Objective To investigate the effect of Kartogenin (KGN) combined with adipose-derived stem cells (ADSCs) on tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in rabbits. Methods After the primary ADSCs were cultured by passaging, the 3rd generation cells were cultured with 10 μmol/L KGN solution for 72 hours. The supernatant of KGN-ADSCs was harvested and mixed with fibrin glue at a ratio of 1∶1; the 3rd generation ADSCs were mixed with fibrin glue as a control. Eighty adult New Zealand white rabbits were taken and randomly divided into 4 groups: saline group (group A), ADSCs group (group B), KGN-ADSCs group (group C), and sham-operated group (group D). After the ACL reconstruction model was prepared in groups A-C, the saline, the mixture of ADSCs and fibrin glue, and the mixture of supernatant of KGN-ADSCs and fibrin glue were injected into the tendon-bone interface and tendon gap, respectively. ACL was only exposed without other treatment in group D. The general conditions of the animals were observed after operation. At 6 and 12 weeks, the tendon-bone interface tissues and ACL specimens were taken and the tendon-bone healing was observed by HE staining, c-Jun N-terminal kinase (JNK) immunohistochemical staining, and TUNEL apoptosis assay. The fibroblasts were counted, and the positive expression rate of JNK protein and apoptosis index (AI) were measured. At the same time point, the tensile strength test was performed to measure the maximum load and the maximum tensile distance to observe the biomechanical properties. Results Twenty-eight rabbits were excluded from the study due to incision infection or death, and finally 12, 12, 12, and 16 rabbits in groups A-D were included in the study, respectively. After operation, the tendon-bone interface of groups A and B healed poorly, while group C healed well. At 6 and 12 weeks, the number of fibroblasts and positive expression rate of JNK protein in group C were significantly higher than those of groups A, B, and D (P<0.05). Compared with 6 weeks, the number of fibroblasts gradually decreased and the positive expression rate of JNK protein and AI decreased in group C at 12 weeks after operation, with significant differences (P<0.05). Biomechanical tests showed that the maximum loads at 6 and 12 weeks after operation in group C were higher than in groups A and B, but lower than those in group D, while the maximum tensile distance results were opposite, but the differences between groups were significant (P<0.05). Conclusion After ACL reconstruction, local injection of a mixture of KGN-ADSCs and fibrin glue can promote the tendon-bone healing and enhance the mechanical strength and tensile resistance of the tendon-bone interface.
ObjectiveTo observe the effect and significance of autologous fibrin clot on tendon-bone healing after anterior cruciate ligament (ACL) reconstruction.MethodsBetween October 2014 and January 2016, 34 patients (34 knees) with ACL injury were enrolled in the study. During ACL reconstruction, autologous fibrin clot was used in 17 cases (trial group) and was not used in 17 cases (control group). The anterior drawer test, Lachman test, and axial displa-cement test were positive in 2 groups before operation. There was no significant difference in gender, age, causes of injury, injury side, disease cause, and preoperative knee joint activity, Lysholm score, and American Hospital for Special Surgery (HSS) score between 2 groups (P>0.05), with comparable. The results of anterior drawer test, Lachman test, and axial displacement test were recorded and compared between 2 groups after operation. The knee joint activity, Lysholm score, and HSS score were used to evaluate the knee function recovery at 6, 24, and 48 weeks after operation; the graft signal intensity, graft signal to noise ratio, bone tunnel expansion, and graft tendon-bone node T2 value were measured.ResultsAll patients were followed up 48 weeks. Surgical incision healed at stage I. No joint infection and joint adhesion occurred. The drawer test, Lachman test, and axial shift test were negative in 2 groups. At 6, 24, and 48 weeks after operation, the Lysholm score of trial group was significantly higher than that of control group (P<0.05); there was no significant difference in knee joint activity between 2 groups (P>0.05). The HSS score of trial group was significantly higher than that of control group at 24 and 48 weeks (P<0.05), but no significant difference was found at 6 weeks (P>0.05). MRI measu-rement showed that there was significant difference in graft signal intensity, bone tunnel expansion, and graft signal to noise ratio between 2 groups at 6, 24, and 48 weeks after operation (P<0.05). There was no significant difference in graft tendon-bone node T2 value between 2 groups (P>0.05) at 48 weeks after operation, but difference was significant at 6 and 24 weeks (P<0.05).ConclusionAutologous fibrin clot can effectively enhance graft revascularization, and accelerate the process of tendon-bone healing after ACL reconstruction.
