to 50%.2-5 Such unsatisfactory outcomes have stimulated the development of a variety of techniques designed to augment repairs with an additional biological stimulus through synovial rasping, trephination, or application of fibrin clot or plateletrich plasma.6-11 Newer techniques with increasing cost and complexity are also being tested in animal models, including local application of exogenous growth factors12,13 or stem cells.14-17 The potential role for marrow stimulation (MS) in meniscal repair augmentation, however, has not been independently investigated. Interestingly, compared with meniscus tears repaired in ligamentously stable knees, tears repaired concurrently with anterior cruciate ligament (ACL) reconstruction are more likely to heal, with success rates of 90% and higher.2,5,18 Although the mechanism behind this phenomenon remains incompletely understood, several theories have
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been proposed.19 Perhaps the most intriguing is that of MS, the concept that marrow contents including progenitor cells and growth factors released from the tibia and femur with tunnel preparation enhance the environment for meniscal healing.19,20 Based on this concept, MS has been advocated by Freedman et al.20 for enhancing meniscus repairs in stable knees as well. MS represents a simple and inexpensive technique that does not involve the acquisition of peripheral blood or other donor tissue and does not require the surgical interposition of clot material between tear edges. No direct evidence to support the use of any MS technique in treatment of meniscus injury is found in the literature, however. Furthermore, to our knowledge, MS is not widely used or accepted, possibly because of a lack of supporting evidence. Thus, the purpose of this study was to critically evaluate the effect of MS on the extent of healing as well as the local biological environment after meniscal injury in ligamentously stable knees in an established rabbit model. We hypothesized that the release of marrow contents into the stable knee joint after meniscus injury would result in improved quantity and quality of reparative tissue and increased expression of proregenerative cytokines at the injury site.
Methods Meniscal Injury and Marrow Stimulation The following experimental protocols were approved by the local Institutional Animal Care and Use Committee. Eighteen skeletally mature 3.5- to 4.5-kg female New Zealand White rabbits were used for the study. Under general anesthesia, the right and left knees of each rabbit were accessed through a medial parapatellar arthrotomy. Hemostasis was achieved with electrocautery. The knee joint was then maximally flexed and a reproducible 1.5-mm-diameter fullthickness cylindrical defect was produced in the avascular inner two-thirds21 of the anterior portion of the medial meniscus in each knee using a biopsy punch11,14 (Miltex, York, PA) (Fig 1). In the right knees, a 2.4-mm Kirschner wire was then drilled 2 cm proximally into the femoral canal from a starting point slightly anterior to the apex of the intercondylar notch, and marrow contents were observed spilling into the knee (Fig 2). The left knees served as controls and no drilling was performed. Capsule and skin were closed in layers with absorbable suture. Rabbits were allowed to move freely in their cages. At 1-, 4-, and 12-week endpoints (n ¼ 6 rabbits, 12 knees each), the rabbits were killed and the medial meniscus from each knee was harvested for histomorphometric, histologic, and immunohistologic analyses.
Fig 1. Reproducible avascular zone meniscal injury. The left and right knee joints of each rabbit were approached through a medial parapatellar arthrotomy, and the anterior horn of the medial meniscus was identified. A 1.5-mm punch biopsy was used to create a reproducible defect in the avascular inner two-thirds of the anterior horn of the medial meniscus in all knees.
Tissue Preparation Meniscus specimens were grossly inspected and photographed, then fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin. Each specimen was then cut into sections 5-mm thick in the radial plane through the center of the original defect. Sections for histologic and histomorphometric analyses were stained with Safranin-O/Fast Green. Quantity and quality of regenerated meniscal tissue were evaluated as described next. Histomorphometry: Tissue Quantity Analysis The quantity of reparative tissue bridging the meniscal defect was evaluated using random tissue sections obtained from the central portion of the defect as shown in Fig 3. To define the original defect, horizontal lines were drawn connecting the superior and inferior surfaces of the meniscus at the borders of the 1.5-mm biopsy. Tissue regeneration was evaluated within the region bounded by these lines and the adjacent native
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quantity of reparative tissue was then expressed as the ratio of reparative tissue area to the entire defect area (R/D). Complete full-thickness bridging of the meniscal defect would result in a value of 100%, and incomplete bridging would result in a value less than 100%. Histology: Tissue Quality Analysis An established quantitative scoring system was used, evaluating 3 dimensions of reparative meniscal tissue quality as described in Table 1.11,14 Sections to be scored were stained at neutral pH with Safranin-O, which identifies areas rich in proteoglycan, an important component of normal meniscal extracellular matrix. Histologic scoring was performed by 2 independent investigators (M.D.D. and M.H.) blinded to treatment category.
Fig 2. MS technique. In the right knees, a 2.4-mm Steinman pin was introduced 2 cm into the intramedullary canal of the distal femur from an entry point just anterior to the apex of the intercondylar notch. This resulted in a standardized entry portal for marrow contents to spill into the knee joint.
meniscal tissue. The area of the original defect (D) and the area occupied by reparative tissue inside the defect (R) were calculated using Osteomeasure XP digital imaging software (OsteoMetrics, Decatur, GA). The
Immunohistochemistry Radial meniscal sections from experimental and control knees of each rabbit in the 1-week group were also evaluated using immunohistochemistry to test for the presence of growth factors involved in meniscal cell proliferation and extracellular matrix synthesis, including transforming growth factor (TGF)b1, insulinlike growth factor I (IGF1), bone morphogenetic protein (BMP)-2, and platelet-derived growth factor (PDGF)-A. One-week specimens were studied for this endpoint because growth factor quantities peak roughly 1 week after injury.6 Unstained sections were deparaffinized, hydrated, and immersed in hydrogen peroxide to quench endogenous peroxidase activity. Slides were next washed in phosphate-buffered saline (PBS) and incubated for 1 hour in 1.5% blocking serum to block nonspecific staining. Specimens were then incubated overnight at 4 C with the following primary antibodies: TGFb-1 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), IGF-1 (1:100 dilution; Santa Cruz Biotechnology), BMP-2 (1:200 dilution; Santa Cruz Biotechnology), and PDGF-A (1:200 dilution; Santa Cruz Biotechnology). Sections were next washed with
Fig 3. Method for calculating the quantity of reparative tissue. Using radial sections taken through the center of the defect, the area of the entire defect (D, black dotted line, left) and the area of the reparative tissue (R, black dotted line, right) were measured. The reparative tissue area/defect area ratio (R/D) was used to quantify the amount of meniscal tissue bridging the defect in each knee. Complete full-thickness bridging would result in a value of 100% and incomplete or partial-thickness bridging would result in a value less than 100%.
