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First published on September 12, 2005, doi:10.1177/0363546505278702
This version was published on January 1, 2006
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The American Journal of Sports Medicine 34:32-42 (2006)
© 2006 American Orthopaedic Society for Sports Medicine

Long-term Outcome for Large Meniscal Defects Treated With Small Intestinal Submucosa in a Dog Model

James L. Cook, DVM, PhD*,{dagger}, Derek B. Fox, DVM, PhD{dagger}, Prasanna Malaviya, PhD{ddagger}, James L. Tomlinson, DVM, MVSc{dagger}, Keiichi Kuroki, DVM, PhD{dagger}, Cristi Reeves Cook, DVM, MS{dagger} and Stephanie Kladakis, PhD§

From the {dagger} Comparative Orthopaedic Laboratory, University of Missouri, Columbia, Missouri, {ddagger} DePuy Orthopaedics Inc, Warsaw, Indiana, and § DePuy Biologics, Raynham, Massachusetts

* Address correspondence to James L. Cook, DVM, PhD, Comparative Orthopaedic Laboratory, University of Missouri, 379 East Campus Drive, Columbia, MO 65211 (e-mail: cookjl{at}missouri.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Background: Large meniscal defects are a common problem for which current treatment options are limited.

Hypothesis: Treatment of posterior medial meniscal defects in dogs with small intestinal submucosa is superior to partial meniscectomy in terms of clinical limb function, chondroprotection, and amount and type of new tissue in the defect.

Study Design: Controlled laboratory study.

Methods: A total of 51 mongrel dogs underwent medial arthrotomy with creation of standardized meniscal defects. The dogs were divided into groups based on defect treatment: small intestinal submucosa meniscal implant (n = 29) or meniscectomy (n = 22). The dogs were assessed for lameness by subjective scoring after surgery and sacrificed at 3, 6, or 12 months and assessed for articular cartilage damage, gross and histologic appearance of the operated meniscus, amount of new tissue in the defect, equilibrium compressive modulus of meniscal tissue, and relative compressive stiffness of articular cartilage.

Results: Dogs in the meniscectomy groups were significantly (P < .001) more lame than dogs treated with small intestinal submucosa. Joints treated with small intestinal submucosa had significantly (P <.001) less articular cartilage damage, based on india ink staining, than did those treated with meniscectomy. Menisci receiving small intestinal submucosa had more tissue filling in the defects than did menisci receiving no implants, and this new tissue was more mature and meniscus-like and better integrated with remaining meniscus.

Conclusion: Small intestinal submucosa scaffolds placed in large meniscal defects resulted in production of meniscus-like replacement tissue, which was consistently superior to meniscectomy in amount, type, and integration of new tissue; chondroprotection; and limb function in the long term.

Clinical Relevance: Small intestinal submucosa implants might be useful for treatment of large posterior vascular meniscal defects in humans.

Key Words: meniscus • small intestinal submucosa • meniscectomy • tissue engineering • dog


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
The meniscal-deficient knee is a common problem facing orthopaedic surgeons. Approximately 1 million meniscal surgeries are performed each year in the United States alone.24 Although repair of meniscal lesions is possible in selected cases, the poor healing capacity of the tissue often dictates removal of damaged tissue, making meniscectomy the most common treatment for meniscal injury. Loss of meniscal tissue can severely diminish load bearing, shock absorption, congruency, stability, lubrication, and nutrition functions of the knee. These losses result in lack of protection of articular cartilage of the femoral and tibial condyles and resultant osteoarthritis.2,4,6,7,13,23,34,36,37 So, although meniscectomy provides pain relief and return to function, the loss of meniscal tissue results in long-term dysfunction and secondary osteoarthritis.2,4,6,7,13,23,34,36,37 Two consistent findings are present in published studies evaluating the long-term results of meniscectomy: (1) meniscectomy causes or exacerbates long-term articular cartilage injuries based on gross, histologic, imaging (radiographic and MRI), and functional analyses; and (2) the amount of remaining meniscus influences the rate and severity of the pathologic changes.2,4,6,7,13,34,36,37

The size, shape, and composition of the menisci play roles in determining the structural and material properties of the tissue. These material properties are vital for ensuring the biomechanical function of the menisci.3,14,15,21,22,29,35 Knee menisci are exposed to tensile, shear, and compressive stresses. These stresses are counteracted by swelling pressures and "hoop function" of the tissue, which depend on extracellular matrix composition and shape. The menisci transmit significant portions of weightbearing load during activity, and loss of meniscal tissue results in significant alterations in biomechanical function. One study reported that resection of 16% to 34% of the meniscus resulted in more than 3-fold increases in contact forces in the knee.29 Numerous studies report that even partial meniscectomies result in significant articular cartilage and bone changes within 5 to 15 years.2,4,6,7,13,34,36,37 These studies conclude that the degree of pathologic change in the joint is directly proportional to the amount of tissue loss.2,4,6,7,13,34,36,37

As stated, the nature and degree of meniscal lesions often require resection of large amounts of meniscal tissue. Therefore, when a meniscal injury dictates the surgical removal of significant portions of the meniscus, replacement with tissue of normal size, shape, and composition would appear to be the optimal objective when considering the long-term function of the knee. Currently, there are multiple strategies for addressing this objective, including meniscal allografts, biologic scaffolds for tissue-engineered replacement tissue, and biologic stimuli for meniscal tissue regeneration.

