Biomaterials Processing
Establishment of novel meniscal scaffold structures using polyglycolic and poly-L-lactic acids
Journal of Biomaterials Applications 2017, Vol. 32(2) 150–161 ! The Author(s) 2017 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328217713631 journals.sagepub.com/home/jba
Tomohiko Murakami1, Shuhei Otsuki1, Kosuke Nakagawa1, Yoshinori Okamoto1, Tae Inoue2, Yuki Sakamoto2, Hideki Sato2 and Masashi Neo1
Abstract The purpose of this study was to evaluate various types of meniscus scaffolds that mimic the meniscus structure, and to establish a novel cell-free meniscus scaffold with polyglycolic acid or poly-L-lactic acid. Four types of scaffolds were implanted into Japanese white rabbits: poly-L-lactic acid sponge poly-L-lactic acid, PGA-coated PLLA sponge, PGA lamination, and film-coated PGA lamination. Samples were harvested at 8 and 12 weeks after implantation, and a compression stress test was performed. The meniscus size and Ishida scores were evaluated for regenerated tissue. Immunohistochemistry was analyzed by anti-type I, II and X collagen antibodies to investigate the structure of the regenerated tissue, and by anti-iNOS antibody to investigate the inflammatory tissue of the meniscus. The cell nuclei of lymphocytes and foreign body multinucleated giant cells were counted in hematoxylin and eosin staining. Modified Mankin scores for cartilage degeneration were used for assessment after Safranin-O/Fast Green staining. The biomechanical test showed that l- and film-coated PGA lamination exhibited greater strength than s- and PGA-coated PLLA sponge. At 12 weeks, the size of meniscus and the Ishida score in implanted film-coated PGA lamination were improved significantly compared with the defect groups. The type II collagen staining intensity in the PGA lamination lamination is significantly higher than the defect at eight weeks. The staining intensity of iNOS and number of lymphocytes significantly increased in sponge poly-L-lactic acid at eight weeks, and increased in p-PLLA at 12 weeks. Foreign body multinucleated giant cells in implantation groups appeared, especially at eight weeks. The Mankin score for film-coated PGA lamination was significantly lower than for the defect at 12 weeks. Novel meniscal scaffolds especially PGA should possess not only biological but also biomechanical functions. In conclusions, film-coated PGA lamination was the beneficial property for meniscus scaffold from the points of better biomechanical function, good regeneration, and less inflammation with chondroprotective effects. Keywords Meniscectomy, meniscus repair, meniscus scaffold, polyglycolic acid, poly-L-lactic acid
Introduction Meniscus plays a key role in knee homeostasis for force transmission, shock absorption, joint stability, lubrication, and proprioception.1,2 Surgical intervention is considered for injured meniscus with mechanical symptoms and pain. Owing to poor healing, especially at the avascular inner zone of the meniscus, partial meniscectomy is recognized as a well-tolerated treatment option.3 However, several articles revealed that meniscectomy accelerated the degeneration of articular cartilage, knee instability, and ultimately progression to knee osteoarthritis.4–6 Restoration of tissue biomechanical
function after meniscal injury is also a great challenge. Meniscus repair should be considered the first-line option for meniscus therapy; however, this surgery is only suited for a limited number of traumatic meniscus injuries,7 and over 90% are treated with partial meniscectomy.8 The tibiofemoral contact area after total 1 2
Osaka Medical College, Osaka, Japan QOL Research Center Laboratory, Gunze Limited, Kyoto, Japan
Corresponding author: Shuhei Otsuki, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569-8686, Japan. Email:
[email protected]
Murakami et al. meniscectomy was reported to decrease by approximately 50%, leading to an overall 2-3-fold increase in contact force.9 These results suggest that the optimal meniscus scaffold must mimic tissue structure for mechanical behavior. Insertion of a meniscal scaffold is considered another option for meniscus preservation and has been developed during the past 10 years.10 Recently, several preclinical reports of synthetic polymers have described the repair of meniscal defects.11,12 Although some meniscus scaffolds have been used in clinical study, problems are still remained in regard to surgical handling and tissue strength13 and long degradation period.14 Therefore, bioresorbable material with good biomechanical property is desired. We focused on polyglycolic acid (PGA)15 and poly16 L-lactic acid (PLLA), which are currently used in human surgery (e.g. suture thread and bone fixation).17 The purpose of this study was to evaluate various types of meniscus scaffolds that mimic the meniscus structure in terms of a smooth surface and extracellular matrix components, and to establish a novel cell-free meniscus scaffold with PGA or PLLA.