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Table 1. Histologic Tissue Quality Score Category Reparative tissues with bonding Bilaterally bonds with surrounding meniscus. Partially bonds with surrounding meniscus. No bond with surrounding meniscus. Existence of fibrochondrocytes Fibrochondrocytes exist diffusely in the reparative tissues. Fibrochondrocytes are localized in the reparative tissues. No fibrochondrocytes in the reparative tissues. Staining with Safranin-O Densely stained with safranin-O. Faintly stained with safranin-O. Not stained with safranin-O.
Points 2 1 0 2 1 0 2 1 0
Reprinted with permission.11
PBS, incubated with a biotinylated secondary antibody (Santa Cruz Biotechnology) for 30 minutes, and washed with PBS again. Finally, sections were incubated with “AB” enzyme reagent (Santa Cruz Biotechnology) for 30 minutes, followed by peroxidase substrate for 1.5 minutes. Sections were then washed, dehydrated, and sealed with Permount mounting medium (Thermo Scientific, Pittsburgh, PA). Controls with only PBS/ blocking serum, primary antibody, or secondary antibody showed no background staining. Statistical Analysis On the basis of an a priori power analysis, a sample size of 6 rabbits at each endpoint was selected to achieve a 90% power of detecting a 3-point difference between the mean tissue quality scores of MS and control groups, assuming 1.5-point SDs in each.11 The Wilcoxon signed rank test was used to compare quantity and quality of reparative tissue in MS and control meniscal defects at each of the 3 time endpoints. Statistical analysis was performed using R statistics software, Version 2.12.2 (The R Foundation for Statistical Computing, http://www.R-project.org). P < .05 was considered statistically significant.
Results Marrow Stimulation Increases Quantity of Meniscal Tissue Bridging the Site of Injury A gross representation of the meniscal tissue bridging the 1.5-mm cylindrical defects 1 week after injury is shown in Fig 4. In control knees, grossly visible fullthickness defects remained in 5 of 6 specimens 1 week after the injury. Conversely, in MS knees, such defects remained in only 1 of 6 specimens. At 4 weeks, fullthickness defects remained in 3 of 6 control knees and only 1 of 6 MS knees. By 12 weeks, full-thickness defects remained in 1 of 6 control knees and 0 of 6 MS knees.
Fig 4. MS promotes early healing (macroscopic observation). Macroscopic photographs show the appearance of defects within the anterior horn of the medial meniscus from each control (left) and MS (right) knee 1 week after injury. The specimens in which full-thickness grossly visible defects remained are denoted by an asterisk.
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Fig 5. MS promotes early meniscal healing (microscopic observation). (A) Representative low- and high-power images of regenerated meniscus sections stained with Safranin-O at 1, 4, and 12 weeks after meniscus injury. Scale bar: 200 mm. (B) Representative low- and high-power images of normal rabbit meniscus section stained with Safranin-O. Scale bar: 200 mm. (C) Reparative tissue area/defect area ratios are displayed as mean with SD (error bars) for MS and control groups at each endpoint. *P < .05 between MS and control groups at each period. (D) Results of histological scoring system for repaired meniscus. The scores are displayed as mean with SD (error bars). *P < .05, **P < .10 between MS and control groups at each endpoint.
A more detailed microscopic analysis of the quantity of tissue bridging the defect, as seen on radial histologic sections, was also performed (Fig 5A and 5B). The quantity of tissue bridging the defect in both MS and control knees increased with time from 1 to 4 weeks and 4 to 12 weeks. The average proportion of defect filling, however, was consistently higher in MS knees than in controls at each endpoint, with differences reaching statistical significance at 1 week and 4 weeks (P < .05) (Table 2 and Fig 5C).
Marrow Stimulation Enhances Quality of Meniscal Tissue Bridging the Site of Injury The quality of tissue bridging the defect was superior in knees treated with MS, as shown by greater mean tissue quality scores at each endpoint. Tissue quality scores increased with time in both experimental and control knees from 1 to 4 weeks and 4 to 12 weeks. Average scores were consistently higher, however, in MS knees compared with control knees at each endpoint, reaching statistical significance at 1 week
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Table 2. Reparative Tissue Area/Defect Area Ratio (range, 0e1) 1 week 4 weeks 12 weeks
Mean SD Mean SD Mean SD
Control
MS
P-value
0.30 0.19 0.53 0.17 0.77 0.39
0.55 0.08 0.71 0.16 0.96 0.05
.016 .047 .156
(P ¼ .03) and approaching statistical significance at 4 weeks (P ¼ .08) (Table 3, Fig 5D). In addition, average values for each of the 3 subscores comprising the composite tissue quality score (tissue bonding, existence of fibrochondrocytes, and staining with SafraninO) were higher at each endpoint in MS knees compared with controls. Interobserver variability for the Tissue Quality Score was noted to be quite low, with an intraclass correlation coefficient of .97. Proregenerative Cytokines are Present in Injured and Healing Tissue After Meniscal Injury In both experimental and control knees, immunostaining for TGFb-1, IGF-1, and PDGF-A was diffusely positive along the borders of the defect and within tissue bridging the defect 1 week after injury (Fig 6). By comparison, staining was minimally positive within the substance of the uninjured meniscus. Staining appeared similar, however, in MS and control specimens 1 week after injury. Finally, staining for BMP-2 was negative in intact and healing tissue in both treatment groups.