Objective To investigate the effect of autologous osteochondral tissue and periosteum transplantation on tendon-bone healing of rotator cuff in rabbits. Methods Twenty-four male New Zealand white rabbits were randomly divided into autologous osteochondral tissue and periosteum transplantation group (experimental group, n=12) and simple suture group (control group, n=12). Both groups were subjected to acute supraspinatus tendon injury and repaired with corresponding techniques. At 4, 8, and 12 weeks after operation, 4 specimens from each group were taken from the right shoulder joint for histological examination (HE staining, Masson staining, and Safranin O-fast green staining), and the left shoulder was subjected to biomechanical tests (maximum tensile load and stiffness). Results Both groups of animals survived until the completion of the experiment after operation. At 4 weeks after operation, both groups showed less collagen fibers and disorder at the tendon-bone junction. At 8 weeks, both groups showed reduced inflammation at the tendon-bone junction, with more organized and denser collagen fibers and chondrocytes. The experimental group showed better results than the control group. At 12 weeks, the experimental group showed typical tendon-bone transition structure, with increased generation of collagen fibers and chondrocytes, and the larger cartilage staining area. Both groups showed an increase in maximum tensile load and stiffness over time (P<0.05). The stiffness at 4 weeks and the maximum tensile load at 4, 8, and 12 weeks in the experimental group were superior to control group, and the differences were significant (P<0.05). There was no significant difference in stiffness at 8, 12 weeks between the two groups (P>0.05). Conclusion Autologous osteochondral tissue and periosteum transplantation can effectively promote the fiber and cartilage regeneration at the tendon-bone junction of rotator cuff and improve the biomechanical effect of shoulder joint in rabbits.
ObjectiveTo explore the effect of silk fibroin/poly(L-lactic acid-co-e-caprolactone) [SF/P(LLA-CL)] nanofibrous scaffold on tendon-bone healing of rabbits.MethodsSF/P(LLA-CL) nanofibrous scaffold was fabricated by electrospinning methods. The morphology of the scaffold was observed by scanning electron microscope (SEM). Pre-osteoblasts MC3T3-E1 cells were seeded on the scaffold and cultured for 1, 3, and 5 days. Cell adhesion and proliferation were also observed by SEM. Meanwhile, twenty-four New Zealand white rabbits were randomly divided into the autogenous tendon group (control group) and the autogenous tendon wrapped with SF/P(LLA-CL) scaffold group (experimental group), with twelve rabbits in each group. An extra-articular model was established, the effect was evaluated by histological examination and mechanical testing.ResultsThe morphology of SF/P(LLA-CL) nanofibrous scaffold was random, with a diameter of (219.4±66.5) nm. SEM showed that the MC3T3-E1 cells seeded on the scaffold were in the normal shape, growing well, and proliferating with time course. The results of histological examination showed that inflammatory cells infltrated into the graft-host bone interface at 6 weeks after operation in both groups. Besides, the width of interface showed no significant difference between groups. At 12 weeks after operation, protruding new bone tissue could be observed at the interface in the experimental group, while scar tissue but no new bone tissue could be seen at the interface in the control group. Mechanical testing showed that there was no significant difference in the failure load and the stiffness between groups at 6 weeks after operation (P>0.05). The failure load and the stiffness in the experimental group were significantly higher than those in the control group at 12 weeks after operation (P<0.05).ConclusionThe SF/P(LLA-CL) nanofibrous scaffold has good cell biocompatibility and can effectively promote tendon-bone healing, thus providing new method for modifying graft for ACL reconstruction in the clinical practice.
ObjectiveTo review the bioactive strategies that enhance tendon graft healing after anterior cruciate ligament reconstruction (ACLR), and to provide insights for improving the therapeutic outcomes of ACLR. Methods The domestic and foreign literature related to the bioactive strategies for promoting the healing of tendon grafts after ACLR was extensively reviewed and summarized. ResultsAt present, there are several kinds of bioactive materials related to tendon graft healing after ACLR: growth factors, cells, biodegradable implants/tissue derivatives. By constructing a complex interface simulating the matrix, environment, and regulatory factors required for the growth of native anterior cruciate ligament (ACL), the growth of transplanted tendons is regulated at different levels, thus promoting the healing of tendon grafts. Although the effectiveness of ACLR has been significantly improved in most studies, most of them are still limited to the early stage of animal experiments, and there is still a long way to go from the real clinical promotion. In addition, limited by the current preparation technology, the bionics of the interface still stays at the micron and millimeter level, and tends to be morphological bionics, and the research on the signal mechanism pathway is still insufficient.ConclusionWith the further study of ACL anatomy, development, and the improvement of preparation technology, the research of bioactive strategies to promote the healing of tendon grafts after ACLR is expected to be further promoted.