Current indications for meniscal allografts include large, irreparable meniscal defects resulting in loss of meniscal function with concurrent pain and instability. On the basis of experimental findings, meniscal allografts initially improve the biomechanical function of the knee, providing improvements in maximum joint pressure, mean pressure, and contact area compared with the knee that has undergone a meniscectomy.1,26,32 Patient satisfaction with the procedure is greater than 75%, and approximately 90% of patients would have the procedure performed again if necessary.5,18,19,27,30,33 Although further research is needed before conclusions about the long-term effects of meniscal allografts can be definitively stated, it appears that meniscal allografting is currently the most appropriate treatment for meniscal lesions that involve the entire extent of the tissue, requiring total meniscectomy. However, currently available materials and techniques do not allow for complete regeneration of functional meniscal tissue, and sizing problems and concerns about disease transmission further limit the usefulness of meniscal allografting.

The only commercially available scaffold for meniscal replacement approved for use is the Collagen Meniscus Implant (CMI) (ReGen Biologics, Redwood City, Calif).28,31 The CMI is approved for use in Europe, Australia, and Chile, and it is currently under regulatory review in Canada. Current indications are for use in medial meniscal defects that involve more than 25% of the radial dimension of the meniscus and have an intact peripheral rim. It has been reported that the use of CMI results in improvements in pain, activity, and self-assessment scores 1 and 2 years after implantation into irreparable medial meniscal defects.28,31 Second-look arthroscopic evaluations performed 6 or 12 months after surgery revealed meniscal-like tissue that filled on average 77% of the original meniscal defect.28 Based on arthroscopic assessment and radiographic evaluations, articular cartilage of the femoral and tibial surfaces maintained integrity without progression of osteoarthritis 2 years after treatment with a CMI.28 Histologic evaluation of biopsies taken from new meniscal tissue was consistent with fibrocartilage matrix formation.28 The CMI appears to provide a suitable biologic implant for inducing meniscal-like tissue formation in large medial meniscal defects involving the anterior, central, and posterior regions while maintaining an intact meniscal rim.

Porcine small intestinal submucosa (SIS) has been used with success to enhance meniscal regeneration in animal models.8,10–12,20 It is a naturally occurring extracellular matrix comprising collagens, proteoglycans, and growth factors derived from the small intestine of pigs. The SIS meniscal implants have been developed to address large meniscal defects involving the posterior horn of the menisci, which occur commonly and are not currently addressed by allografts or CMI. The current standard of care for the meniscal injuries for which our SIS implants were designed is partial meniscectomy. From our previous work using SIS, it appears that this biomaterial works to augment the amount and type of meniscal replacement tissue primarily by providing a 3-dimensional scaffold for clot formation and cell conduction, allowing for new tissue production.10,12,16 In addition, SIS may also have mitogenic properties for some cell types involved in the tissue regeneration that occurs in this application.16,17 Our data suggest that SIS-induced meniscal tissue replacement can be successful by ensuring the following criteria are addressed:

  1. The meniscal defect should extend to the vascular zone.
  2. A source for initial clot formation should be present or created.
  3. The SIS implant should be adequately stabilized in the meniscal defect.
  4. The SIS implant or meniscus should be protected during the initial phases of healing.

When these criteria have been addressed, we have realized excellent outcomes for SIS-treated meniscal defects in terms of amount and type of replacement tissue, limb function, and articular cartilage protection in the short term (up to 6 weeks after surgery).8,10–12 However, long-term studies have not been performed to date. Therefore, the purpose of this study was to determine the long-term (3–12 months) effects of replacement of large vascular posterior meniscal defects with SIS scaffolds in promoting meniscal regeneration and protecting articular cartilage. Our central hypothesis was that SIS-treated vascular posterior medial meniscal defects in dogs would be superior to the current standard of care (partial meniscectomy) in terms of clinical limb function, chondroprotection, and amount and type of new tissue in the defect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Preoperative Evaluation
All procedures were approved by the University Animal Care and Use Committee. Adult (2–5 years of age) conditioned dogs (n = 51), weighing 22.0 to 34.4 kg, were used. The dogs were judged healthy and free from orthopaedic disease, based on packed-cell volume, plasma total solids, blood urea nitrogen levels, heartworm test, complete physical and orthopaedic examination, clinical lameness evaluation, and radiographs of the hips and knees. The dogs were housed individually in runs approved by the Association for Assessment and Accreditation of Laboratory Animal Care and fed a commercially available maintenance diet for the duration of the study.