Discussion The results of this study suggest that MS leads to a modest improvement meniscal healing, in terms of both quantity and quality of tissue bridging a site of meniscal injury, particularly during the first several weeks after injury. First popularized by Steadman in the 1990s, MS with microfracture has become a widely accepted treatment for articular cartilage injuries in not only the knee but also the shoulder, hip, and ankle.22-29 Although it stands to reason that the release of pluripotent cells and growth factors into the joint with MS may also aid in healing of other intra-articular injuries, there has been little investigation into this theory with regard to meniscal healing. Table 3. Tissue Quality Score (range, 0e6) 1 week 4 weeks 12 weeks
Mean SD Mean SD Mean SD
Control
MS
P-value
0.3 0.3 2.8 1.9 4.5 2.6
1.8 1.0 4.3 1.5 5.9 0.2
.029 .084 .211
Fig 6. Immunohistochemistry for proregenerative cytokines. In both MS and control knees, immunohistochemistry assays demonstrated positive staining for TGFb-1, IGF-1, and PDGFA, in meniscal tissue bordering the site of injury and within the reparative tissue 1 week after injury. Staining for TGFb-1 is shown here in sections from a knee treated with MS and a control knee, with each image including intact tissue on the left and healing tissue within the meniscal defect on the right. Arrows indicate defect border. Scale bar: 100 mm.
The meniscus is composed of a dense fibrocartilage matrix and sparse population of meniscal fibrochondrocytes. Only the peripheral 10% to 30% of the meniscus is vascularized, and tears occurring in the more central portions show poor healing capacity even after close approximation with suture repair.2-5,19 Recent studies using a variety of techniques aimed at enhancing the biological environment for meniscal healing suggest that biological factors may be of greater importance than repair technique.6-17,30-32 Meniscal rasping and trephination,6-9 direct application of cytokine-rich substances such as fibrin clot or platelet-rich plasma,11,30,31 and even preliminary stem cellebased therapies in animal models14-16,32 have been applied to meniscal defects, leading to improved laboratory and clinical results. MS may represent yet another tool capable of enhancing healing rates and further improving clinical outcomes. Indirect evidence in support of this concept is found in the superior healing rates in meniscus tears repaired concurrently with ACL reconstruction and associated release of marrow contents with tunnel preparation.2,5,18 The results of this study provide the first direct evidence that MS alone does accelerate meniscal healing, leading to improvements in both quantity and quality of tissue bridging the site of meniscal injury during the first several weeks after injury. In terms of quantity, the volume of tissue bridging the injury site is critical for both strength of the repair and restoration of a smooth contour for articulation with the adjacent chondral surfaces. MS knees were found to have a greater quantity of tissue bridging the repair site compared with control knees at each endpoint, with differences reaching statistical significance at 1 and 4 weeks. In addition, gross inspection of 1-week specimens revealed complete partial-thickness coverage of the defect in 5 of 6 MS knees, compared with only 1 of
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6 control knees. This is likely a result of more abundant clot formation within the injury site in MS knees, which may serve as a biological scaffold that is remodeled with new fibrocartilage over the next several weeks. Quality of tissue traversing the defect was evaluated using a 3-component scoring system11,14 that assesses for the presence of cells resembling meniscal fibrochondrocytes, density of staining for proteoglycan, and the extent of bonding between intact meniscus and regenerated tissue. The first 2 of these components reflect the similarity or lack thereof between the reparative tissue and native meniscus. The third, extent of bonding, reflects the integrity of the repair and may serve as an indicator of repair strength, which could not be directly assessed in this model. At all endpoints, each of the 3 subscores, as well as the cumulative tissue quality scores, were higher in MS knees than in control knees, with these differences reaching or approaching significance at early and midterm follow-up. By 12 weeks, defects in both MS and control groups were found to heal relatively well in most cases. Although overall scores remained higher in MS knees, differences were not statistically significant. Thus, the effect of MS seems to be most beneficial within the first several weeks of treatment in the present model. This may be of clinical importance, as surgeons often limit weight bearing and/or range of motion during the first several weeks after repair. Acceleration of the repair process during this early period of protection may better prepare the meniscus to tolerate increased loading with resumption of activity in the subsequent weeks and months, when the risk if reinjury is greatest.3 Several in vitro experiments have implicated specific growth factors such as TGFb, IGF-1, PDGF, and BMP-2 in promoting meniscal healing and regeneration.33-39 For example, Stewart et al.37 found that PDGF, IGF-1, and TGFb increased ovine meniscal fibrochondrocyte proliferation and type I collagen synthesis when cultured on polyglycolic acid scaffolds. Similarly, Bhargava et al.34 found that BMP-2 and PDGF increased bovine meniscal cell migration and DNA synthesis in vitro. In animal studies, however, the role of these cytokines is less clear. Kim et al.,39 for example, found that TGF-b1 and VEGF expression was lower in rabbit knees treated with autologous bone marrow injection after posterior cruciate injury than in control knees treated without additional biological stimulus, despite superior ligament healing in the bone marroweinjection groups. In addition, a clinically relevant sheep model evaluating the use of VEGF-impregnated suture in meniscal repair showed no benefit over conventional suture.35 Similar to these 2 studies, the results of this experiment suggest that although proregenerative cytokines appear to play a role in meniscal healing and regeneration in vivo, additional factors are critical as well. In