Objective To evaluate the effect of biodegradable magnesium alloy materials in promoting tendon-bone healing during rotator cuff tear repair and to investigate their potential underlying biological mechanisms.Methods Forty-eight 8-week-old Sprague Dawley rats were taken and randomly divided into groups A, B, and C. Rotator cuff tear models were created and repaired using magnesium alloy sutures in group A and Vicryl Plus 4-0 absorbable sutures in group B, while only subcutaneous incisions and sutures were performed in group C. Organ samples of groups A and B were taken for HE staining at 1 and 2 weeks after operation to evaluate the safety of magnesium alloy, and specimens from the supraspinatus tendon and proximal humerus were harvested at 2, 4, 8, and 12 weeks after operation. The specimens were observed macroscopically at 4 and 12 weeks after operation. Biomechanical tests were performed at 4, 8, and 12 weeks to test the ultimate load and stiffness of the healing sites in groups A and B. At 2, 4, and 12 weeks, the specimens were subjected to the following tests: Micro-CT to evaluate the formation of bone tunnels in groups A and B, HE staining and Masson staining to observe the regeneration of fibrocartilage at the tendon-bone interface after decalcification and sectioning, and Goldner trichrome staining to evaluate the calcification. Immunohistochemical staining was performed to detect the expressions of angiogenic factors, including vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2), as well as osteogenic factors at the tendon-bone interface. Additionally, immunofluorescence staining was used to examine the expressions of Arginase 1 and Integrin beta-2 to assess M1 and M2 macrophage polarization at the tendon-bone interface. The role of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in tendon-bone healing was further analyzed using real-time fluorescence quantitative PCR. Results Analysis of visceral sections revealed that magnesium ions released during the degradation of magnesium alloys did not cause significant toxic effects on organs such as the heart, liver, spleen, lungs, and kidneys, indicating good biosafety. Histological analysis further demonstrated that fibrocartilage regeneration at the tendon-bone interface in group A occurred earlier, and the amount of fibrocartilage was significantly greater compared to group B, suggesting a positive effect of magnesium alloy material on tendon-bone interface repair. Additionally, Micro-CT analysis results revealed that bone tunnel formation occurred more rapidly in group A compared to group B, further supporting the beneficial effect of magnesium alloy on bone healing. Biomechanical testing showed that the ultimate load in group A was consistently higher than in group B, and the stiffness of group A was also greater than that of group B at 4 weeks, indicating stronger tissue-carrying capacity following tendon-bone interface repair and highlighting the potential of magnesium alloy in enhancing tendon-bone healing. Immunohistochemical staining results indicated that the expressions of VEGF and BMP-2 were significantly upregulated during the early stages of healing, suggesting that magnesium alloy effectively promoted angiogenesis and bone formation, thereby accelerating the tendon-bone healing process. Immunofluorescence staining further revealed that magnesium ions exerted significant anti-inflammatory effects by regulating macrophage polarization, promoting their shift toward the M2 phenotype. Real-time fluorescence quantitative PCR results demonstrated that magnesium ions could facilitate tendon-bone healing by modulating the PI3K/AKT signaling pathway. ConclusionBiodegradable magnesium alloy material accelerated fibrocartilage regeneration and calcification at the tendon-bone interface in rat rotator cuff tear repair by regulating the PI3K/AKT signaling pathway, thereby significantly enhancing tendon-bone healing.
Objective To explore the biosafety of a novel elastic internal tension-relieving augmentation tape (hereinafter referred to as “novel tension-relieving tape”) for posterior cruciate ligament (PCL) reconstruction and its regulatory effects on graft healing and tendon-bone integration via a Diannan miniature pig PCL reconstruction model. Methods Eighteen female Diannan miniature pigs aged 6-8 months were randomly assigned to tension-relieving group and non-tension-relieving group, with 9 pigs per group. One hind limb of each pig was randomly selected as the experimental limb for PCL reconstruction (n=9): autologous tendon combined with the novel tension-relieving tape was applied in tension-relieving group, while only autologous tendon was used in non-tension-relieving group; the contralateral hind limb was served as self-normal control. Postoperative general conditions of animals were observed, and complete blood count (CBC) tests were performed. Knee joint specimens were harvested at 3, 6, and 12 months postoperatively for gross observation and Marburger Arthroscopy Score (MAS) evaluation. Toluidine blue staining, safranin O-fast green staining, hematoxylin-eosin (HE) staining, collagen type Ⅰ/Ⅲ immunohistochemistry staining, and Sirius red staining were conducted to assess tendon-bone interface healing. HE staining of liver, lung, spleen, and the tissues around tendon-bone implantation area were performed to evaluate the biosafety of the material. Results All animals survived to the experimental endpoint. The indicators of CBC tests of both groups before operation and at different time points after operation, including white blood cell counting, hemoglobin, platelet counting, and neutrophil percentage, remained within normal physiological ranges, with no significant intergroup differences (P>0.05). No difference was found in histological scores of liver, lung, and spleen between groups (P>0.05). No giant cell aggregation, chronic inflammatory granuloma, or tissue erosion and degradation was observed in soft tissues around the implantation area, which indicated that the novel tension-relieving tape exhibited favorable biocompatibility without systemic or local toxic reactions. At 3 and 6 months postoperatively, the tension-relieving group obtained superior MAS score, tendon-bone healing score, nuclear morphological score, collagen type Ⅰ immunohistochemical score, and collagen volume fraction (P<0.05) as well as more regularly arranged elastic fibers compared with non-tension-relieving group, whereas no intergroup difference was detected in collagen type Ⅲ immunohistochemical score (P>0.05). At 12 months postoperatively, the degree of graft ligamentization and tendon-bone interface integration were comparable between the two groups, and no significant intergroup difference was observed in all above indicators (P>0.05). Conclusion The novel tension-relieving tape possesses reliable biosafety. It can effectively accelerate early graft collagen remodeling and tendon-bone interface healing after PCL reconstruction, and improve early knee joint stability.