Materials and methods Experimental design We prepared four types of scaffolds to implant into Japanese white rabbits: PLLA sponge (s-PLLA), PGA-coated PLLA sponge (p-PLLA), PGA lamination (l-PGA), and film-coated PGA lamination (f-PGA). Compression stress tests were performed to determine the strength of each scaffolds. Thirty-six knees of Japanese white rabbits were divided with six groups and evaluated with meniscus size, histological regeneration and inflammation, and chondroprotective property at 8 and 12 weeks after implantation.
Fabrication of PLLA and PGA scaffolds Commercial PLLA pellets (Mw of 200,000–280,000) were dissolved (final concentration 5 wt.%) in dioxane for making PLLA sponge. PGA felt, which was soft non-woven fabric in clinical use (‘‘NEOVEIL; GUNZE LIMITED, Kyoto, Japan), and P (LA/caprolactone (CL)) copolymer films (a polyester with a 50:50 molar composition of lactate and e-caprolactone), which were made for melt extruding, were used in the scaffolds material.
Scaffold constructure with combinations of PLLA and PGA Four types of scaffolds were prepared: PLLA sponge (s-PLLA), PGA-coated PLLA sponge (p-PLLA), PGA
151 lamination (l-PGA), and film-coated PGA lamination (f-PGA) (Figure 1(a)). The s-PLLA scaffold was prepared by lyophilizing a PLLA solution in 1,4-dioxane and subsequently curing it at 70 C for 12 h. The p-PLLA scaffold was prepared by covering the upper and lower surface of PLLA scaffold samples with PGA felt. The l-PGA scaffold was configured to alternately laminate a PGA felt layer and P(LA/CL) film layer. The f-PGA scaffold was prepared by hot-pressing with P(LA/CL) and e-caprolactone films on the upper and lower surfaces of l-PGA. All scaffolds were cylindrical, approximately 3.0 mm in diameter and 3.0 mm thick.
Scanning electron microscopy All scaffolds were imaged at 200-fold magnification using a TM-1000 Miniscope (Hitachi, Tokyo, Japan) by each one (Figure 1(a), scale bar ¼ 250 mm). The surfaces of all scaffolds were sputter-coated with gold using a JFC 1500 Quick Auto Coater (JEOL, Tokyo, Japan) before scanning electron microscopic observations at 15 kV.
Mechanical tests Mechanical characterization under 50% unconfined compressive stress was performed with all scaffolds. Analyses were performed on an EZ Graph testing machine (Shimadzu, Tokyo, Japan) with a 5 kN load cell at a strain rate of 1 mm/min (n ¼ 6).18
Animals The Animal Research Committee of Osaka Medical College approved all experiments (No. 26101). In this study, 18 skeletally mature Japanese white rabbits (6 months old, mean weight 3.2 kg; range 3.0–3.4 kg) without degenerative change were obtained from the Oriental Yeast Company (Tokyo, Japan).
Surgical procedures Rabbits were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg; Somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan) and a subcutaneous injection of 1% lidocaine hydrochloride (Xylocaine; AstraZeneca, Osaka, Japan). Surgery was performed under sterile conditions as described previously.19 Briefly, a medial parapatellar incision was made, and the medial meniscus was disclosed. A 2-mm fullthickness cylindrical defect was made at the anterior region of the inner two-thirds of the medial meniscus by using a biopsy punch, and each scaffold was implanted. After surgery, the rabbits were permitted
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Figure 1. (a) Candidates for the novel meniscus scaffold. Four types of scaffold were established: poly-L-lactic acid (PLLA) sponge (s-PLLA), PLLA-coated polyglycolic acid (p-PGA), PGA lamination (l-PGA), and e-caprolactone and PLA film-coated PGA lamination (f-PGA). Electron microscopic pictures are shown (upper: arrow, center: arrow head) (scale bar ¼ 250 mm). (b) Biomechanical test of scaffold materials. The compression force required to cause a 50% decrease in thickness for scaffold samples. Compressive force at the 50% value is significantly higher in the polyglycolic acid (PGA) than the poly-L-lactic acid (PLLA) scaffolds (n ¼ 3, p < 0.01, *p < 0.05).
cage (60 cm 60 cm 40 cm) activity without any immobilization while they were monitored for infections and other complications. Implants with PGA were known to be almost absorbed within 12 weeks.16 Moreover, we evaluated rabbits’ menisci at eight weeks in our previous research19 and Ishida evaluated rabbit’s menisci at 4, 8, and 12 weeks.20 Therefore, rabbit menisci, including scaffolds, were harvested 8 and 12 weeks after implantation.
