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Abstract

Background

Complex meniscal lesions often require meniscectomy with favorable results in the short term but a high risk of early osteoarthritis subsequently. Partial meniscectomy treated with meniscal substitutes may delay articular cartilage degeneration.

Purpose

To evaluate the status of articular cartilage by T2 mapping after meniscal substitution with polyurethane scaffolds enriched with mesenchymal stem cells (MSC) and comparison with acellular scaffolds at 12 months.

Methods

Seventeen patients (18-50 years) with past meniscectomies were enrolled in 2 groups: (1) acellular polyurethane scaffold (APS) or (2) polyurethane scaffold enriched with MSC (MPS). Patients in the MPS group received filgrastim to stimulate MSC production, and CD90+ cells were obtained and cultured in the polyurethane scaffold. The scaffolds were implanted arthroscopically into partial meniscus defects. Concomitant injuries (articular cartilage lesions or cartilage lesions) were treated during the same procedure. Changes in the quality of articular cartilage were evaluated with T2 mapping in femur and tibia at 12 months.

Results

In tibial T2 mapping, values for the MPS group increased slightly at 9 months but returned to initial values at 12 months (P > 0.05). In the APS group, a clear decrease from 3 months to 12 months was observed (P > 0.05). This difference tended to be significantly lower in the APS group compared with the MPS group at the final time point (P = 0.18). In the femur, a slight increase in the MPS group (47.8 ± 3.4) compared with the APS group (45.3 ± 4.9) was observed (P > 0.05).

Conclusion

Meniscal substitution with polyurethane scaffold maintains normal T2 mapping values in adjacent cartilage at 12 months. The addition of MSC did not show any advantage in the protection of articular cartilage over acellular scaffolds (P > 0.05).

Keywords

meniscal implant meniscal substitute polyurethane scaffold meniscectomy mesenchymal stem cells knee articular cartilage

Introduction

The knee is one of the most frequently injured joints in the human body. In 15% of all knee injuries either one of the menisci is involved.¹ Previously reported incidence rates for hospital admission after meniscal injury vary between 0.35 and 0.7 per 1000 person-years.² Knee osteoarthritis (OA) shares many risk factors and biological processes with degenerative meniscal tears. OA affects 27% of women and 21% of men, with an increase in incidence for both groups from the age of 45 years. According to an arthroscopic study, there is a 40% coincidence rate of meniscal injuries in those with knee OA. Furthermore, people who had previously partial or total meniscectomies are at risk of developing OA subsequently.³

The meniscus plays a pivotal role in load bearing and load transmission to the cartilage and the subchondral bone.⁴ After removal of the meniscus, the contact area decreases by 40% medially and by as much as 52% laterally.^5,6 Meniscus injuries occur either traumatically and are often associated with an articular cartilage lesion (ACL) rupture, or chronically as a result of structural weakening due to aging and wear.^7,8 Repair strategies allow preservation of the meniscal tissue when the rupture is not complex and is located in the vascular area.⁹ However, a high number of complex lesions cannot be repaired and usually require partial or total meniscectomy as the current standard of care, and favorable results are observed in the short term following a partial meniscectomy. However, the risk of early OA has been reported due to the decreased contact area when the menisci are missing and subsequent mechanical overload of the underlying articular cartilage. Dhollander et al. reported a decrease in intraarticular contact area of approximately 75% and an increase in peak contact pressure by approximately 235% after a total meniscectomy.¹⁰ Comparable results were found in a study by Ahmed and Burke.^4,11 Roos et al. reported a long-term clinical study with a follow-up of 21 years of patients after total meniscectomy compared with matched controls. They confirmed that the increased pressure seen in biomechanical studies led to radiographic evidence of OA with a relative risk factor of 14.¹²

Several long-term studies have shown that partial meniscectomy treated with meniscal substitutes may delay degeneration but not prevent it.¹³ A study of 136 patients who underwent partial meniscectomy for isolated meniscal tears showed a re-operation rate of 22.8% at 8.5 years, and 53% of patients had osteoarthritic radiographic changes compared with only 22% in the unaffected control knee.¹⁴ Meniscal substitutes have been used to relieve symptoms in patients who underwent partial meniscectomies.^15,16 The objective of these implants is to provide a better functionality of the meniscus, reduce the level of pain in young patients, and delay the onset of OA. Meniscal substitution with a polyurethane scaffold (Actifit, Orteq Sports Medicine, London, UK) has been shown to be an effective treatment by providing significant pain relief, and functional improvement in the short and medium term.¹⁷ Using a different material, a polycarbonate urethane, Shriram et al. demonstrated that the contact pressure on the articular cartilage and implant displacement were sensitive to the stiffness of the polycarbonate urethane meniscal implant. According to their study, increasing the material stiffness of the meniscal implant led to elevated contact pressures on the articular cartilage, which increased the risk of physiological damage to the articular cartilage.¹⁴ It is believed that the size of the implant is correlated with these findings.¹⁸

To the best of our knowledge, the evaluation of cartilage protection or delay in deterioration by T2 mapping in patients following meniscectomy and treatment with a polyurethane scaffold has not been described before. Furthermore, the clinical use of mesenchymal stem cell (MSC) with a polyurethane scaffold has not been reported. Therefore, we decided to develop the present study to evaluate the changes in articular cartilage quality after meniscectomy treated with acellular polyurethane scaffolds and compare them to MSC-enriched scaffolds at 12 months.