Ultrasonographic evaluation of meniscal tissue was performed once during the week before surgery. Both knees were imaged using an ultrasound machine (Logic 500, General Electric Medical Systems, Milwaukee, Wis) and a 13 MHz transducer. Ultrasonographic evaluation of menisci included the assessment of subjective appearance and the determination of a cross-sectional area (CSA). Cross-sectional images of the cranial, central, and caudal regions of the medial menisci were obtained and recorded. Subjective assessment was based on size, shape, and echogenicity. Measurements of meniscal base (b) (proximal to distal extents) and height (h) (medial to lateral extents) were determined to the nearest 0.01 mm.12

Surgical Instrumentation
On the day of surgery, the dogs were premedicated with xylazine (0.5 mg/kg intramuscularly) and morphine (0.5 mg/kg intramuscularly), anesthetized with thiopental (10–20 mg/kg intravenously), and maintained with isoflurane in oxygen. Using an aseptic technique, a medial approach with osteotomy of the origin of the medial collateral ligament and medial arthrotomy was performed on one randomly assigned knee of each dog. A standardized partial meniscectomy was created in the posterior portion of the medial meniscus (Figure 1Go) using a cut template designed and made by DePuy Orthopaedics Inc (Warsaw, Ind). The dimensions of the cut template were 10 mm in longitudinal length and 5 mm in radial depth. The dimensions of the meniscal resection matched the size of the wedge portion of the final SIS implant and extended to the vascular zone of the meniscus.





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  		Figure 1. A, cut template; B, defect outline; C, meniscal defect.

 
The dogs were randomly assigned to 1 of the following treatment groups (Figure 2Go):





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  		Figure 2. A, small intestinal submucosa repair technique; B, small intestinal submucosa implant; C, meniscectomy.

 
SIS meniscal implant (n = 29)—a 3-dimensional SIS implant was sutured into the meniscal defect using 5-0 prolene. The SIS meniscal implant consisted of wedge-shaped SIS foam encased within a laminated 20-layer wedge. The SIS laminates at the inner edge of the wedge implant were folded over to eliminate delamination at the implant apex.
Meniscectomy (n = 22)—the meniscal defect received no implant.

The implant was stabilized in the defect site with sutures of 5-0 prolene. Sutures of 5-0 prolene were also placed at the margin of resection in the meniscectomy group to standardize the treatment and mark the site of original resection. The medial collateral ligament was reattached by means of 2.7-mm screw fixation of the osteotomy site. Nonweightbearing slings were placed on the operated limb of each dog. Analgesics (morphine or aspirin) were administered to the dogs at the time of extubation and then as necessary to control signs of pain. The dogs were recovered and returned to their individual kennels.

Clinical Evaluation
The dogs were observed daily, and rectal temperature, pulse rate, and respiratory rate, as well as appetite, attitude, and activity level, were recorded. The dogs were restricted to cage rest after surgery. The slings were maintained, based on daily observation, and changed as needed until they were removed 3 weeks after surgery. Soft-padded, bivalved casts were placed on the operated limbs after sling removal and were maintained for 3 weeks. Clinical lameness evaluation was performed in blinded fashion by 2 veterinary orthopaedic surgeons every 4 weeks after surgery by observing the dogs at a trot and scoring their operated limb function using the following scale:

0 no observable lameness
1 intermittent, mild weightbearing lameness with little if any change in gait
2 consistent, mild weightbearing lameness with little change in gait
3 moderate weightbearing lameness; obvious lameness with noticeable change in gait
4 severe weightbearing lameness; "toe-touching" only
5 nonweightbearing

The dogs were sacrificed 3 months (n = 11; SIS group = 5, meniscectomy group = 6), 6 months (n = 16; SIS group = 12, meniscectomy group = 4), or 12 months (n = 24; SIS group = 12, meniscectomy group = 12) after surgery.

Gross Evaluation
After sacrifice, both knees of each dog (n = 51) were examined. The tibial plateau and femoral condyles were photographed. Measurements for determination of CSA were made (see below). For knees designated for histologic analysis, the entire medial meniscus from both knees of each dog was collected and photographed, along with a millimeter-scale marker, before placement in formalin for histologic processing. After removal of the medial meniscus, the medial femoral and tibial condyles of both knees from each dog were painted with india ink, washed after 60 seconds with tap water, and photographed.