Morphologic analysis To evaluate inflammation and the size of the meniscus, macrophotographs were obtained using a COOLPIX P310 camera (Nikon, Tokyo, Japan). The surface area of the medial meniscus was plotted against a scale and analyzed by Image J version 1.46 software (National Institutes of Health, Bethesda, MD, USA).19 Inflammation was defined as an increase in
Murakami et al. redness and synovial hyperplasia around the meniscus and scaffold.
Histological analysis After the medial meniscus was resected from the knee, radial sections were made at the anterior region. The meniscus was fixed in 10% neutral buffered formalin. Paraffin-embedded samples were sectioned at 5 mm, deparaffinized in xylene, passed through an ethanol series, stained with Safranin-O/Fast Green, and placed in xylene for clear penetration. Histological assessment of regenerated tissue was performed with an Ishida score that included three components: tissue bonding, existence of fibrochondrocytes, and SafraninO/Fast Green staining score (range 0–6; higher score indicates better regeneration).19,20 Two orthopedic surgeons blinded to the group allocations assessed the surface area and Ishida score of each sample.
Counting of lymphocytes and foreign body multinucleated giant cells Tissue sections were stained with hematoxylin and eosin (H&E) for cell morphological analysis using Harris’ H&E Staining Protocol. Nuclei are stained blue and the cytoplasm red.21 Three different images adjacent to the implant were obtained randomly at 400 magnification from the H&E stained sections. The total numbers of lymphocytes and foreign body multinucleated giant cells (FBMGCs) were counted. FBMGCs were defined as large cells containing more than two nuclei.
Immunohistochemical analysis Immunohistochemistry involved staining by anti-type I collagen antibody (BD Bioscience, Franklin Lakes, NJ, USA), anti-type II collagen antibody (the Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and anti-type X collagen antibodies (Abcam, Cambridge, UK) to investigate the structure of the regenerated tissue. An anti-inducible nitric oxide synthase (iNOS) antibody (Merck Millipore, Darmstadt, Germany) was used to investigate the inflammatory tissue content of the meniscus and scaffold. Sections were washed with phosphate-buffered saline (PBS) and blocked with skim milk at room temperature for 10 min. The primary antibodies were diluted 1:100 as type I collagen and iNOS, 1:50 as type II collagen, and 1:500 as type X collagen. These primary antibodies were incubated overnight at 4 C along with negative controls (without the primary antibody). After washing with PBS, sections were incubated with biotinylated anti-mouse immunoglobulin G (Vector
153 Laboratories, Burlingame, CA, USA) for type II collagen, with biotinylated anti-mouse immunoglobulin M (Vector Laboratories, Burlingame, CA, USA) for type X collagen, and with biotinylated anti-rabbit immunoglobulin G (Vector Laboratories, Burlingame, CA, USA) for type I collagen and iNOS as a secondary antibody for 60 min at room temperature. After washing with PBS, all sections were incubated with a streptavidin-horseradish peroxidase conjugate (Vector Laboratories) for 10 min at room temperature. After again washing with PBS, sections were reacted with the diaminobenzidine (DAB) Chromogen/Substrate Kit (ScyTek Laboratories, Logan, Utah, USA) for 5 min. After rinsing with PBS, the slides were counterstained with hematoxylin and mounted. All immunohistochemical staining was performed at the same time.19 Photomicrographs were obtained with an optical BX53 microscope (Olympus Optical, Tokyo, Japan) connected to a DP22 digital color camera (Olympus). Each slide was evaluated by using multiple digital pictures; one microscopic field was equivalent to one picture. Images were obtained at 2560 1920 pixels (resolution: 1 mm ¼ 590 pixels) under standard lighting conditions.