Methods

The study was performed in accordance with Good Clinical Practice and the Declaration of Helsinki. It was approved by the ethics committee of the National Institute of Rehabilitation “Luis Guillermo Ibarra Ibarra,” Mexico City, Mexico. Written informed consent was obtained from all patients for this prospective, longitudinal, comparative, and analytic study. Patients with a history of previous partial irreparable meniscal injury or partial (or subtotal) meniscectomy were included.

In the original design, only 11 patients were included to be treated with the MSC-enriched polyurethane scaffold. However, 6 young patients diagnosed with pathologies (ACL reconstruction or cartilage repair) treatment were found to have complex meniscal lesions that required meniscectomy in the index procedure and were therefore treated with acellular polyurethane scaffolds.

The patients were between 18 and 50 years old, had a history of chronic symptomatic meniscectomy (>25% to 90%) or acute meniscectomy performed during ACL reconstruction or any cartilage repair techniques, clinical and radiographic alignment, and body mass index (BMI) <30. Patients with OA grade IV evaluated by X-ray, history of septic arthritis, hypersensitivity to any implant component (Actifit) or to filgrastim, personal or familiar history of neoplasia, and presence of a hematopoietic system pathology were excluded. Patients were divided in 2 groups: (1) polyurethane scaffold enriched with mobilized MSCs (MPS) and (2) acellular polyurethane scaffold (APS).

Mesenchymal Stem Cell Mobilization

Patients enrolled in the MPS group received a daily subcutaneous injection of 300 µg of G-CSF (Filgrastim, Amgen, Thousand Oaks, CA) in the deltoid area for 3 consecutive days. The objective of G-CSF was to increase the pool of MSC in the peripheral blood stream. In order to observe the behavior of leukocytes, granulocytes, monocytes, and lymphocytes, a blood count control and hematic cell count were performed from day 1 to day 4. A daily questionnaire was performed to detect side effects that could be caused by the filgrastim application as headache, arthralgia, sternal pain, iliac crest pain, and fatigue.

Mobilized Mesenchymal Stem Cells Harvesting

At day 4, 400 mL of peripheral blood was obtained from a brachial vein. The blood unit was separated to obtain an average of 50 mL of the leukocyte fraction (“buffy coat”) ( Fig. 1A ).

Figure 1.

Peripheral blood sample with mobilized mesenchymal stem cells after granulocyte colony stimulating factor application. (A) Blood unit was separated from the leukocyte fraction “buffy coat.” (B) Mononuclear cells were isolated from the buffy coat in an automated equipment (Biosafe). (C) Staining of mononuclear cells with anti-CD90 coupled to a superparamagnetic pearl. (D) Separation of the CD90+ cells by the temporary union of the super-magnetic pearls; the cells go through an electromagnetic column designed by Miltenyi Biotec. (E) Harvest of CD90+ cells with some remaining erythrocytes. (F) Counting CD90+ cells in the Scepter equipment (Merck Millipore). (G) Hydration of scaffold by positive pressure with DMEM medium. (H) Seeding of the CD90+ cells in the polyurethane scaffold and sealing with based fibrin glue (Baxter).

Isolation of Mononuclear Cells from the Buffy Coat

In the Biotechnology Unit of our institution, the buffy coat was processed under sterile conditions inside a class 100 laminar flow hood with a cell separation kit (CS-900) to separate the mononuclear cells from the white fraction in a Biosafe Sepax 2 cell separation system (GE Life Sciences, Marlborough, MA). This system consists of a fully automatic, closed-loop, sterile mononuclear cell separator that allows a mononuclear isolation of the blood components through optical sensors. The content is transferred from the collector bag to a 50 mL tube and then centrifuged for 10 minutes at 300g to begin the process of mononuclear cell count ( Fig. 1B ).

Isolation of CD90+ Cells

CD90+ cells were isolated from the mononuclear cells after centrifugation ( Fig. 1C ). Ten microliters of mononuclear cells were taken, mixed in a 1-mL tube with 990 µL of marking solution, and read in an automated cell counter (Scepter, Millipore Sigma, Burlington, MA). Once the number of mononuclear cells had been established, the content was divided into three 15-mL tubes to which 300 µL of anti-CD90+ antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) was added. The 15-mL tubes were incubated during 20 minutes at 4°C using an orbital agitator (MACSmix Tube Rotator, Miltenyi). After incubation, the manual cell separation kit was assembled inside a laminar flow hood and the LS cell separation columns (Miltenyi) were placed. The cell/antibody sample was passed through the column with magnetic beads. At the end of passing the sample through the column, this column was removed from the separation system and placed in a 15-mL tube ( Fig. 1D ). The tube was centrifuged at 300g for 10 minutes and an aliquot of 10 µL was taken and then processed. The cell count was then repeated with an automated counter (Scepter) ( Fig. 1E ).

Characterization by Flow Cytometry

To establish their immunophenotype, an aliquot of 2.5 × 10⁴ cells in 100 µL of PBS were incubated with 5 µL of the antibody suspension for 30 minutes at 4°C. The monoclonal antibodies applied were CD34, CD45, CD90, CD117, CD73, CD166, CD105, and CD47 (BD Biosciences, San Diego, CA). The samples and/or unlabeled controls were included for each antibody and used to set the electronic gates on the flow cytometer. All data were obtained in a BD FACSCalibur flow cytometer and analyzed with CellQuest PRO software (BD Biosciences) with a mean of 20,000 events ( Fig. 1F ).