Unexposed radiographic film was placed over femoral and tibial condyles and cut to match the surface area of the condyle. The areas of india ink staining were outlined using a permanent marker. Tracings of the india ink–stained tibial and femoral condyles were evaluated without knowledge of dog number or treatment group. The tracings were scanned using a computer software program (Image-Pro Plus, Media Cybernetics, Carlsbad, Calif), and the percentage of the total area of the tibial and the femoral condyles that stained was calculated and recorded as the percentage area of cartilage damage (%ACD). The %ACD was determined for the tibial and femoral condyles, separately and together, for each dog. The tibial and femoral condyles were then harvested and placed in formalin for histologic processing.

Biomechanical Testing
All 6-month and 12-month dogs were evaluated for relative compressive stiffness of the medial femoral condyle of both the operated and contralateral knees, and knees designated for histologic assessment were evaluated for relative compressive stiffness of the medial tibial condyles of both knees. Compressive stiffness of the cartilage was determined for each condyle using an arthroscopic cartilage stiffness testing device (Artscan 1000, Artscan Oy, Helsinki, Finland).9,21,23 Multiple readings (minimum of 8) were obtained at consistent locations on the weightbearing surface of each condyle. Artscan software was then used to choose 3 compressive stiffness measurement curves in which the applied force was consistently held at 10 N for a minimum of 2 seconds, as recommended, to determine the mean relative compressive stiffness value for each of the 3 curves. The mean of the 3 readings was determined and used as the relative compressive stiffness value for each condyle. Mean values are reported as relative compressive stiffness (reaction force in N).

For knees designated for meniscal biomechanical testing (n = 18), the disarticulated joints were shipped on ice to DePuy Orthopaedics Inc. The limbs were stored at –80°C until testing (within 1 month). Although the meniscal tissue was still frozen, a 2-mm-diameter explant was extracted from the regenerated tissue (implanted meniscus-defect site [IM-D]) and tissue from the same anatomical location in the posterior horn on the contralateral, unoperated medial menisci (CM-D) using a biopsy punch. Explants from the anterior horn of each meniscus (IM-A and CM-A) were also obtained for testing in the same manner. The sample numbers tested were as follows:

Each explant was thawed to room temperature in a proteinase inhibitor solution (10 mM N-ethylmaleimide, 5 mM benzamidine, 1 mM phenylmethanesulfonyl fluoride, and 2 mM EDTA in phosphate buffered saline, pH 7). The explants were trimmed to obtain 1-mm-thick discs with parallel faces. These discs were then tested in proteinase inhibitor solution under unconfined compression with step-wise cumulative strain of 5%, 10%, 15%, and 20% (0.1% strain/s, 30 min of stress relaxation) using the DDL 200R materials testing machine (DDL Inc, Eden Prairie, Minn). Stress-strain curves were obtained based on equilibrium stress values. Tissue modulus was calculated from the slope of the linear region of the stress-strain plot.

Area of Replacement Tissue
Cross-sectional area measurements were calculated using direct measurements of operated (n = 51) and unoperated (contralateral) (n = 51) medial menisci. Digital calipers were used to measure the menisci in situ to determine the axial-to-abaxial distance (h) and the superior-to-inferior distance (b) at the anterior, middle, and posterior aspects of the sites where the original defect was made or at the same locations on the unoperated menisci. The CSA was then determined using the formula for an isosceles triangle (b · h/2) for each site, and the mean of values for the 3 sites was used for calculating the cross-sectional percentage of the original area (CSA%). The CSA% was calculated using the following formula: (replacement CSA/contralateral CSA) · 100.

Photographs of the operated menisci (n = 51) obtained at the time of surgery and at postmortem examination were outlined to obtain a value for total surface area (TSA) of each. Using image analysis software (ImagePro), the area of the meniscus at the defect site before meniscectomy (original TSA) and the area of remaining meniscus and regenerated tissue at the defect site at the time of sacrifice (replacement TSA) were calculated. The total surface area percentage (TSA%) was determined by using the following formula: (replacement TSA/original TSA) · 100.

Histologic Assessment
After routine histologic processing, 5-µm sections were cut from each meniscus, femoral condyle, and tibial condyle designated for histologic assessment (n = 33; 3 months, SIS = 5, meniscectomy = 6; 6 months, SIS = 6, meniscectomy = 4; 12 months, SIS = 6, meniscectomy = 6) and were stained with hematoxylin and eosin (H&E). All sections from each tissue type were cut in cross-section for evaluation. Histologic assessment was performed by one investigator (J.L.C.) who was blinded to dog number and treatment group. The H&E stained sections were subjectively evaluated for the amount and character of meniscal replacement tissue and the amount and severity of articular cartilage damage.

