Takuya Tokunaga

FGF-2 Stimulates the Growth of Tenogenic Progenitor Cells to Facilitate the Generation of Tenomodulin-Positive Tenocytes in a Rat Rotator Cuff Healing Model

Background: Fibroblast growth factor (FGF)–2 has the potential to enhance tendon-to-bone healing after rotator cuff (RC) injury. Hypothesis: FGF-2 stimulates tenogenic differentiation of progenitors to improve the biomechanical strength and histological appearance of repaired RCs in rats. Study Design: Controlled laboratory study. Methods: Adult male Sprague-Dawley rats (N = 156) underwent unilateral surgery to repair the supraspinatus tendon to insertion sites. The FGF-2-treated group (gelatin hydrogel containing 5 μg of FGF-2) and a control group (gelatin hydrogel only) were compared to investigate the effects of FGF-2 at 2, 4, 6, 8, and 12 weeks postoperatively. Biomechanical testing was performed at 6 and 12 weeks. Semiquantitative histological analysis and immunohistochemical analysis for the proliferating cell nuclear antigen (PCNA) were performed, and the expression of tendon-related markers, including Scleraxis (Scx) and Tenomodulin (Tnmd), was monitored by real-time reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. SRY-box containing gene 9 (Sox9) expression was monitored by RT-PCR and immunohistochemical analysis. At 2 and 4 weeks, immunohistochemical analysis for mesenchymal stem cell (MSC) markers was also performed. Results: The FGF-2-treated group demonstrated a significant improvement in mechanical strength at 6 and 12 weeks and significantly higher histological scores than the control group at ≥4 weeks. The average incidence of PCNA-positive cells was significantly higher at 2 and 4 weeks, and more cells expressing MSC markers were detected at the insertion site in the FGF-2-treated group. The expression level of Scx increased significantly in the FGF-2-treated group from 4 to 8 weeks, while the Tnmd level increased significantly from 4 to 12 weeks postoperatively. The localization of Tnmd overlapped with the locations of reparative tissues accompanying collagen fibers with an aligned orientation. Sox9 expression was significantly upregulated at 4 weeks in the FGF-2-treated group. Conclusion: FGF-2 promotes growth of the tenogenic progenitor cells, which participate in tendon-to-bone healing, resulting in biomechanical and histological improvement of the repaired RC. Clinical Relevance: These findings provide clues regarding the clinical development of regenerative repair strategies for RC injury.

Effect of Postoperative Passive Motion on Rotator Cuff Reconstruction With Acellular Dermal Matrix Grafts in a Rat Model

Background: Although postoperative rehabilitation is critical for rotator cuff tendon-to-bone healing and shoulder function recovery, no standardized protocol has been established. Hypothesis: Postoperative immediate passive motion is detrimental to tendon-to-bone remodeling and tendon maturation after rotator cuff acellular dermal matrix (ADM) grafting, although postoperative delayed passive motion does no harm. Study Design: Controlled laboratory study. Methods: Male Sprague-Dawley rats underwent rotator cuff reconstruction with ADM grafts. Their shoulders were immobilized for 2 weeks thereafter. The rats were assigned to 3 different rehabilitation protocols: (1) immobilization without passive motion (nonpassive motion [N-PM], controls), (2) immobilization with immediate passive motion (I-PM), and (3) immobilization with delayed passive motion (D-PM). Specimens obtained 2, 6, and 12 weeks postoperatively were analyzed histologically, and semiquantitative histomorphological measurements of collagen organization, vascularity, and cellularity were obtained; the area of interest was divided into 2 zones, the midsubstance of the graft and the graft-bone interface. Another set of samples taken at 12 weeks was subjected to biomechanical analysis. Results: At 2 weeks, there was no significant difference among the groups in terms of semiquantitative histomorphological measurements of collagen organization, vascularity, and cellularity. At 6 weeks, collagen organization at the insertion site was significantly poorer in I-PM than in N-PM and D-PM rats (P = .0095). At 12 weeks, collagen organization at the insertion site and midsubstance of ADM grafts was also significantly poorer in I-PM rats (P = .0125 and P = .0018, respectively), and ultimate load-to-failure was lower in this group (P = .0043). Conclusion: While postoperative immediate passive motion was detrimental to remodeled tendon-to-bone healing and to the tendon maturation of ADM grafts placed in the rotator cuff tendon defects, delayed passive motion did no harm. Clinical Relevance: For patients with 6-week immobilization after rotator cuff reconstruction, we recommend that early passive motion be started no sooner than 3 weeks after surgery. Immediate early passive motion should be avoided.