Culturing of CD90+ Cells

An aliquot of 2.5 × 10⁴ CD90+ cells were isolated from the mobilized peripheral blood cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Technologies, Carlsbad, CA), 20% autologous serum, and 1% antibiotic-antimycotic (Gibco). Cells were cultured in an incubator at 37°C with 5% CO₂ in 75 cm² culture flasks for 15 days.

Cell Seeding in Polyurethane Scaffold

After 15 days of culture, cells were seeded in the polyurethane meniscal scaffold by adding approximately 20 × 10⁶ cells ( Fig. 1G ). The surface of the scaffold was sealed in order to avoid cell loss with the use of fibrin glue covering all the implant surfaces ( Fig. 1H ). Then was left in culture media in the incubator for 2 days to promote cell adhesion. The scaffold was kept in sterile conditions and sent to the operating room for arthroscopic implantation ( Fig. 2 ).

Figure 2.

Cell seeding in the polyurethane meniscal scaffold. (A) Harvesting of the cells from the cultivation of the preexpanded CD90+. (B) Aliquots 3.3 × 10⁶ cells suspended in 200 µL of fiber glue using a 300 µL micropipette tip. (C,) Injection of the cells within approximately 5 mm of the scaffold. (E) Coating of all faces of the scaffold with fibrin glue. (F) The scaffold was submerged and kept in culture for 2 days. (G) Positive calcein staining (green), for the cells within the scaffold after 2 days in cellular culture.

Surgical Technique: Arthroscopic Implantation

All cases were performed arthroscopically under regional anesthesia by the same surgeon (AOM) via standard anterolateral and anteromedial portals. Routine knee arthroscopy was performed documenting chondral surfaces, ligament stability, and state of the meniscus to confirm the appropriate indication for meniscal substitution. At the time of the index surgery, remnants or damaged sections of the previous meniscectomy were excised, leaving a residual meniscal rim in the vascular zone and straight walls in the adjacent native meniscal tissue. The size of the defect was measured by arthroscopy, then the implant was trimmed on the back table 10 mm larger than the defect size to prevent over-tensioning of the sutures when fixed to native meniscus ( Fig. 3A ). The scaffold was delivered into the joint through a 10-mm cannula and sutured to the capsule and native meniscus borders. For the posterior horn, all-inside fixation with nonabsorbable suture (Fast-Fix, Smith & Nephew, Andover, MA) was used. For fixation of the scaffold in the meniscus body, an inside-out technique with absorbable PDS suture and a protector meniscus device (Arthrex, Naples, FL) was used. For the anterior horn, an outside-in fixation with PDS suture was used with a micro-suture lasso device (Arthrex, Naples, FL) ( Fig. 3B to F ).

Figure 3.

Surgical technique of medial meniscus substitution in the posterior horn with polyurethane implant enriched with MSCs. (A) Defect size is estimation with a flexible ruler. (B, C) Once the implant is trimmed in the back table this is introduced into the joint using arthroscopic forceps to push the implant into the defect area. (D, E) Implant is fixed to the adjacent healthy meniscal tissue with horizontal stiches (all inside technique) and vertical ones to hold it to the capsule and meniscal rim. (F) Pump flow is closed, and blood clot is formed over the sutured area. Notice that a PDS suture is placed in the implant to keep the tips outside the joint in order to do not lose the implant inside the joint.

Cartilage status in the tibia plateau and femoral condyle was assessed with the International Cartilage Repair Society (ICRS) scoring system. For patients in the APS group, the scaffold was opened in the operating room without any processing. For the patients in the MPS group, the cultured scaffold was transported from the biotechnology laboratory to the operating room in sterile conditions soaked in DMEM culture media with 10% autologous serum and 1% antibiotic-antimycotic.

Rehabilitation Protocol

All patients received postoperatively a knee brace locked from full extension to 90° of flexion for 6 weeks, which was removed 3 to 4 times per day to perform self-assisted passive range-of-motion exercises. During the first 4 weeks, the range of motion was limited from 0° to 90°, then allowed to increase to 130°. After 6 weeks, the brace was unlocked and worn for comfort only. Walking was allowing but with non–weight-bearing during the first 2 weeks. At 3 to 4 weeks, transitioning from partial to total weight-bearing was allowed. After 12 weeks, muscle strengthening and unrestricted activity was performed.

Imaging Evaluation

Magnetic resonance imaging (MRI) evaluation was performed using T2 mapping at 3, 6, 9, and 12 months postoperatively using a 1.5 Tesla clinical imaging system (GE Healthcare, Milwaukee WI), using an 8-channel HD knee array (GE Healthcare). The cartilage quality in the involved compartment was evaluated by 2 independent radiologists using T2 mapping (FuncTool 4.5.1, GE Healthcare) to identify deterioration signs of the articular cartilage adjacent to the meniscal scaffold. Those values were calculated by taking 2 regions of interest (ROI): weight-bearing area of the femoral condyle (FC) and tibial plateau (TP) in sagittal planes ( Figs. 4 and 5 ).

Figure 4.

Polyurethane implant enriched with MSCs and sealed with fibrin glue. (A) Implant is manipulated carefully to prevent cell and scaffold damage. (B, C) After arthroscopic estimation of the size of the defect area, the implant is measured in the back table and trimmed 10 mm larger than the required size.