Statistical Analyses
All statistical analyses were performed using a computer software program (Sigma Stat, San Rafael, Calif). Data from each group at each sacrifice time point were combined, and means ± SD and SEM were determined. A t test or 1-way analysis of variance (ANOVA) was performed to determine differences between groups with respect to each outcome measure of continuous data (CSA%, TSA%, %ACD, relative compressive stiffness, tissue modulus) at each time point. An ANOVA on ranks was performed to determine differences between both treatment groups with respect to each outcome measure of categorical data (lameness scores) at each time point. Significance was set at P <.05. When significant differences among groups were obtained, an all pairwise multiple comparison was performed to determine which groups were different from each other. Differences within a group with respect to each assay at different collection times were analyzed in a similar manner, when appropriate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
All dogs were judged to be healthy, with no evidence of musculoskeletal abnormality, at the start of the study. All menisci were judged to be of sufficient size for creation of the standardized defect and of normal echogenicity based on preoperative ultrasonographic assessment. All dogs survived, and no surgical complications were noted for the duration of the study. Dogs in the meniscectomy group were significantly (P <.001) more lame than dogs in the SIS group 3, 6, and 12 months after surgery. The degree of lameness was significantly (P <.05) lower at 12 months than at 3 months for dogs in the SIS group. No significant differences were noted among lameness scores within the meniscectomy group over time (P = .944) (Figure 3Go).



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  		Figure 3. Mean ± SD lameness scores for dogs in each group 3 months (n = 51), 6 months (n = 40), and 12 months (n = 24) after surgery. Dogs in the meniscectomy group were significantly (P <.001) more lame than were dogs in the SIS group at all time points (analysis of variance on ranks).

 
Gross Evaluation
Meniscal defects treated with SIS had more replacement tissue present with a more mature appearance than that seen in defects in the meniscectomy group. Subjectively, replacement tissue filling of the meniscal defects was more consistent and mature in the SIS group (Table 1Go). New meniscal tissue appeared firmly attached to the remaining meniscus in SIS-treated defects, and the margin of initial resection was not distinct. New tissue present in the meniscectomy group defects ranged from mature and well integrated to immature and friable. Representative images of menisci from each group at each sacrifice time point are shown in Figure 4Go.


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  		TABLE 1 Subjective Evaluation of Gross Defect Fill and Tissue Appearance From Dogs in Each Group 3, 6, and 12 Months After Surgery
 


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  		Figure 4. Gross appearance of operated menisci from dogs in each group 3, 6, and 12 months after surgery. Meniscal defects treated with SIS had more replacement tissue present than that seen in defects in the meniscectomy group, and this meniscal tissue appeared mature and firmly attached to remaining meniscus in SIS-treated defects. The maturity of the tissue appeared to increase over time in both groups.

 
Area Measurements
At 3 months after surgery, the SIS group had significantly (P = .05) higher CSA% values than did the meniscectomy group. At 6 months after surgery, SIS-treated menisci had significantly (P <.001) more tissue regeneration than did those in the meniscectomy group, as determined by area measurements. One year after surgery, the CSA% values for the SIS group (mean ± SD, 104.3 ± 42.7) and the meniscectomy group (mean ± SD, 89.6 ± 21.2) were not significantly different (P = .298, t test, power = .061). However, TSA% values were significantly (P <.001, t test) lower in the meniscectomy group compared with the SIS group at this 12-month time point (Table 2Go).


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  		TABLE 2 Mean ± SEM Cross-sectional Area Percentage (CSA%) and Total Surface Area Percentage (TSA%) Measurements From Dogs in Each Group 3, 6, and 12 Months After Surgery
 
Articular Cartilage Damage
There were no significant differences in %ACD among the treatment groups 3 months after surgery. At 6 months after surgery, all SIS-grafted joints had significantly (P <.001) less articular cartilage damage than did those in the meniscectomy group. One year after surgery, the meniscectomy group had significantly (P <.001, t test) more articular cartilage damage than did the SIS group (Figure 5Go). There was significantly (P <.05) more articular cartilage damage in the meniscectomy dogs 1 year after surgery compared with 6 months after surgery. No other significant temporal differences in %ACD were noted.



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  		Figure 5. Mean ± SD percentage area of cartilage damage (%ACD); femoral %ACD and tibial %ACD values were added to determine the total amount of damage. Significant (P <.001) differences between groups were present 6 and 12 months after surgery (t test).

 
Articular Cartilage Compressive Stiffness
For the 6-month time point, there were no significant differences between the 2 treatment groups for operated or nonoperated condyles. For the 12-month time point, articular cartilage on the femoral condyles was significantly (P <.014, ANOVA) more stiff in the contralateral unoperated (control) knees (5.0 ± 1.1) than in both SIS-treated (3.7 ± 1.2) and meniscectomy-treated (2.8 ± 1.6) knees. For the tibial condyles, contralateral unoperated (control) articular cartilage was significantly (P = .009, ANOVA) more stiff than meniscectomy cartilage but not significantly different from SIS cartilage. The compressive stiffness values for the SIS group and the meniscectomy group were not significantly different from one another for either location (power > .74) (Figure 6Go).