Quantification of immunohistochemical staining The immunohistochemical staining intensities were determined using Image J digital image analysis software. Photomicrographs were coded and analyzed in a blinded fashion. Briefly, the area to be analyzed was selected, and the image was submitted to ‘‘color deconvolution,’’ an analytical procedure written as a ‘‘plugin’’ for Image J software, using the hematoxylin and DAB built-in vector.22 Color deconvolution generated three panels: cell staining with hematoxylin, or DAB staining only, and background. The final DAB intensity varied from 0 (white, negative expression) to 255 (dark brown, highest expression).23
Evaluation of femoral cartilage For assessments of chondroprotective effects of the scaffolds, the medial femoral condyle was stained with Safranin-O/Fast Green, and the degree of cartilage degradation was scored using the modified Mankin system.24,25 Briefly, the distal femur was fixed with 4% phosphate-buffered paraformaldehyde for 48 h, decalcified with 10% ethylene diamine tetraacetic acid for 10 days, and then embedded in paraffin. Paraffin sections at 4 mm thickness of the medial femoral condyle were stained with Safranin-O/Fast Green, as well as meniscus. The modified Mankin score identifies a range of changes from 0 (normal fibrocartilage) to 14 (severe degeneration). The structure category was used
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to evaluate changes in the surface irregularities and depth of the clefts. The cell category was used to evaluate changes in cellularity. The Safranin-O staining category was used to evaluate the production of proteoglycan (Figure 7(e)).
separated by a minimum of two weeks. Reliabilities for measurements of interval data were assessed with the intraclass correlation coefficient (ICC).
Results Mechanical tests
Statistical analyses All data analyses were performed with JMP Pro version 11.2.0 (SAS 196 Institute Japan, Tokyo, Japan). The results are reported as means standard deviation. Dunn’s multiple comparisons method was employed to analyze differences between groups. Differences with p values of less than 0.05 were considered significant. Intra- and inter-observer reliabilities for the Ishida scores, modified Mankin scores, and nuclei counts were assessed by two independent blinded observers (TM, SO) for all samples. For intra-observer reliabilities, scoring and counting were performed at two occasions
The biomechanical test showed that the compression force causing a 50% decrease in thickness exceeded 40 MPa for both l-PGA (42.09 18.10 MPa) and f-PGA (53.44 9.05 MPa). This was greater strength than that of either s-PLLA (2.09 0.20 MPa, p < 0.01) or p-PLLA (2.32 0.13 MPa, p < 0.01) (Figure 1(b)).
Morphological analysis Macroscopic inflammation was evident in s-PLLA and p-PLLA, especially synovial hyperplasia, which was located at the outer meniscus and scaffold.
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Figure 2. Macrophotographs to evaluate inflammation and size of the meniscus. (a) Dotted circling lines shown meniscus and asterisk shows the implanted scaffold. Macroscopic inflammation (arrows) is evident in the poly-L-lactic acid (PLLA) and polyglycolic acid (PGA)-coated PLLA groups. Note especially the synovial hyperplasia that is located at the outer meniscus and scaffold (scale bar ¼ 5 mm). (b) The surface area in film-coated laminate PGA (f-PGA) scaffolds is increased significantly compared with the control defect at 12 weeks (n ¼ 3, p ¼ 0.03, *p < 0.05).
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Figure 3. Histological analysis of Safranin-O/Fast Green staining. (a,b) At eight weeks, poly-L-lactic acid (PLLA) scaffolds are maintained. (c,d) At 12 weeks, scaffolds containing polyglycolic acid (PGA) are replaced with original tissue (scale bar ¼ (a,c) 500 mm, (b,d) 50 mm). (e) The Ishida score shows that film-coated laminate PGA (f-PGA) is significantly improved compared to the control meniscus defect at 12 weeks (n ¼ 3, p < 0.01, *p < 0.05).
Macroscopic inflammation was not detected in l-PGA and f-PGA at 8 and 12 weeks (Figure 2(a)). Moreover, the surface size in f-PGA was significantly increased compared with defect (p ¼ 0.03) (Figure 2(b)).