Figure 5.

Evaluation of the cartilage status after meniscal substitution by T2 mapping. In the acellular group the tendency in the tibial plateau was toward decrease significantly from 3 to 12 months. Cartigram values in the MSCs group slightly increased at 9 months but at 12 months returns to the initial measurements. This difference tends to be significantly lower in acellular patients than cellular implants at final outcome (P = 0.18).

Clinical Evaluation

Before surgery and postoperatively (3, 6, 9 and 12 months), all patients underwent clinical follow-up examinations and Lysholm functional scores were used to assess the outcome.

Statistical Analysis

Data were analyzed with SPSS v22 (IBM, Armonk, NY). Descriptive statistical analysis was performed for demographic variables in both groups. An intragroup analysis was performed for T2 mapping using a 2-way variance analysis (Friedman test) for related samples. The sphericity of the variance-covariance matrix was evaluated first by the Mauchly sphericity test, taking W < 0.05 to reject the sphericity hypothesis. In the case the sphericity hypothesis was rejected, the univariate statistical analysis of Greenhouse-Geisser was used. In contrast, when W > 0.05 (spherical variance), the multivariate contrast was applied with the statistical analysis of the Hotelling trace. Each follow-up between intervention groups was also compared with the nonparametric Mann-Whitney U test. A value of P < 0.05 was taken as the level of significance for all the tests.

Results

Demographics

A total of 17 patients were included in the study; 13 men and 4 women with a mean age of 36.0 years (±7.6; range 21-47). Eleven patients (64.70%) received the polyurethane scaffold enriched with MSC (MPS), and 6 patients (35.3%) were treated with acellular polyurethane scaffolds (APS). In the APS group, all 6 patients were men (100%), while in the MPS group, 7 cases (63. 6%) were men and 4 cases (36.4%) women. Nine right knees (52.9%) and 8 left knees were treated (47.1%). Preoperatively, demographic characteristics of all patients were analyzed by gender. As noted above, the intervention groups differed significantly in the distribution of patients by gender since in the APS group 100% of patients were male compared with 63.6% in the MPS group (P = 0.04). They also differed in the distribution; in APS group 83.3% knees were right versus 36.4% in the MPS group (P = 0.05) ( Table 1 ).

Table 1.

Preoperative Characteristics of the Patients According to Gender.

Variable	Gender	Mean	SD (±)	P
Age	Female	32.75	11.55	0.62
Age	Male	37.08	6.26	0.62
Weight	Female	69.50	17.31	0.13
Weight	Male	81.85	8.69	0.13
BMI	Female	27.25	5.93	0.47
BMI	Male	27.10	2.89	0.47

P < 0.05 are significantly different.

Demographic characteristic were evaluated before surgery by gender; no significant difference was observed in age, weight, and body mass index between men and women.

Influence of Gender on Scaffold Size and Basal T2 Mapping

The mean post-meniscectomy time in the APS group was 31.8 months compared with 14.2 months in the MPS group (P = 0.09). Analyzing further by gender, men had longer post-meniscectomy times (22.27 ± 21.7 months) compared with women (14.0 ±6.16 months) but the difference was not statistically significant (P = 0.48). The defect size and resulting scaffold size were significantly larger in men (1.74 ± 4.59, 4.91 ± 1.76) than in women (1.65 ± 4.20, 2.37 ± 0.75) (P = 0.001). The mean scaffold size in the APS group was 55 mm compared with 35 mm in MPS group (P = 0.8). Basal T2 mapping did not show a significant difference when evaluated by gender in either femoral condyle (male 47.92 ± 4.41, female 51.42 ± 8.84, P = 0.78) or tibial plateau (male 44.19 ± 4.75, female 42.09 ± 5.05, P = 0.62). However, the age of the patients correlated with the femoral T2 mapping showing a positive coefficient (0.471), meaning that the older the patient, the higher the initial femoral cartigram (P = 0.05).

Concomitant Lesions

A total of 6 patients received concomitant ACL reconstruction (35.29%)—4 patients from the APS group (66.7%) and 2 patients from the MPS group (18.2%) (P = 0.04). Three of the 6 patients with ACL reconstruction had additional cartilage lesions in the medial femoral condyle that required concomitant treatment. Two patients were treated with MSC seeded in a polyglycolic acid (PGA) mesh (Neoveil, Gunze Ltd., Tokyo, Japan) and one patient was treated with microfracture technique (MFx). The meniscal lesions in these patients were also in the medial compartment. The 2 patients with cartilage lesions treated with MSCs + PGA mesh were in the APS group, while the patient treated with MFx was in the MPS group.

The total number of patients with cartilage lesions in both groups was 10 (58.82%)—2 in the APS group (11.76%) corresponding to 33.3% of the patients in the APS group, and 8 patients (47.05%) in the MPS group corresponding to 72.72% of this group. Chondral lesions were classified according to the ICRS score system, observing that 5 lesions (29.4%) were grade 4 (concomitant repair technique was performed), 3 lesions (33.3%) were grade 3, and 1 lesion (11.1%) was grade 2. The mean size of cartilage lesions was 9.5 mm in the APS group and 10.72 mm in the MPS group.