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  		Figure 6. Mean ± SD relative compressive stiffness values for medial femoral and tibial condyles 1 year after surgery. Articular cartilage on the femoral condyles was significantly (P <.014) more stiff in the controls than in the SIS and meniscectomy groups, and on the tibial condyles, control cartilage was significantly (P = .009) more stiff than cartilage in the meniscectomy group but not cartilage in the SIS group.

 
Biomechanical Testing of Menisci
For both the 6-month and 12-month meniscal tissue testing, sufficient regenerated tissue was not consistently available for testing from the IM-D location data for the meniscectomy group. New tissue in the defect of dogs in the meniscectomy group was either not of sufficient quantity to obtain an appropriate test sample or was so fragile that sample preparation was not possible. Therefore, all samples from the IM-D location in this group were excluded from testing to avoid skewing the data by only using a subset of samples.

For the 6-month time point, the compressive modulus of the regenerated tissue in the SIS group was significantly lower compared with tissue at the same site in the contralateral meniscus (mean IM-D vs CM-D, 26.1 ± 6.2 kPa vs 158.2 ± 27.8 kPa, P <.005). Compressive moduli of tissue from the anterior horns of implanted and contralateral knees were also significantly different (mean IM-A vs CM-A, 72.6 ± 10.9 kPa vs 162.8 ± 30.1 kPa, P <.05), implying a significant limb-favoring effect.

For 12-month data, the compressive modulus was not statistically different between the SIS and meniscectomy groups for the 3 locations (IM-A, CM-A, CM-D) of the meniscus that were tested. However, the regenerated tissue (IM-D) equilibrium modulus in the SIS implant was significantly less (P <.05) than the equilibrium modulus for the contralateral medial posterior meniscus (CM-D) (Figure 7Go). Although the compressive modulus of the regenerated tissue was lower as compared with that of meniscal tissue from the same location in the contralateral menisci, this finding was significantly confounded because of the change in tissue modulus of the contralateral menisci apparently resulting from shifting weight to the nonoperated limb during the postoperative period. To accommodate for this effect, a normalization strategy was chosen (IM-D/IM-A x CM-A/CM-D), indicating that at 6 months, the normalized tissue modulus of the regenerated tissue in SIS-treated defects was 37% of the contralateral side, and at 1 year was 55% of the contralateral side. These data could not be determined for the meniscectomy group because of the inability to consistently obtain IM-D tissues for appropriate testing.



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  		Figure 7. SIS implant and meniscectomy variation of aggregate modulus for separate meniscal regions (mean ± SEM); n = 6 for all groups except SIS IM-D (n = 5), meniscectomy CM-D (n = 4), and meniscectomy CM-D (n = 4). IM-D, ipsilateral (treated) meniscus defect site; IM-A, ipsilateral meniscus anterior site; CM-D, contralateral (nontreated) meniscus defect site (no defect made); CM-A, contralateral meniscus anterior site.

 
Histologic Assessment
Meniscal defects treated with SIS were more consistent in terms of amount, type, and integration of new tissue compared with those in the meniscectomy group. Subjectively, replacement tissue varied from very small amounts of unorganized fibrous tissue in the meniscectomy group to extensive, organized tissue that appeared very meniscus-like in the SIS group. Cell density was highest in the 3-month SIS-treated defects. Extracellular matrix was more prevalent and organized in the 6-month sections. Replacement tissue was most abundant and most mature in the 6-month groups and, subjectively, the SIS group showed more consistent meniscal tissue regeneration in terms of tissue amount and organization. Attachment and incorporation of new tissue to remaining meniscus were good to excellent in the vast majority of SIS-treated sections. Sections from the meniscectomy group showed little to no replacement tissue. When present, this tissue was unorganized and poorly integrated.

One year after surgery, meniscal defects treated with SIS were more consistent in terms of amount, type, and integration of new tissue compared with meniscal defects left untreated. New tissue in the defects of SIS-treated dogs was consistently meniscus-like with respect to cell and extracellular matrix characteristics and was well integrated with remaining native meniscus. In general, menisci from dogs in the meniscectomy group had little new tissue in the defect, and new tissue was not as meniscus-like, mature, or well integrated compared with those in the SIS group. However, new tissue in the meniscectomy group was subjectively more mature at 12 months compared with tissue in this group at 3 or 6 months, as expected. Representative photomicrographs of menisci from each group at each sacrifice time point are shown in Figure 8Go.



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  		Figure 8. Histologic appearance of operated menisci from dogs in each group 3, 6, and 12 months after surgery. New tissue varied from small amounts of unorganized fibrous tissue in the meniscectomy group to extensive, organized tissue that appeared meniscus-like in the SIS group. Meniscal defects treated with SIS were judged to be more consistent in amount, type, and integration of new tissue compared with meniscal defects left untreated.