Histological analysis Safranin-O/Fast Green staining showed that s-PLLA scaffolds were left, especially at eight weeks (Figure 3(a) and (b)), whereas scaffolds containing l-PGA were replaced with original tissue at 12 weeks (Figure 3(c) and (d)). The Ishida score showed that f-PGA scaffolds significantly improved the meniscus defect at 12 weeks (p < 0.01) (Figure 3(e)). The ICCs of intra- and inter-observer reliabilities of all measurement were excellent (ICC > 0.84; range, 0.84–0.96). At eight weeks, significantly more lymphocytes were found in s-PLLA than in the defect (p ¼ 0.02)
(Figure 4(a), (b), (e)). At 12 weeks, significantly more lymphocytes were found in p-PLLA than in f-PGA (p ¼ 0.03) (Figure 4(c) to (e)). FBMGCs infiltrated the encapsulated tissues along the scaffolds. The numbers of FBMGCs adjacent to the scaffolds tended to increase in the s-/p-PLLA groups compared to the l-/ f-PGA groups, but there were no significant differences (Figure 4(a) to (d, (f)). The ICCs of intra- and interobserver reliabilities of all measurement were excellent (ICC > 0.89; range, 0.89–0.97). There are no significant differences about the type I (Figure 5(a), (b) and (e)) and type X collagen (Figure 6(a), (b), (e)) among groups. The type II collagen staining intensity in the l-PGA lamination is significantly higher than the defect at eight weeks (p ¼ 0.03) (Figure 5(c), (d), (f)). The iNOS staining intensity in the s-PLLA group is significantly higher than the defect at eight weeks (p¼ 0.03) and the
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Figure 4. Hematoxylin and eosin staining at 8 and 12 weeks. Lymphocytes (arrow heads) and foreign body multinucleated giant cells (FBMGCs) (arrows) are evident around scaffolds at (a, b) 8 and (c, d) 12 weeks (scale bar ¼ (a, c) 500 mm, (b, d) 50 mm). (e) There are significantly more lymphocytes in the poly-L-lactic acid sponge (s-PLLA) than the defect group at eight weeks (n ¼ 3, p ¼ 0.02, *p < 0.05), and in the polyglycolic acid (PGA)-coated PLLA (p-PLLA) than film-coated PGA lamination (f-PGA) group at 12 weeks (n ¼ 3, p ¼ 0.03, *p < 0.05). (f) The numbers of FBMGCs adjacent to the scaffolds tended to increase in the s-/p-PLLA groups compared to the l-/f-PGA groups, but there are no significant differences, (n ¼ 3, p > 0.05).
p-PLLA group is higher than sham at 12 weeks (p ¼ 0.03) (Figure 6(c), (d), (f)).
Analysis of femoral cartilage The Mankin score for the defect was significantly higher than for sham at both 8 and 12 weeks (p ¼ 0.02) (Figure 7(a) to (d), (f)). The score for fPGA was significantly lower than for the defect at 12 weeks (p ¼ 0.04) (Figure 7(c), (d), (f)). The ICCs of intra- and inter-observer reliabilities of all measurement were excellent for all parameters (ICC > 0.82; range 0.82–0.96), which indicated good reproducibility in the measurements.
Discussion A cell-free scaffold has the advantage of one-step surgery without donor site morbidity, in contrast to cell-
based surgical treatment. The results of the current study suggest that f-PGA showed not only biological regeneration without inflammation but also a good biomechanical property for meniscus scaffolds. There are currently two types of cell-free scaffolds available for the meniscus: one is based on animal type I collagen (CMI; Ivy Sports Medicine, Grafeling, Germany) and the other is polyurethane (Actifit; Orteq, London, UK). Both are reported to yield good clinical outcomes.26,27 CMI scaffolds appear to be safe with successful long-term outcomes,10 although some issues remain with regard to surgical handling and tissue strength for suturing during implantation.13 Actifit might provide improved handling in arthroscopic treatment with good clinical outcomes,26 whereas the degradation period has still to be investigated.14 We had reported the synergistic effect of a type I collagen scaffold (PELNAC) and infrapatellar fat pad implantation for meniscus repair.19 Although the
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Figure 5. Immunohistochemistry of type I, II collagen. (a, b, e) There are no significant differences in the type I collagen staining intensity. (c, d, f) The type II collagen staining intensity in the film-coated PGA lamination (f-PGA) is significantly higher than the defect at 12 weeks. (n ¼ 3, p ¼ 0.03, *p < 0.05) ((a to d) scale bar ¼ 500 mm).
meniscus defect exhibited both macrographical and histological healing in the previous research, this scaffold was difficult to handle with water. PGA is an absorbable reinforcement that is used for humans and is applied to regions with short healing times. In the past decade, biodegradable materials such as PLLA and PGA have been preferred, especially in intraarticular procedures.28 In addition, poly-(lactic-co-glycolic acid) (PLGA),29 polyurethane,30 polyester carbon,31 and polycaprolactone32 have been used. Advantages of a PGA scaffold include effective delivery and anchoring of cells because of its highly porous structure. Novel meniscal scaffolds should possess not only biological but also biomechanical functions.33 We established here that PGA lamination for high strength,
and a P(LA/CL) and e-caprolactone film coating for better biomechanical intensity, yields an improved product. The compressive elastic modulus for human meniscus is between 0.09 and 0.16 MPa,34 and the circumferential tensile elastic modulus for human meniscus is between 60 and 160 MPa.35,36 Our biomechanical test showed that PGA scaffolds with/without film had greater strength. Moreover, histology of the femoral condyle showed that the f-PGA scaffold provided better chondroprotection than the other scaffolds, indicating that f-PGA induced better mechanical condition than any other scaffolds. Meniscal cells are known to synthesize and maintain extracellular matrix (ECM) similar to chondrocytes.37
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Figure 6. Immunohistochemistry of type X collagen, and inducible nitric oxide synthase (iNOS). (a, b, e) There are no significant differences in the type X collagen staining intensity. (c, d, f) The iNOS staining intensity in the poly-L-lactic acid sponge (s-PLLA) group is significantly higher than the control defect at eight weeks (p ¼ 0.03) and polyglycolic acid-coated PLLA scaffold (p-PLLA) group is higher than sham at 12 weeks (n ¼ 3, p ¼ 0.03, *p < 0.05) ((a to d) scale bar ¼ 500 mm).