Quality of Adjacent Cartilage Evaluated with T2 Mapping after Meniscal Substitution

The evolution of tibial cartilage means-adjusted for age, height, months of meniscectomy, and scaffold size showed no significant differences between treatment groups. It was noted that tibial T2 mapping in the MPS group increased slightly at 9 months (42.6 ± 3.1) but decreased at 12 months (43.3 ± 4.2) to the same level as at 3 months (43.3 ± 3.3). In the APS group, T2 mapping in the tibial plateau showed a significant decrease from 3 to 12 months (45.2 ± 6.1 vs. 41.4 ± 4.1). This difference tends to be significantly lower in acellular patients (41.4 ± 4.1) compared with MSCs implants (43.3 ± 3.3) at the final time point (P = 0.18) ( Fig. 4 ).

Analyzing only the male patients, in the APS group at 12 months, the mean of the T2 mapping in tibial plateau was significantly lower compared with the male MPS patients (41.4 ± 4.1 vs. 43.3 ± 4.2, P = 0.01). In femoral T2 mapping, the differences were few between treatments and evolved in a similar way over time. Nonetheless, at 12 months it increased in the MPS group compared with the APS group (47.8 ± 3.4 vs. 45.3 ± 4.9, respectively). In both groups, persistent higher values of T2 mapping in the femoral condyle compared with the tibial plateau for men and women were observed ( Table 2 ).

Table 2.

Quality of Adjacent Cartilage Evaluated with T2 Mapping after Meniscal Substitution.

Gender	T2 mapping	3 Months	6 Months	9 Months	12 Months
Acellular polyurethane implant
Males	Femoral	47.3 ± 3.3	46.7 ± 2.9	45.4 ± 3.4	45.3 ± 4.9
Males	Tibial	45.2 ± 6.1	42.1 ± 2.1	42.4 ± 4.2	41.4 ± 4.1
P		0.59	0.04	0.30	0.19
MSCs polyurethane implant
Males	Femoral	48.4 ± 5.3	45.9 ± 5.0	46.5 ± 3.5	47.8 ± 3.4
Males	Tibial	43.3 ± 3.3	42.0 ± 4.3	42.6 ± 3.1	43.3 ± 4.2
P		0.01	0.12	0.02	0.02
Females	Femoral	51.4 ± 8.8	50.7 ± 5.6	55.3 ± 10.0	46.6 ± 3.8
Females	Tibial	42.0 ± 5.0	42.1 ± 6.3	47.8 ± 12.0	42.0 ± 3.7
P		0.06	0.03	0.29	0.23

The t2 mapping values of the femoral condyle and tibial plateau were compared intragroup and between groups by gender. The values were significantly lower in the tibial plateau compare to femoral condyle in the PIA (polyurethane implant, acellular) group at 6 months. Similar results were observed in PIM (polyurethane implant, MSCs) group for males at 3, 9, and 12 months, while in females those differences were reflected only at 3 and 6 months in the same group.

Comparison of Intergroup Variables

Due to the small sample size, it was not possible to perform a multivariate analysis to control post hoc the influence of age, gender, size, knee side, presence or absence of ACL injury, months of meniscectomy, and scaffold size on the evolution of the T2 mapping at 12 months. Therefore, only a step-by-step analysis was carried out considering some of the variables as a whole ( Table 3 ).

Table 3.

Comparison of Variables between Groups (PIA vs. PIM).

Analyzed Variable	Group		P
Analyzed Variable	Acellular Implant (n = 6)	MSCs Implant (n = 11)	P
Age	33.1 ± 5.9	37.6 ± 8.2	0.26
Male gender	6 (100%)	7 (63.6%)	0.04
Weight	84.6 ± 10.4	75.8 ± 11.9	0.14
Body mass index	26.8 ± 3.6	27.2 ± 3.7	0.82
Right knee	5 (83.3%)	4 (36.4%)	0.05
ACL injury	4 (66.7%)	2 (18.2%)	0.04
Presence of cartilage injury	2 (33.3%)	8 (72.7%)	0.11
Cartilage injury size	9.5 ± 77.7	10.7 ± 12.2	0.36
Months of meniscectomy	31.8 ± 30.7	14.2 ± 5.3	0.09
Implant size (cm)	5.5 ± 1.4	3.7 ± 1.9	0.08
Tibial cartigram (ms)	41.8 ± 3.6	43.9 ± 5.9	0.50
Femoral cartigram (ms)	43.7 ± 4.6	47.6 ± 3.7	0.13

P < 0.05 are significantly different.

There was significant difference between groups in gender, knee side, ACL reconstruction, months of meniscectomy, and implant size. Values were higher acellular patients compare with the patients with MSCs implants (P < 0.05).

Patients in the MPS group were older (37.6 ± 8.2 years) than those in acellular group (33.1 ± 5.9 years), but this difference was not statistically significant (P = 0.26). There was a statistically significant gender difference between groups (P = 0.04). All patients in the APS group were male (100%), and in the MPS group, the males accounted for 63.6% of the cases and females 36.4%.

Weight and BMI were not significantly different between groups (P > 0.05). The number of operated right knees in the APS group (83.3%) was significantly higher than in the MPS group (36.4%) (P = 0.05). Meniscal substitution without any other concomitant treatment was performed only in one patient in every group. The presence of ACL reconstruction in the APS group was significantly higher (66.7%) compared with the MPS group (18.2%) (P = 0.04).