 
Histologic sections of medial femoral and tibial condyles demonstrated a variety of appearances ranging from normal articular cartilage to severe erosive and degenerative changes. Subjectively, the histologic appearance of the articular cartilage corresponded well to %ACD data for each group. Representative photomicrographs of articular cartilage are shown in Figure 9Go.



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  		Figure 9. Histologic appearance of articular cartilage from operated knees 1 year after surgery showing normal cartilage, superficial fibrillation, and full-thickness erosion. The histologic appearance subjectively corresponded well to articular cartilage damage scores, with dogs in the meniscectomy group showing the most severe changes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
The SIS scaffolds placed in posterior vascular meniscal defects resulted in the production of functional meniscus-like replacement tissue. The meniscal replacement tissue in SIS-grafted meniscal defects is consistently superior to meniscectomy in terms of amount, type, and integration of new tissue; chondroprotection; and limb function as early as 3 weeks after surgery8,10,11 and continuing up to 1 year after surgery. Evidence for maintenance of superior joint and limb function in SIS-treated meniscal defects compared to meniscal defects receiving no treatment is provided in this study. Dogs receiving SIS grafts had significantly better limb function as assessed by lameness scores than did dogs that had meniscectomies. Dogs treated with SIS implants also had significantly less articular cartilage damage in the operated knee 1 year after surgery. The relative articular cartilage compressive stiffness values for dogs treated with SIS were not different from normal articular cartilage, whereas values for meniscectomy knees were significantly lower than for normal cartilage. In addition, corresponding meniscal tissue from the contralateral, nonoperated knees of dogs in the meniscectomy group was stiffer than that of dogs in the SIS group. Taken together, these data suggest that SIS treatment of meniscal defects in dogs results in significant improvements in long-term limb function and joint health compared to partial meniscectomy.

The mechanism for SIS-associated improvements in limb and joint function likely involves induction of meniscus-like tissue regeneration within the defect. Grafting of meniscal defects with SIS resulted in increased tissue regeneration in the defects as assessed by objective area measurements, gross appearance, and histologic assessment. Both the amount of tissue in the defect and the nature of the tissue in the defect likely influenced this outcome. Not only was the amount of tissue in SIS-grafted defects significantly greater than in meniscectomy defects, but also tissue in SIS-grafted defects was more mature (amount, type, and organization of cells and extracellular matrix), better integrated to remaining meniscus, and achieved a more normal shape compared to tissue in meniscectomy defects. These morphologic factors influence the material properties of the tissue and may be important for interpreting biomechanical data from this study. Although the biomechanical properties of this new tissue are still inferior to those of native meniscus 1 year after grafting, the properties appear sufficient to provide relative chondroprotection and appropriate limb function. The increase in compressive modulus in the SIS group from 6 to 12 months (26.1 kPa at 6 months; 58.18 kPa at 12 months) is evidence of improved mechanical properties of the regenerated tissue over time. The improved mechanical properties can be attributed to the maturation of the newly formed tissue observed by histologic assessment. Direct comparisons of the biomechanical properties of regenerated tissues from the SIS and meniscectomy groups could not be made in this study because the amount of regenerated tissue in the meniscectomy defects was too small to allow for consistent creation of explants for appropriate testing. Although it is tempting to infer poor biomechanical properties of regenerated tissue in the meniscectomy group based on tissue amount, associated lameness and articular cartilage damage, and gross and histologic appearance, this conclusion cannot be drawn from these data. However, the biomechanical data suggest that native meniscal tissue surrounding the defect maintained its mechanical properties in the SIS-treated knees compared with the meniscectomy group 1 year after surgery.

It is interesting that the compressive modulus in the anterior horn of the untreated contralateral medial menisci of dogs in this study was significantly higher than that in the corresponding region of menisci in which the posterior horn was treated with SIS or meniscectomy. In addition, the compressive modulus in both horns of the untreated contralateral menisci of dogs in the meniscectomy group was higher than for SIS-treated dogs. These data suggest that the untreated contralateral limb is loaded to a higher degree than the treated limb, causing an alteration in the material properties of the menisci, and this result occurs to a greater degree in the meniscectomy-treated dogs compared with the SIS-treated dogs. This statement is supported by the lameness scoring data from this study and by previously reported data in dogs.25 This finding should be taken into account when analyzing and interpreting data regarding the material properties of treated menisci when the contralateral limb is used as a control.

Based on the corresponding data from lameness scores, amount of tissue in the defect, and amount of articular cartilage damage, it seems likely that the increased amount of meniscal tissue present with SIS treatment resulted in greater chondroprotection, which in turn resulted in better limb function. Although lameness scoring is a subjective method of assessment of limb function, our previous studies have shown that lameness scores correlate well with objective force plate data.12 The postoperative management protocol used for this study was designed to provide adequate protection of the SIS implants and newly forming tissue during the critical early time points through a nonweightbearing protocol (sling for 3 weeks), followed by controlled weightbearing with minimal knee range of motion protocol (cast for 3 weeks). The protocol used in this animal model was adapted to mimic the anticipated postoperative rehabilitation protocol to be used in people based on currently used protocols for similar treatment techniques (allografts and CMI).