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the present study, positive staining for 3 relevant growth factors was seen in the reparative tissue of both MS and control knees 1 week after injury, indicating that upregulation of proregenerative cytokines may represent an inherent response to acute meniscal injury even in the absence of MS. This suggests, however, that an additional healing stimulus resulting from the release of marrow contents was responsible for differences in the quality and quantity of reparative tissue observed. Potential explanations include an increased intra-articular population of pluripotent mesenchymal progenitor cells, which are known to adhere to sites of meniscal injury and stimulate meniscal healing and regeneration in animal models.14-16 In addition, hemarthrosis in MS knees may lead to clot formation within the defect, providing a scaffold for cellular adhesion and differentiation, as well as subsequent formation of appropriate extracellular matrix.30,31 The location of subchondral bone penetration used for MS in this study (distal femoral chondral surface anterior to the apex of the intercondylar notch) was selected for its ease of access, reproducibility, and direct approach to the intramedullary space to ensure adequate marrow drainage into the joint. In clinical practice, however, this location is undesirable, as it results in damage to the distal femoral articular cartilage. In addition, the 2.4-mm tunnel diameter was selected, in part, because this size is comparable in scale to the femoral tunnel diameter in an anterior cruciate ligament (ACL) reconstruction, but such a relatively large tunnel may not be necessary in clinical practice. More clinically acceptable methods of MS include the creation of multiple smaller perforations in either the intercondylar notch wall as proposed by Freedman et al.20 or the medial or lateral cortex of the femur or tibia (depending on tear location), but neither technique has been studied critically. Limitations of this study include differences between rabbit and human meniscus structure and regenerative capacity,21 our inability to directly assess mechanical strength of meniscal healing using this model, and the lack of comparison between the effects of MS and other repair adjuncts such as rasping, trephination, or addition of fibrin clot, exogenous growth factors, or stem cells. In addition, the meniscal injury (cylindrical defect) used in this experiment was not sutured and differs from those seen clinically, which are typically linear tears. Such substantial differences mandate caution when extrapolating these results into the clinical setting. The chosen model was selected despite these limitations because it confers several advantages. Creation of a cylindrical defect allows for a quantitative assessment of the extent of defect bridging, as well as the quality of tissue within the defect. Furthermore, it produces a stable, highly reproducible injury, which minimizes side-to-side differences and negates the need
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for attempted suture repair and introduction of associated variables. One additional limitation warranting further discussion is the fact that this study did not investigate other potential explanations for superior healing rates observed in meniscal repairs performed simultaneously with ACL reconstruction. Aside from the effects of MS, other possible explanations include differences in tear acuity or symptomatology between patients with isolated meniscal tears and those with concomitant ACL injury (selection bias).19 For example, patients with both ACL and meniscal injuries may seek treatment earlier than those with isolated meniscus tears, resulting in a greater number of acute meniscus tears being treated with ACL reconstruction, thus leading to greater potential for healing.3 Second, a patient seeking treatment for an isolated meniscus tear is presumably symptomatic from the meniscus tear itself, whereas a patient with an associated ACL injury may have an asymptomatic meniscus tear that is repaired and remains asymptomatic whether it heals or not, thus resulting in a clinically successful repair. Finally, there may be underlying differences in the quality of a meniscus that tears in isolation and a meniscus that requires a major joint subluxation to tear. In other words, a meniscus that tears in isolation may be of inferior quality and thus less likely to mend after suture repair. In addition to the effects of MS, some or all of these factors may play a role in the superior healing rates observed in meniscal repairs performed with ACL reconstruction.
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Conclusions
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The results of this study suggest that MS leads to modest improvements in quality and quantity of reparative tissue bridging a meniscal defect, particularly during the early recovery period.
15.
Acknowledgment The authors thank Lin Bustamante and Chaitali Mukherjee for their assistance with specimen sectioning and staining.
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chondral defects of the knee: Average 11-year follow-up. Arthroscopy 2003;19:477-484. Arnoczky SP, Warren RF, Spivak JM. Meniscal repair using an exogenous fibrin clot. An experimental study in dogs. J Bone Joint Surg Am 1988;70:1209-1217. Henning CE, Lynch MA, Yearout KM, Vequist SW, Stallbaumer RJ, Decker KA. Arthroscopic meniscal repair using an exogenous fibrin clot. Clin Orthop Relat Res 1990;252:64-72. Mizuno K, Muneta T, Morito T, et al. Exogenous synovial stem cells adhere to defect of meniscus and differentiate into cartilage cells. J Med Dent Sci 2008;55:101-111. Becker R, Pufe T, Kulow S, et al. Expression of vascular endothelial growth factor during healing of the meniscus in a rabbit model. J Bone Joint Surg Br 2004;86:1082-1087. Bhargava MM, Attia ET, Murrell GA, Dolan MM, Warren RF, Hannafin JA. The effect of cytokines on the proliferation and migration of bovine meniscal cells. Am J Sports Med 1999;27:636-643. Lietman SA, Hobbs W, Inoue N, Reddi AH. Effects of selected growth factors on porcine meniscus in chemically defined medium. Orthopedics 2003;26:799-803. Pangborn CA, Athanasiou KA. Growth factors and fibrochondrocytes in scaffolds. J Orthop Res 2005;23:1184-1190. Stewart K, Pabbruwe M, Dickinson S, Sims T, Hollander AP, Chaudhuri JB. The effect of growth factor treatment on meniscal chondrocyte proliferation and differentiation on polyglycolic acid scaffolds. Tissue Eng 2007;13:271-280. Tumia NS, Johnstone AJ. Platelet derived growth factorAB enhances knee meniscal cell activity in vitro. Knee 2009;16:73-76. Kim E, Jeong HJ, Park SJ, et al. The effect of intra-articular autogenous bone marrow injection on healing of an acute posterior cruciate ligament injury in rabbits. Arthroscopy 2011;27:965-977.