Cartilage lesions in the APS group were found in 2 patients (33.3%) compared with 8 patients (72.7%) in MPS group (P = 0.11). However, the 2 patients with cartilage lesions in APS group were scored as ICRS-4 and were repaired by MFx in one patient and MSC+PGA scaffold in the second patient. Three patients (30%) in the MPS group had also cartilage injuries ICRS-4 that were treated with MFx. Patients in the APS group had significantly more months post-meniscectomy (31.8 ± 30.7 months) than those with MSCs (14.2 ± 5.3 months) (P = 0.09). There was a correlation with the time of meniscectomy, and the required size of the implant was significantly larger in more chronic patients than in those with less months of meniscectomy (PIA 5.5 ± 1.4 months, PIM 3.7 ± 1.9 months, P = 0.08).

Lysholm Scale Analysis

In both groups, Lysholm scores increased significantly from preoperative values to 12 months. In the APS group, the increase was 26.5% (66.6 vs. 84.3, P = 0.008) while in the MPS group it was 11.2% (75.0 vs. 83.4, P = 0.01). However, there was no statistically significant difference between groups all time points ( Table 4 ). However, the initial Lysholm scores correlated very strongly with scaffold size (correlation coefficient = −0.494, P = 0.04) and with chondral lesion size (correlation coefficient = −0.595, P = 0.11). Since larger scaffold sizes correspond to males, gender may be a possible confounding variable. It should be noted that the initial Lysholm scores were significantly lower in men (66.8 ± 22.1) compared with women (89.2 ± 14. 2) (P = 0.04). Due to an analysis of variance and covariance adjusting the change in Lysholm scores from preoperative values to 12 months, comparison between groups by gender could only be carried out for males and showed that, at 12 months, Lysholm scores were lower in the PIA group (86. 9 ± 5.4) compared with the PIM group (88.5 ± 5.0), but this difference was not statistically significant (P = 0.82). No significant difference was observed between men and women in the MPS group at 12 months (P = 0.07).

Table 4.

Clinical Evolution Evaluated with Lysholm Comparing Acellular versus MSCs Implant.

Time	T2 Mapping	Mean	P
Preoperative	Acellular	66.6 ± 16.2	0.47
Preoperative	MSCs	75.0 ± 25.4	0.47
3 months	Acellular	64.0 ± 21.4	0.57
3 months	MSCs	69.3 ± 3.3	0.57
6 months	Acellular	83.8 ± 10.9	0.63
6 months	MSCs	86.6 ± 11.5	0.63
9 months	Acellular	87.1 ± 11.6	0.90
9 months	MSCs	87.9 ± 11.8	0.90
12 months	Acellular	84.3 ± 9.7	0.91
12 months	MSCs	83.4 ± 18.4	0.91

In the clinical analysis, Lysholm scale increased significantly from preoperative to 12 months in both groups (P > 0.05). However, there was no significant difference in the evolution between groups at any time.

Implant Failures

Implant failure needing reoperation and postoperative complications were evaluated in the clinical assessment. Any required reoperation or persistence of preoperative symptoms (pain and functional impairment) was likely caused by a mechanical failure of the device or nonintegration of the scaffold with the meniscal remnant. No implant failures were observed at 12 months.

Discussion

Untreated meniscal injuries progressively destabilize the knee and ultimately lead to degenerative osteoarthritic changes. Therefore, meniscus preservation and regeneration remains essential to maintain the functional integrity of the knee joint.^19,20 Partial meniscal replacement appears to have a significant beneficial effect in relieving pain, enhancing knee function, and delaying articular cartilage deterioration.²¹ Interest has been growing in the use of stem cells to replace meniscal tissue.^22,23 These typically involve a stem cell–seeded scaffold to regenerate meniscus-like tissue. Studies have demonstrated meniscal regeneration using seeded scaffolds in rats, rabbits, and pigs.^24-26 Lu et al. demonstrated a substantial improvement in meniscal healing compared with controls, which essentially exhibited no healing.²⁶

In this study, we investigated whether a polyurethane meniscal scaffold alone or in combination with autologous MSCs obtained from peripheral blood would enhance healing, promote meniscus-like tissue ingrowth, and preserve the articular adjacent cartilage as evaluated by T2 mapping on both articular surfaces. In the tibial plateau of the MPS group, values increased slightly at 9 months but returned to the initial values at 12 months (P > 0.05), while in the APS group, a clear decrease from 3 months to 12 months was observed (P > 0.05). This difference tended to be significantly lower in the APS group compared with the MPS group loaded at 12 months (P = 0.18). In the femur, few differences were observed between treatments. A slight increase at 12 months in the MPS group compared with the APS group was noted (47.8 ± 3.4 vs. 45.3 ± 4.9, P > 0.05). However, is important to note that initial T2 mapping in the tibial plateau was always lower than the femur in both groups. In the APS group, the tibial-femoral difference was only significant at 6 months (P = 0.04). It is important to consider the evaluation by gender, as only men were operated in the APS group, whereas in the MPS group significant tibial-femoral differences in T2 mapping were maintained from 3 to 12 months in both genders. In the case of femoral T2 mapping, the comparisons were biased because females had higher values than males.

We found that the meniscal replacement either alone or with addition of MSCs achieve significant and encouraging improvement in knee scores when compared with baseline values (P < 0.05). However, in the evaluation of clinical results at the endpoint, there was no significant difference between groups (P > 0.05). Our prospective study has an obvious source of bias in that 10 of the 17 patients underwent another procedure in addition to the scaffold implantation. Thus, although clinical benefits were recorded, we cannot determine with certainty that they were directly related to the scaffold, ligament repair, cartilage repair, or the addition of MSC to the meniscal scaffold.