In this study, SIS treatment accomplished the objective of chondroprotection. It is important to note that dogs incur significant articular cartilage damage within 3 weeks of partial meniscectomy when adequate meniscal function is not restored.8,10–12 Therefore, protection of articular cartilage for more than 3 weeks after surgery can be considered a significant, long-term benefit. In the present study, 6 and 12 months after surgery, SIS-grafted dogs had significantly less articular cartilage damage than meniscectomy dogs as assessed by objective measurement and subjective histologic appearance. These data provide further evidence that the presence of the SIS meniscal implant helps to maintain long-term joint health as compared to partial meniscectomy.

The authors recognize the limitations of the present study. As mentioned, it was not possible to assess the material properties of new tissue in the defects of dogs in the meniscectomy group. In addition, menisci from the contralateral limb of each dog were used for biomechanical testing controls, which proved to be limited in providing normal values because of differences in limb loading. Therefore, the data only allow us to make conclusions regarding the material properties of SIS-associated regenerative tissue with respect to normalized data and between SIS and meniscectomy groups with respect to remaining native meniscal tissue. Future studies should include control tissue from nontreated dogs and methodology for testing of material properties that account for limited tissue. Another limitation of this study is the lack of objective histologic data. Articular cartilage abmormalities and meniscal tissue character and composition were assessed by subjective evaluation only. Although it would be ideal to include comprehensive, quantitative outcome measures for these 2 parameters, currently available methodologies (eg, histomorphometry, immunohistochemistry, scoring systems) are semiquantitative and extremely labor intensive for this type of study. The authors suggest that the objective measures used, articular cartilage damage quantification and tissue area measurements, provide comprehensive, objective data that address the stated study objectives. Lastly, this study employed open arthrotomy via detachment of the medial collateral ligament in dogs. Although the technique for arthroscopic implantation of SIS grafts has been successfully developed and performed in human cadavers, differences in SIS-induced meniscal regeneration related to anatomy, biomechanics, and surgical approach have not been determined.

These data suggest that SIS-induced regeneration of meniscus-like tissue is successful when initial fixation of the SIS scaffold is stable, the SIS scaffold has direct access to a rich blood supply and source of cells, hemarthrosis for fibrin clot formation on the scaffold is ensured, and the SIS scaffold and regenerative tissue are protected during the initial postoperative period (6 weeks). Therefore, defects that extend into the vascular zone of the meniscus appear to be appropriate indications for SIS treatment based on data from this animal model. In addition, a source of blood for clot formation via hemarthrosis from open arthrotomy, hemorrhage from meniscal vessels, or induction of hemorrhage via trephination of meniscus or subchondral bone appears to be necessary for successful SIS-enhanced meniscal regeneration. When these criteria are met, SIS appears to consistently result in excellent functional tissue regeneration in dogs. However, to the authors’ knowledge, no currently available treatment for meniscal defects, including these SIS scaffolds, results in complete restitution of normal meniscus tissue and function. Therefore, clinical studies will need to be performed to determine the effects of SIS scaffolds on the natural history of the meniscectomized knee in humans.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
Placing SIS scaffolds in posterior vascular meniscal defects resulted in production of meniscus-like replacement tissue, which was consistently superior to meniscectomy in terms of amount, type, and integration of new tissue; chondroprotection; and limb function in the long term. Previous studies have reported that retaining or replacing as much meniscal tissue as is possible is critical for maintaining or reestablishing biomechanical properties and function of the knee while preventing osteoarthritic changes.2,4,6,7,13,23,34,36,37 The treatment of vascular posterior meniscal defects in dogs using SIS appears to accomplish these objectives for up to 1 year after surgery.

These data indicate that SIS implants might be useful for treatment of large posterior vascular meniscal defects in humans.


   ACKNOWLEDGMENTS
 
The authors thank Dr Steven Arnoczky, Dr Juri Ota, and Mr Tyler Haskins for their help with this work. This work was funded by DePuy Orthopaedics Inc and DePuy Biologics Inc—Johnson & Johnson Companies. J. L. C. and D.B.F.are consultants for DePuy Orthopaedics Inc and DePuy Biologics Inc, P. M. is an employee of DePuy Orthopaedics Inc, and S. K. is an employee of DePuy Biologics Inc.


   FOOTNOTES
 
Portions of these data were presented at the 50th annual meeting of the Orthopaedic Research Society, San Francisco, California, March 7-10, 2004.

One or more of the authors has declared a potential conflict of interest: James Cook and Derek Fox are consultants for DePuy, and Prasanna Malaviya and Stephanie Kladakis are employees of DePuy.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
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