Purpose: To critically evaluate the effect of marrow stimulation (MS) on the extent of healing and the local biological environment after meniscal injury in ligamentously stable knees in a rabbit model. Methods: A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in 18 New Zealand White rabbits (36 knees). In right knees (MS knees), a 2.4-mm Steinman pin was drilled into the apex of the femoral intercondylar notch and marrow contents were observed spilling into the joint. Left knees served as controls. Rabbits were killed in 3 groups (n ¼ 6 rabbits each) at 1, 4, and 12 weeks with meniscal harvest and blinded histomorphometric and histologic evaluation using an established 3-component tissue quality score (range, 0 to 6). One-week specimens were also evaluated for the presence of proregenerative cytokines using immunohistochemistry. Results: The mean proportion of the avascular zone defect bridged by reparative tissue was greater in MS knees than in controls at each endpoint (1 week, 55% v 30%, P ¼ .02; 4 weeks, 71% v 53%, P ¼ .047; 12 weeks, 96% v 77%, P ¼ .16). Similarly, there was a consistent trend toward superior tissue quality scores in knees treated with MS compared with controls (1 week, 1.8 v 0.3, P ¼ .03; 4 weeks, 4.3 v 2.8, P ¼ .08; 12 weeks, 5.9 v 4.5, P ¼ .21). No statistically significant differences, however, were observed at the 12-week endpoint. Increased staining for insulin-like growth factor I, transforming growth factor-b, and platelet-derived growth factor was observed in regenerated tissue, compared with native meniscal tissue, in all specimens at 1 week. Staining density for all growth factors was similar, however, in reparative tissue of MS and control knees. Conclusions: The results of this study suggest that marrow stimulation leads to modest improvements in quality and quantity of reparative tissue bridging a meniscal defect, particularly during the early recovery period. Clinical Relevance: Clinical evaluation of marrow stimulation techniques designed to enhance healing in isolated meniscus repair surgery may be indicated.
eniscal fibrocartilage is a largely avascular tissue with limited inherent healing capacity.1 Conventional meniscal suture repair techniques approximate torn tissue edges but do little to enhance the local biological environment, often leading to failure. For
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From the Department of Orthopaedic Surgery, Scott and White Memorial Hospital (M.D.D., B.N.R., Z.T.H., R.E.R.); Texas A&M Institute for Regenerative Medicine (M.H.); Department of Systems Biology and Translational Medicine, Scott & White Memorial Hospital (H.W.S.); Department of Surgery, Texas A&M University Health Science Center (D.C.J., B.T.), Temple, Texas, U.S.A.; and Tokyo Medical and Dental University (M.H.), Tokyo, Japan. The authors report that they have no conflicts of interest in the authorship and publication of this article. Received November 29, 2011; accepted June 25, 2012. Address correspondence to Matthew D. Driscoll, M.D., Department of Orthopaedic Surgery, Scott and White Memorial Hospital, Temple, TX, 76502, U.S.A. E-mail: [email protected] Ó 2013 by the Arthroscopy Association of North America 0749-8063/11790/$36.00 http://dx.doi.org/10.1016/j.arthro.2012.06.023
example, reported rates of clinical failure for isolated meniscus tears repaired in ligamentously stable knees range from 24% to 50%.2-5 Such unsatisfactory outcomes have stimulated the development of a variety of techniques designed to augment repairs with an additional biological stimulus through synovial rasping, trephination, or application of fibrin clot or plateletrich plasma.6-11 Newer techniques with increasing cost and complexity are also being tested in animal models, including local application of exogenous growth factors12,13 or stem cells.14-17 The potential role for marrow stimulation (MS) in meniscal repair augmentation, however, has not been independently investigated. Interestingly, compared with meniscus tears repaired in ligamentously stable knees, tears repaired concurrently with anterior cruciate ligament (ACL) reconstruction are more likely to heal, with success rates of 90% and higher.2,5,18 Although the mechanism behind this phenomenon remains incompletely understood, several theories have
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been proposed.19 Perhaps the most intriguing is that of MS, the concept that marrow contents including progenitor cells and growth factors released from the tibia and femur with tunnel preparation enhance the environment for meniscal healing.19,20 Based on this concept, MS has been advocated by Freedman et al.20 for enhancing meniscus repairs in stable knees as well. MS represents a simple and inexpensive technique that does not involve the acquisition of peripheral blood or other donor tissue and does not require the surgical interposition of clot material between tear edges. No direct evidence to support the use of any MS technique in treatment of meniscus injury is found in the literature, however. Furthermore, to our knowledge, MS is not widely used or accepted, possibly because of a lack of supporting evidence. Thus, the purpose of this study was to critically evaluate the effect of MS on the extent of healing as well as the local biological environment after meniscal injury in ligamentously stable knees in an established rabbit model. We hypothesized that the release of marrow contents into the stable knee joint after meniscus injury would result in improved quantity and quality of reparative tissue and increased expression of proregenerative cytokines at the injury site.
Methods Meniscal Injury and Marrow Stimulation The following experimental protocols were approved by the local Institutional Animal Care and Use Committee. Eighteen skeletally mature 3.5- to 4.5-kg female New Zealand White rabbits were used for the study. Under general anesthesia, the right and left knees of each rabbit were accessed through a medial parapatellar arthrotomy. Hemostasis was achieved with electrocautery. The knee joint was then maximally flexed and a reproducible 1.5-mm-diameter fullthickness cylindrical defect was produced in the avascular inner two-thirds21 of the anterior portion of the medial meniscus in each knee using a biopsy punch11,14 (Miltex, York, PA) (Fig 1). In the right knees, a 2.4-mm Kirschner wire was then drilled 2 cm proximally into the femoral canal from a starting point slightly anterior to the apex of the intercondylar notch, and marrow contents were observed spilling into the knee (Fig 2). The left knees served as controls and no drilling was performed. Capsule and skin were closed in layers with absorbable suture. Rabbits were allowed to move freely in their cages. At 1-, 4-, and 12-week endpoints (n ¼ 6 rabbits, 12 knees each), the rabbits were killed and the medial meniscus from each knee was harvested for histomorphometric, histologic, and immunohistologic analyses.
Fig 1. Reproducible avascular zone meniscal injury. The left and right knee joints of each rabbit were approached through a medial parapatellar arthrotomy, and the anterior horn of the medial meniscus was identified. A 1.5-mm punch biopsy was used to create a reproducible defect in the avascular inner two-thirds of the anterior horn of the medial meniscus in all knees.