The Actifit Study Group reported a multicenter case-series study of 52 patients treated with the Actifit scaffold alone. Significant improvements were recorded for pain and function (IKDC [International Knee Documentation Committee], KOOS [Knee Injury and Osteoarthritis Outcome Score], and Lysholm scores). In addition, the condition of the cartilage (ICRS grade) remained unchanged or improved in 92.5% of patients. We are currently working to record the cartilage condition by second-look arthroscopy and compare it to the condition at the time of scaffold implantation as part of a further analysis.

Our study results are expected as the polyurethane scaffold has already been shown to be beneficial in the short and medium term.²⁷ However, Leroy et al.¹⁵ and Bulgheroni et al.²⁸ reported a decrease in scaffold dimensions over time which leads to concern about the scaffold’s capacity to cover the femoral and tibial articular surfaces. It is hypothesized that this could progressively lead to osteoarthrosis. It is important to note that the results of these studies have been inconclusive, and further long-term studies are required to assess the full clinical potential of these scaffolds.

Our results show that the addition of MSCs to the polyurethane meniscal scaffold shows no additional clinical benefit at 12 months. Nonetheless, we remain optimistic that for a specific patient population, the Actifit scaffold offers a clinical benefit. This conjecture is supported by earlier studies and our preliminary data. In addition, longer term studies are required since no complete clinical, MRI, and T2 mapping assessment were achieved within the current study period of 12 months.

Limitations

The small number of patients, lack of randomization, length of follow-up, and subjectivity of the function of MSC compared with acellular scaffolds because of bleeding during the surgery are the main limitations in this study. The lack of biopsy samples for histologic examination as an outcome measure may be considered as an additional limitation.

Conclusions

Implantation of a polyurethane meniscal scaffold maintains normal T2 mapping values in adjacent cartilage at 12 months. The addition of MSCs did not show any advantage in articular cartilage protection over acellular scaffolds. Clinical improvements are significant from preoperative values to 12 months in both groups, but there is no significant benefit in MSC-loaded scaffolds. Longer term studies are needed to assess the full benefits of MSC and meniscal scaffolds.

Footnotes

Acknowledgments and Funding

This work was supported by the Mexican National Council of Science and Technology (CONACyT, Mexico City, Mexico) Grants SALUD-2011-1-162387, PDCPN-2013-01-215138, and PDCPN-2013-01-216779. We would like to acknowledge and thank Orteq® Sports Medicine (OSM) who donated the Actifit® meniscal scaffolds used in this study. The authors express their gratitude to Ivonne Trigueros-Anaya, Gloria González-Vellano, Leticia Balderas-López, Ana María Godinez-Monroy, Catalina Cano-Alvarado, Carmen González Vellano (Arthroscopy Surgical Nursing team); Mónica Saldaña García, Cecilia Nieto-Gómez, Miguel Antonio Cervera-Bustamante (Blood Bank); and Karina Martinez Rodriguez (Biomedical Engineering) for their assistance in conducting this study.

Declaration of Conflicting Interests

The author(s) declare no conflicts of interest with respect to research, authorship, and/or publication of this article.

Ethics Approval

Ethical approval for this study was obtained from the National Institute of Rehabilitation “Luis Guillermo Ibarra Ibarra,” Mexico City, Mexico approval number 1/11 bis.

Informed Consent

Written informed consent was obtained from all patients for this prospective, longitudinal, comparative, and analytic study.

ORCID iD

Carlos Landa-Solís

References

Majewski

Susanne

Klaus

Epidemiology of athletic knee injuries: a 10-year study. Knee. 2006;13:184-8.

Vrancken

Buma

van Tienen

TG.

Synthetic meniscus replacement: a review. Int Orthop. 2013;37:291-9.

Oosthuizen

Takahashi

Rogan

Snyckers

Vermaak

Jones

, et al. The Knee Osteoarthritis Grading System for arthroplasty. J Arthroplasty. 2019;34:450-55.

Ahmed

Burke

DL.

In-vitro measurement of static pressure distribution in synovial joints—Part I: tibial surface of the knee. J Biomech Eng. 1983;105:216-25.

Ihn

Kim

Park

IH.

In vitro study of contact area and pressure distribution in the human knee after partial and total meniscectomy. Int Orthop. 1993;17:214-8.

Shoemaker

Markolf

KL.

The role of the meniscus in the anterior-posterior stability of the loaded anterior cruciate-deficient knee. Effects of partial versus total excision. J Bone Joint Surg Am. 1986;68:71-9.

Lind

Menhert

Pedersen

AB.

The first results from the Danish ACL Reconstruction Registry: epidemiologic and 2 year follow-up results from 5818 knee ligament reconstructions. Knee Surg Sports Traumatol Arthrosc. 2009;17:117-24.

Binfield

Maffulli

King

JB.

Patterns of meniscal tears associated with anterior cruciate ligament lesions in athletes. Injury. 1993;24:557-61.

Rubman

Noyes

Barber-Westin

SD.

Arthroscopic repair of meniscal tears that extend into the avascular zone. A review of 198 single and complex tears. Am J Sports Med. 1998;26:87-95.

10.

Dhollander

Verdonk

Treatment of painful, irreparable partial meniscal defects with a polyurethane scaffold: midterm clinical outcomes and survival analysis. Am J Sports Med. 2016;44:2615-21.