Meniscal ECM contains mainly water (72%), collagens (22%), and glycosaminoglycans (0.8%).38 Type I collagen accounts for over 90% of the total collagen content. Type II, III, and X collagen are the remaining meniscal collagens.39 Previous report has shown the presence of types I, II, and X collagens in menisci of rabbits.40 Therefore, the mechanisms of meniscal restoration are considered that meniscal cell after penetration into scaffold synthesize ECM including collagen. In the current study, neo tissues after implantation of PLLA or PGA scaffolds was composed of the type I and type X collagen as well as sham group. Moreover, the l-PGA scaffold increased type II collagen at eight weeks. In addition to these results, the Ishida score to
evaluate meniscus cell and glycosaminoglycans showed that f-PGA scaffolds significantly improved the meniscus defect at 12 weeks. These results suggested that PGA scaffold might induce higher ECM including collagen in meniscus for meniscal regeneration. The regeneration of tissue-engineered meniscus in an immunocompetent environment, because of homeostatic and defense mechanisms, usually fails owing to severe inflammation induced by the scaffold and its degradation products.41 The problems with resorbable scaffolds compared to non-resorbable materials involve inflammation.42 Thus, construction of a resorbable polymer scaffold remains a challenge. In the current study, inflammatory-related cells, such as macrophages,
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Figure 7. Histological analysis of Safranin-O/Fast Green staining for femoral cartilage. The defect model showed severe cartilage degeneration at (a, b) 8 and (c, d) 12 weeks, whereas cartilage implanted with scaffolds, especially PGA, is maintained (scale bar ¼ (a, c) 200 mm, (b, d) 50 mm). (e) The modified Mankin scoring system. (f) The Mankin score for the defect was significantly higher than for sham at 8 and 12 weeks (p ¼ 0.02). f-PGA was significantly lower than for the defect at 12 weeks (p ¼ 0.04), suggesting that f-PGA provides better chondroprotection than the other scaffolds (n ¼ 3, *p < 0.05).
lymphocytes, and giant cells, appeared as the immune reaction that had progressed after implanting tissueengineered meniscus.43 Especially, PLLA scaffolds showed engulfment of the scaffold with lymphocytes and FBMGCs at eight weeks. In contrast, no obvious inflammatory reaction could be seen in f-PGA at 12 weeks. Similar to the synovium or articular cartilage,44 meniscus can produce nitric oxide that can be significantly enhanced by cytokines45 or mechanical stress.46 In particular, iNOS is expressed after exposure to inflammatory stimuli.47 Nitric oxide production is accompanied by a decrease in collagen and proteoglycan synthesis, suggesting that lower iNOS levels in meniscus might be important for meniscal regeneration.
The main limitation of this study was that the meniscus defect differed from clinical meniscus injury. Nevertheless, this model is recognized in meniscal research in rabbits. In future studies, arthroscopic treatment with this scaffold should be performed with a large animal model.
Conclusions The present study has demonstrated that the f-PGA was the beneficial property for meniscus scaffold from the points of better biomechanical function, good regeneration, and less inflammation with chondroprotective effects. First, f-PGA was greater strength than PLLA scaffolds. Second, meniscus size and histological score in implanted f-PGA were improved significantly
160 compared with the defect groups, while the staining intensity of iNOS and number of lymphocytes significantly increased in PLLA scaffolds. Last, Mankin score for f-PGA was significantly lower than for the defect at 12 weeks. Therefore, we propose that f-PGA scaffold might be a new method of application for meniscal repair. Acknowledgement We thank Yoshie Seki for her expert technical assistance.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Japan Society for the Promotion of Science, KAKENHI 15K10498 (Kiban C to S.O).
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