Tissue Preparation Meniscus specimens were grossly inspected and photographed, then fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin. Each specimen was then cut into sections 5-mm thick in the radial plane through the center of the original defect. Sections for histologic and histomorphometric analyses were stained with Safranin-O/Fast Green. Quantity and quality of regenerated meniscal tissue were evaluated as described next. Histomorphometry: Tissue Quantity Analysis The quantity of reparative tissue bridging the meniscal defect was evaluated using random tissue sections obtained from the central portion of the defect as shown in Fig 3. To define the original defect, horizontal lines were drawn connecting the superior and inferior surfaces of the meniscus at the borders of the 1.5-mm biopsy. Tissue regeneration was evaluated within the region bounded by these lines and the adjacent native
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quantity of reparative tissue was then expressed as the ratio of reparative tissue area to the entire defect area (R/D). Complete full-thickness bridging of the meniscal defect would result in a value of 100%, and incomplete bridging would result in a value less than 100%. Histology: Tissue Quality Analysis An established quantitative scoring system was used, evaluating 3 dimensions of reparative meniscal tissue quality as described in Table 1.11,14 Sections to be scored were stained at neutral pH with Safranin-O, which identifies areas rich in proteoglycan, an important component of normal meniscal extracellular matrix. Histologic scoring was performed by 2 independent investigators (M.D.D. and M.H.) blinded to treatment category.
Fig 2. MS technique. In the right knees, a 2.4-mm Steinman pin was introduced 2 cm into the intramedullary canal of the distal femur from an entry point just anterior to the apex of the intercondylar notch. This resulted in a standardized entry portal for marrow contents to spill into the knee joint.
meniscal tissue. The area of the original defect (D) and the area occupied by reparative tissue inside the defect (R) were calculated using Osteomeasure XP digital imaging software (OsteoMetrics, Decatur, GA). The
Immunohistochemistry Radial meniscal sections from experimental and control knees of each rabbit in the 1-week group were also evaluated using immunohistochemistry to test for the presence of growth factors involved in meniscal cell proliferation and extracellular matrix synthesis, including transforming growth factor (TGF)b1, insulinlike growth factor I (IGF1), bone morphogenetic protein (BMP)-2, and platelet-derived growth factor (PDGF)-A. One-week specimens were studied for this endpoint because growth factor quantities peak roughly 1 week after injury.6 Unstained sections were deparaffinized, hydrated, and immersed in hydrogen peroxide to quench endogenous peroxidase activity. Slides were next washed in phosphate-buffered saline (PBS) and incubated for 1 hour in 1.5% blocking serum to block nonspecific staining. Specimens were then incubated overnight at 4 C with the following primary antibodies: TGFb-1 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), IGF-1 (1:100 dilution; Santa Cruz Biotechnology), BMP-2 (1:200 dilution; Santa Cruz Biotechnology), and PDGF-A (1:200 dilution; Santa Cruz Biotechnology). Sections were next washed with
Fig 3. Method for calculating the quantity of reparative tissue. Using radial sections taken through the center of the defect, the area of the entire defect (D, black dotted line, left) and the area of the reparative tissue (R, black dotted line, right) were measured. The reparative tissue area/defect area ratio (R/D) was used to quantify the amount of meniscal tissue bridging the defect in each knee. Complete full-thickness bridging would result in a value of 100% and incomplete or partial-thickness bridging would result in a value less than 100%.
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Table 1. Histologic Tissue Quality Score Category Reparative tissues with bonding Bilaterally bonds with surrounding meniscus. Partially bonds with surrounding meniscus. No bond with surrounding meniscus. Existence of fibrochondrocytes Fibrochondrocytes exist diffusely in the reparative tissues. Fibrochondrocytes are localized in the reparative tissues. No fibrochondrocytes in the reparative tissues. Staining with Safranin-O Densely stained with safranin-O. Faintly stained with safranin-O. Not stained with safranin-O.
Points 2 1 0 2 1 0 2 1 0
Reprinted with permission.11
PBS, incubated with a biotinylated secondary antibody (Santa Cruz Biotechnology) for 30 minutes, and washed with PBS again. Finally, sections were incubated with “AB” enzyme reagent (Santa Cruz Biotechnology) for 30 minutes, followed by peroxidase substrate for 1.5 minutes. Sections were then washed, dehydrated, and sealed with Permount mounting medium (Thermo Scientific, Pittsburgh, PA). Controls with only PBS/ blocking serum, primary antibody, or secondary antibody showed no background staining. Statistical Analysis On the basis of an a priori power analysis, a sample size of 6 rabbits at each endpoint was selected to achieve a 90% power of detecting a 3-point difference between the mean tissue quality scores of MS and control groups, assuming 1.5-point SDs in each.11 The Wilcoxon signed rank test was used to compare quantity and quality of reparative tissue in MS and control meniscal defects at each of the 3 time endpoints. Statistical analysis was performed using R statistics software, Version 2.12.2 (The R Foundation for Statistical Computing, http://www.R-project.org). P < .05 was considered statistically significant.
Results Marrow Stimulation Increases Quantity of Meniscal Tissue Bridging the Site of Injury A gross representation of the meniscal tissue bridging the 1.5-mm cylindrical defects 1 week after injury is shown in Fig 4. In control knees, grossly visible fullthickness defects remained in 5 of 6 specimens 1 week after the injury. Conversely, in MS knees, such defects remained in only 1 of 6 specimens. At 4 weeks, fullthickness defects remained in 3 of 6 control knees and only 1 of 6 MS knees. By 12 weeks, full-thickness defects remained in 1 of 6 control knees and 0 of 6 MS knees.
Fig 4. MS promotes early healing (macroscopic observation). Macroscopic photographs show the appearance of defects within the anterior horn of the medial meniscus from each control (left) and MS (right) knee 1 week after injury. The specimens in which full-thickness grossly visible defects remained are denoted by an asterisk.