11.

Baratz

Mengato

Meniscal tears: the effect of meniscectomy and of repair on intraarticular contact areas and stress in the human knee. A preliminary report. Am J Sports Med. 1986;14:270-5.

12.

Roos

Lauren

Adalberth

Roos

Jonsson

Lohmander

LS.

Knee osteoarthritis after meniscectomy: prevalence of radiographic changes after twenty-one years, compared with matched controls. Arthritis Rheum. 1998;41:687-93.

13.

Faunø

Nielsen

AB.

Arthroscopic partial meniscectomy: a long-term follow-up. Arthroscopy. 1992;8:345-9.

14.

Shriram

Kumar

Cui

Lee

YHD

Subburaj

Evaluating the effects of material properties of artificial meniscal implant in the human knee joint using finite element analysis. Sci Rep. 2017;7:6011.

15.

Leroy

Beaufils

Faivre

Steltzlen

Boisrenoult

Pujol

Actifit® polyurethane meniscal scaffold: MRI and functional outcomes after a minimum follow-up of 5 years. Orthop Traumatol Surg Res. 2017;103:609-14.

16.

Faivre

Bouyarmane

Lonjon

Boisrenoult

Pujol

Beaufils

Actifit® scaffold implantation: influence of preoperative meniscal extrusion on morphological and clinical outcomes. Orthop Traumatol Surg Res. 2015;101:703-8.

17.

Gelber

Petrica

Isart

Mari-Molina

Monllau

JC.

The magnetic resonance aspect of a polyurethane meniscal scaffold is worse in advanced cartilage defects without deterioration of clinical outcomes after a minimum two-year follow-up. Knee. 2015;22:389-94.

18.

Schüttler

Haberhauer

Gesslein

Heyse

Figiel

Lorbach

, et al. Midterm follow-up after implantation of a polyurethane meniscal scaffold for segmental medial meniscus loss: maintenance of good clinical and MRI outcome. Knee Surg Sports Traumatol Arthrosc. 2016;24:1478-84.

19.

Arnold

Hirschmann

Verdonk

PC.

See the whole picture: knee preserving therapy needs more than surface repair. Knee Surg Sports Traumatol Arthrosc. 2012;20(2):195-6.

20.

Cook

JL.

The current status of treatment for large meniscal defects. Clin Orthop Relat Res. 2005;(435):88-95.

21.

Rodkey

DeHaven

Montgomery

3rd Baker

Jr Beck

Jr Hormel

, et al. Comparison of the collagen meniscus implant with partial meniscectomy. A prospective randomized trial. J Bone Joint Surg Am. 2008;90:1413-26.

22.

Angele

Johnstone

Kujat

Zellner

Nerlich

Goldberg

, et al. Stem cell based tissue engineering for meniscus repair. J Biomed Mater Res A. 2008;85(2):445-55.

23.

Dutton

Choong

Goh

Lee

Hui

JH.

Enhancement of meniscal repair in the avascular zone using. J Bone Joint Surg Br. 2010;92:169-75.

24.

Izuta

Ochi

Adachi

Deie

Yamasaki

Shinomiya

Meniscal repair using bone marrow-derived mesenchymal stem cells: experimental study using green fluorescent protein transgenic rats. Knee. 2005;12(3):217-23.

25.

Kang

Son

Lee

Park

, et al. Regeneration of whole meniscus using meniscal cells and polymer scaffolds in a rabbit total meniscectomy model. J Biomed Mater Res A. 2006;78(3):659-71.

26.

Cai

Wang

Shi

DH.

Whole meniscus regeneration using polymer scaffolds loaded with fibrochondrocytes. Chin J Traumatol. 2011;14(4):195-204.

27.

Verdonk

Beaufils

Bellemans

Djian

Heinrichs

Huysse

, et al. Successful treatment of painful irreparable partial meniscal defects with a polyurethane scaffold: two-year safety and clinical outcomes. Am J Sports Med. 2012;40:844-53.

28.

Bulgheroni

Regazzola

Mazzola

Polyurethane scaffold for the treatment of partial meniscal tears. Clinical results with a minimum two-year follow-up. Joints. 2013;1:161-6.

First Clinical Application of Polyurethane Meniscal Scaffolds with Mesenchymal Stem Cells and Assessment of Cartilage Quality with T2 Mapping at 12 Months

Abstract

Background

Purpose

Methods

Results

Conclusion

Keywords

Introduction

Methods

Mesenchymal Stem Cell Mobilization

Mobilized Mesenchymal Stem Cells Harvesting

Isolation of Mononuclear Cells from the Buffy Coat

Isolation of CD90+ Cells

Characterization by Flow Cytometry

Culturing of CD90+ Cells

Cell Seeding in Polyurethane Scaffold

Surgical Technique: Arthroscopic Implantation

Rehabilitation Protocol

Imaging Evaluation

Clinical Evaluation

Statistical Analysis

Results

Demographics

Influence of Gender on Scaffold Size and Basal T2 Mapping

Concomitant Lesions

Quality of Adjacent Cartilage Evaluated with T2 Mapping after Meniscal Substitution

Comparison of Intergroup Variables

Lysholm Scale Analysis

Implant Failures

Discussion

Limitations

Conclusions

Footnotes

Acknowledgments and Funding

Declaration of Conflicting Interests

Ethics Approval

Informed Consent

ORCID iD

References