TGF-β1 Improves Biomechanical Strength by Extracellular Matrix Accumulation Without Increasing the Number of Tenogenic Lineage Cells in a Rat Rotator Cuff Repair Model

Background: Transforming growth factor β1 (TGF-β1) positively regulates the tenogenic marker genes scleraxis (Scx) and tenomodulin (Tnmd) in mesenchymal progenitors in vitro. However, little is known about the effect of TGF-β1 on the expression of tenogenic markers during rotator cuff (RC) healing in rats. Hypothesis: TGF-β1 improves the biomechanical properties and histological maturity of reparative tissue in a rat RC repair model by stimulating the growth of tenogenic cells. Study Design: Controlled laboratory study. Methods: Adult male Sprague-Dawley rats (N = 180) underwent unilateral supraspinatus tendon-to-bone surgical repair and were randomly treated with a gelatin hydrogel presoaked in TGF-β1 (100 ng) or phosphate-buffered saline. The effects of TGF-β1 on RC healing were investigated at 2, 4, 6, 8, and 12 weeks postoperatively by immunostaining for proliferating cell nuclear antigen, by real-time reverse transcription polymerase chain reaction and in situ hybridization or immunostaining for enthesis-related markers (SRY-box containing gene 9 [Sox9], Scx, and Tnmd), and by real-time reverse transcription polymerase chain reaction and immunostaining for type I and III collagen. At 6 and 12 weeks postoperatively, biomechanical testing, micro–computed tomography, and biochemical analysis were also performed. At 2 and 4 weeks postoperatively, mesenchymal stem cell–related markers, phospho-Smad2, and matrix metalloproteinase 9 (MMP-9) and MMP-13 were assessed by immunostaining. Results: The TGF-β1-treated group had significantly higher ultimate load to failure and tissue volume at 6 and 12 weeks postoperatively and a higher collagen content at 12 weeks compared with the saline group. Tendon-related gene expression, histological maturity, cell proliferation, and mesenchymal stem cell–related marker immunoreactivity were not affected by exogenously administrated TGF-β1 at all time points. In the TGF-β1-treated group, the percentage of phospho-Smad2-positive cells within the healing tissue increased, whereas the expression of MMP-9 and MMP-13 significantly decreased at 2 and 4 weeks postoperatively. Conclusion: TGF-β1 enhances formation of tough fibrous tissues at the healing site by inhibiting MMP-9 and MMP-13 expression to increase collagen accumulation but without the growth of tenogenic lineage cells. Clinical Relevance: These findings suggest that TGF-β1 could be used for enhancing biomechanical strength after RC surgical repair.

Enhancement of rotator cuff tendon–bone healing with fibroblast growth factor 2 impregnated in gelatin hydrogel sheets in a rabbit model

BACKGROUND Application of fibroblast growth factor 2 (FGF-2) may improve the healing response after rotator cuff (RC) surgical repair. This study aimed to determine whether FGF-2-impregnated gelatin hydrogel sheet (GHS) incorporation into the bony trough on the greater tuberosity facilitates healing after RC surgical repair in rabbits. METHODS We assigned 120 adult male Japanese white rabbits treated with unilateral surgery for supraspinatus tendon repair into the following groups: suture-only group (suture); suture and GHS with phosphate-buffered saline (carrier); suture and GHS with 3 µg of FGF-2 (F3); and suture and GHS with 30 µg of FGF-2 (F30). The effect of FGF-2 was assessed using histologic, biomechanical, and microcomputed tomography evaluations at 2, 6, and 12 weeks. RESULTS At 12 weeks, loose fibrovascular tissues emerged at the repair site in the suture and carrier groups and dense tendon-like tissues in the F3 and F30 groups, which demonstrated significantly higher ultimate load-to-failure and stress-to-failure at 12 weeks than that in the suture and carrier groups. Microcomputed tomography imaging showed ectopic calcification formation in some specimens from each group. Appearances or frequencies were similar among groups. The histologic and biomechanical effects of FGF-2 on RC healing were obvious at ≥6 weeks postoperatively. CONCLUSION FGF-2-impregnated GHS incorporation into the bony trough on the greater tuberosity before RC surgical repair is feasible and results in histologic and biomechanical improvements during RC healing in rabbits. No detrimental effect on ectopic calcification was observed.

Matrix metalloproteinase promotes elastic fiber degradation in ligamentum flavum degeneration

Ligamentum flavum (LF) hypertrophy in lumbar spinal canal stenosis (LSCS) is characterized by a loss of elastic fibers and fibrosis. Chronic inflammation is thought to be responsible for the histological change but the mechanism underlying elastic fiber degradation remains unclear. Given that matrix metalloproteinase (MMP)-2 and -9 have elastolytic activity and are partly regulated by inflammatory cytokines such as interleukin (IL)-6, in this study, we investigated whether MMPs mediate LF degeneration using 52 LF samples obtained during lumbar surgery, including 31 LSCS and 21 control specimens. We confirmed by histological analysis that the LSCS samples exhibited severe degenerative changes compared with the controls. We found that MMP-2 was upregulated in LF tissue from patients with LSCS at the mRNA and protein levels, whereas MMP-9 expression did not differ between the two groups. The MMP-2 level was positively correlated with LF thickness and negatively correlated with the area occupied by elastic fibers. IL-6 mRNA expression was also increased in LF tissue from patients with LSCS and positively correlated with that of MMP-2. Signal transducer and activator of transcription (STAT)3, a component of the IL-6 signaling pathway, was activated in hypertrophied LF tissues. Our in vitro experiments using fibroblasts from LF tissue revealed that IL-6 increased MMP-2 expression, secretion, and activation via induction of STAT3 signaling, and this effect was reversed by STAT3 inhibitor treatment. Moreover, elastin degradation was promoted by IL-6 stimulation in LF fibroblast culture medium. These results indicate that MMP-2 induction by IL-6/STAT3 signaling in LF fibroblasts can degrade elastic fibers, leading to LF degeneration in LSCS.