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Fig 5. MS promotes early meniscal healing (microscopic observation). (A) Representative low- and high-power images of regenerated meniscus sections stained with Safranin-O at 1, 4, and 12 weeks after meniscus injury. Scale bar: 200 mm. (B) Representative low- and high-power images of normal rabbit meniscus section stained with Safranin-O. Scale bar: 200 mm. (C) Reparative tissue area/defect area ratios are displayed as mean with SD (error bars) for MS and control groups at each endpoint. *P < .05 between MS and control groups at each period. (D) Results of histological scoring system for repaired meniscus. The scores are displayed as mean with SD (error bars). *P < .05, **P < .10 between MS and control groups at each endpoint.
A more detailed microscopic analysis of the quantity of tissue bridging the defect, as seen on radial histologic sections, was also performed (Fig 5A and 5B). The quantity of tissue bridging the defect in both MS and control knees increased with time from 1 to 4 weeks and 4 to 12 weeks. The average proportion of defect filling, however, was consistently higher in MS knees than in controls at each endpoint, with differences reaching statistical significance at 1 week and 4 weeks (P < .05) (Table 2 and Fig 5C).
Marrow Stimulation Enhances Quality of Meniscal Tissue Bridging the Site of Injury The quality of tissue bridging the defect was superior in knees treated with MS, as shown by greater mean tissue quality scores at each endpoint. Tissue quality scores increased with time in both experimental and control knees from 1 to 4 weeks and 4 to 12 weeks. Average scores were consistently higher, however, in MS knees compared with control knees at each endpoint, reaching statistical significance at 1 week
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Table 2. Reparative Tissue Area/Defect Area Ratio (range, 0e1) 1 week 4 weeks 12 weeks
Mean SD Mean SD Mean SD
Control
MS
P-value
0.30 0.19 0.53 0.17 0.77 0.39
0.55 0.08 0.71 0.16 0.96 0.05
.016 .047 .156
(P ¼ .03) and approaching statistical significance at 4 weeks (P ¼ .08) (Table 3, Fig 5D). In addition, average values for each of the 3 subscores comprising the composite tissue quality score (tissue bonding, existence of fibrochondrocytes, and staining with SafraninO) were higher at each endpoint in MS knees compared with controls. Interobserver variability for the Tissue Quality Score was noted to be quite low, with an intraclass correlation coefficient of .97. Proregenerative Cytokines are Present in Injured and Healing Tissue After Meniscal Injury In both experimental and control knees, immunostaining for TGFb-1, IGF-1, and PDGF-A was diffusely positive along the borders of the defect and within tissue bridging the defect 1 week after injury (Fig 6). By comparison, staining was minimally positive within the substance of the uninjured meniscus. Staining appeared similar, however, in MS and control specimens 1 week after injury. Finally, staining for BMP-2 was negative in intact and healing tissue in both treatment groups.
Discussion The results of this study suggest that MS leads to a modest improvement meniscal healing, in terms of both quantity and quality of tissue bridging a site of meniscal injury, particularly during the first several weeks after injury. First popularized by Steadman in the 1990s, MS with microfracture has become a widely accepted treatment for articular cartilage injuries in not only the knee but also the shoulder, hip, and ankle.22-29 Although it stands to reason that the release of pluripotent cells and growth factors into the joint with MS may also aid in healing of other intra-articular injuries, there has been little investigation into this theory with regard to meniscal healing. Table 3. Tissue Quality Score (range, 0e6) 1 week 4 weeks 12 weeks
Mean SD Mean SD Mean SD
Control
MS
P-value
0.3 0.3 2.8 1.9 4.5 2.6
1.8 1.0 4.3 1.5 5.9 0.2
.029 .084 .211
Fig 6. Immunohistochemistry for proregenerative cytokines. In both MS and control knees, immunohistochemistry assays demonstrated positive staining for TGFb-1, IGF-1, and PDGFA, in meniscal tissue bordering the site of injury and within the reparative tissue 1 week after injury. Staining for TGFb-1 is shown here in sections from a knee treated with MS and a control knee, with each image including intact tissue on the left and healing tissue within the meniscal defect on the right. Arrows indicate defect border. Scale bar: 100 mm.
The meniscus is composed of a dense fibrocartilage matrix and sparse population of meniscal fibrochondrocytes. Only the peripheral 10% to 30% of the meniscus is vascularized, and tears occurring in the more central portions show poor healing capacity even after close approximation with suture repair.2-5,19 Recent studies using a variety of techniques aimed at enhancing the biological environment for meniscal healing suggest that biological factors may be of greater importance than repair technique.6-17,30-32 Meniscal rasping and trephination,6-9 direct application of cytokine-rich substances such as fibrin clot or platelet-rich plasma,11,30,31 and even preliminary stem cellebased therapies in animal models14-16,32 have been applied to meniscal defects, leading to improved laboratory and clinical results. MS may represent yet another tool capable of enhancing healing rates and further improving clinical outcomes. Indirect evidence in support of this concept is found in the superior healing rates in meniscus tears repaired concurrently with ACL reconstruction and associated release of marrow contents with tunnel preparation.2,5,18 The results of this study provide the first direct evidence that MS alone does accelerate meniscal healing, leading to improvements in both quantity and quality of tissue bridging the site of meniscal injury during the first several weeks after injury. In terms of quantity, the volume of tissue bridging the injury site is critical for both strength of the repair and restoration of a smooth contour for articulation with the adjacent chondral surfaces. MS knees were found to have a greater quantity of tissue bridging the repair site compared with control knees at each endpoint, with differences reaching statistical significance at 1 and 4 weeks. In addition, gross inspection of 1-week specimens revealed complete partial-thickness coverage of the defect in 5 of 6 MS knees, compared with only 1 of
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6 control knees. This is likely a result of more abundant clot formation within the injury site in MS knees, which may serve as a biological scaffold that is remodeled with new fibrocartilage over the next several weeks. Quality of tissue traversing the defect was evaluated using a 3-component scoring system11,14 that assesses for the presence of cells resembling meniscal fibrochondrocytes, density of staining for proteoglycan, and the extent of bonding between intact meniscus and regenerated tissue. The first 2 of these components reflect the similarity or lack thereof between the reparative tissue and native meniscus. The third, extent of bonding, reflects the integrity of the repair and may serve as an indicator of repair strength, which could not be directly assessed in this model. At all endpoints, each of the 3 subscores, as well as the cumulative tissue quality scores, were higher in MS knees than in control knees, with these differences reaching or approaching significance at early and midterm follow-up. By 12 weeks, defects in both MS and control groups were found to heal relatively well in most cases. Although overall scores remained higher in MS knees, differences were not statistically significant. Thus, the effect of MS seems to be most beneficial within the first several weeks of treatment in the present model. This may be of clinical importance, as surgeons often limit weight bearing and/or range of motion during the first several weeks after repair. Acceleration of the repair process during this early period of protection may better prepare the meniscus to tolerate increased loading with resumption of activity in the subsequent weeks and months, when the risk if reinjury is greatest.3 Several in vitro experiments have implicated specific growth factors such as TGFb, IGF-1, PDGF, and BMP-2 in promoting meniscal healing and regeneration.33-39 For example, Stewart et al.37 found that PDGF, IGF-1, and TGFb increased ovine meniscal fibrochondrocyte proliferation and type I collagen synthesis when cultured on polyglycolic acid scaffolds. Similarly, Bhargava et al.34 found that BMP-2 and PDGF increased bovine meniscal cell migration and DNA synthesis in vitro. In animal studies, however, the role of these cytokines is less clear. Kim et al.,39 for example, found that TGF-b1 and VEGF expression was lower in rabbit knees treated with autologous bone marrow injection after posterior cruciate injury than in control knees treated without additional biological stimulus, despite superior ligament healing in the bone marroweinjection groups. In addition, a clinically relevant sheep model evaluating the use of VEGF-impregnated suture in meniscal repair showed no benefit over conventional suture.35 Similar to these 2 studies, the results of this experiment suggest that although proregenerative cytokines appear to play a role in meniscal healing and regeneration in vivo, additional factors are critical as well. In
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the present study, positive staining for 3 relevant growth factors was seen in the reparative tissue of both MS and control knees 1 week after injury, indicating that upregulation of proregenerative cytokines may represent an inherent response to acute meniscal injury even in the absence of MS. This suggests, however, that an additional healing stimulus resulting from the release of marrow contents was responsible for differences in the quality and quantity of reparative tissue observed. Potential explanations include an increased intra-articular population of pluripotent mesenchymal progenitor cells, which are known to adhere to sites of meniscal injury and stimulate meniscal healing and regeneration in animal models.14-16 In addition, hemarthrosis in MS knees may lead to clot formation within the defect, providing a scaffold for cellular adhesion and differentiation, as well as subsequent formation of appropriate extracellular matrix.30,31 The location of subchondral bone penetration used for MS in this study (distal femoral chondral surface anterior to the apex of the intercondylar notch) was selected for its ease of access, reproducibility, and direct approach to the intramedullary space to ensure adequate marrow drainage into the joint. In clinical practice, however, this location is undesirable, as it results in damage to the distal femoral articular cartilage. In addition, the 2.4-mm tunnel diameter was selected, in part, because this size is comparable in scale to the femoral tunnel diameter in an anterior cruciate ligament (ACL) reconstruction, but such a relatively large tunnel may not be necessary in clinical practice. More clinically acceptable methods of MS include the creation of multiple smaller perforations in either the intercondylar notch wall as proposed by Freedman et al.20 or the medial or lateral cortex of the femur or tibia (depending on tear location), but neither technique has been studied critically. Limitations of this study include differences between rabbit and human meniscus structure and regenerative capacity,21 our inability to directly assess mechanical strength of meniscal healing using this model, and the lack of comparison between the effects of MS and other repair adjuncts such as rasping, trephination, or addition of fibrin clot, exogenous growth factors, or stem cells. In addition, the meniscal injury (cylindrical defect) used in this experiment was not sutured and differs from those seen clinically, which are typically linear tears. Such substantial differences mandate caution when extrapolating these results into the clinical setting. The chosen model was selected despite these limitations because it confers several advantages. Creation of a cylindrical defect allows for a quantitative assessment of the extent of defect bridging, as well as the quality of tissue within the defect. Furthermore, it produces a stable, highly reproducible injury, which minimizes side-to-side differences and negates the need
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for attempted suture repair and introduction of associated variables. One additional limitation warranting further discussion is the fact that this study did not investigate other potential explanations for superior healing rates observed in meniscal repairs performed simultaneously with ACL reconstruction. Aside from the effects of MS, other possible explanations include differences in tear acuity or symptomatology between patients with isolated meniscal tears and those with concomitant ACL injury (selection bias).19 For example, patients with both ACL and meniscal injuries may seek treatment earlier than those with isolated meniscus tears, resulting in a greater number of acute meniscus tears being treated with ACL reconstruction, thus leading to greater potential for healing.3 Second, a patient seeking treatment for an isolated meniscus tear is presumably symptomatic from the meniscus tear itself, whereas a patient with an associated ACL injury may have an asymptomatic meniscus tear that is repaired and remains asymptomatic whether it heals or not, thus resulting in a clinically successful repair. Finally, there may be underlying differences in the quality of a meniscus that tears in isolation and a meniscus that requires a major joint subluxation to tear. In other words, a meniscus that tears in isolation may be of inferior quality and thus less likely to mend after suture repair. In addition to the effects of MS, some or all of these factors may play a role in the superior healing rates observed in meniscal repairs performed with ACL reconstruction.
5.
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9.
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11.
12.
13.
Conclusions
14.
The results of this study suggest that MS leads to modest improvements in quality and quantity of reparative tissue bridging a meniscal defect, particularly during the early recovery period.
15.
Acknowledgment The authors thank Lin Bustamante and Chaitali Mukherjee for their assistance with specimen sectioning and staining.
16.
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