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Enhancement of tendon-to-bone healing after anterior cruciate ligament reconstruction using bone marrow-derived mesenchymal stem cells genetically modified with bFGF/BMP2.

ABSTRACT: Many strategies, including various growth factors and gene transfer, have been used to augment healing after anterior cruciate ligament (ACL) reconstruction. The biological environment regulated by the growth factors during the stage of tendon-bone healing was considered important in controlling the integrating process. The purpose of this study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) genetically modified with bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) on healing after ACL reconstruction. BMSCs were infected with an adenoviral vector encoding BMP2 (AdBMP2) or bFGF (AdbFGF). Then, the infected BMSCs were surgically implanted into the tendon-bone interface. At 12 weeks postoperatively, the formation of abundant cartilage-like cells, smaller tibial bone tunnel and significantly higher ultimate load and stiffness levels, through histological analysis, micro-computed tomography and biomechanical testing, were observed. In addition, the AdBMP2-plus-AdbFGF group had the smallest bone tunnel and the best mechanical properties among all the groups. The addition of BMP2 or bFGF by gene transfer resulted in better cellularity, new bone formation and higher mechanical property, which contributed to the healing process after ACL reconstruction. Furthermore, the co-application of these two genes was more powerful and efficient than either single gene therapy.

SUBMITTER: Chen B

PROVIDER: S-EPMC4865959 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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Project description:BackgroundWe previously demonstrated, in a rat anterior cruciate ligament (ACL) graft reconstruction model, that the delayed application of low-magnitude-strain loading resulted in improved tendon-to-bone healing compared with that observed after immediate loading and after prolonged immobilization. The purpose of this study was to determine the effect of higher levels of strain loading on tendon-to-bone healing.MethodsACL reconstruction was carried out in a rat model in three randomly assigned groups: high-strain daily loading beginning on either (1) postoperative day one (immediate-loading group; n = 7) or (2) postoperative day four (delayed-loading group; n = 11) or (3) after prolonged immobilization (immobilized group; n = 8). Animals were killed two weeks after surgery and micro-computed tomography (micro-CT) and biomechanical testing of the bone-tendon-bone complex were carried out.ResultsThe delayed-loading group had greater tissue mineral density than either the immediate-loading or immobilized group (mean [and standard deviation], 813.0 ± 24.9 mg/mL compared with 778.4 ± 32.6 mg/mL and 784.9 ± 26.4 mg/mL, respectively; p < 0.05). There was a trend toward greater bone volume per total volume fraction in both the immobilized and the delayed-loading group compared with the immediate-loading group (0.24 ± 0.03 and 0.23 ± 0.06 compared with 0.20 ± 0.05; p = 0.06). Trabecular thickness was greater in the immobilized group compared with the immediate-loading group (106.5 ± 23.0 μm compared with 72.6 ± 10.6 μm; p < 0.01). There were no significant differences in failure load or stiffness between the immobilized group and either high-strain cyclic-loading group.ConclusionsImmediate application of high-strain loading appears to have a detrimental effect on healing in this rat model. Any beneficial effects of delayed loading on the healing tendon-bone interface (after a brief period of immobilization) may be offset by the detrimental effects of excessive strain levels or by the detrimental effects of stress deprivation on the graft.Clinical relevanceThe timing and magnitude of mechanical load on a healing rat ACL reconstruction graft may have important implications for postoperative rehabilitation. Avoidance of exercises that cause high graft strain in the early postoperative period may lead to improved tendon-to-bone healing in humans.

Project description:BackgroundScar tissue formation at the tendon-bone interface caused by excessive inflammation leads to insufficient healing strength, while the phagocytic clearance of dying cells (efferocytosis) has profound consequences on macrophage polarisation and the inflammatory response. Modulating the inflammatory microenvironment may have satisfactory curative effects in patients with anterior cruciate ligament reconstruction (ACLR).MethodsBone marrow-derived macrophages (BMDMs) and polymorphonuclear leukocytes (PMNs) were harvested from bone marrow. The effects of milk fat globulin protein E8 (MFG-E8) on macrophage polarisation were compared with those of M1 and M2 macrophages induced by conventional methods. The BMDMs and apoptotic PMNs co-culture system was used to assess the efficiency of efferocytosis. The biological functions of MFG-E8 in tendon-bone healing by regulating macrophage efferocytosis and polarisation were further investigated using a rat ACLR model.ResultsBMDMs and PMNs were successfully isolated. Compared to conventional induction methods, MFG-E8 alone did not significantly induce macrophage M1 or M2 polarisation, but it could partially reverse the expression of inducible nitric oxide synthase (iNOS) in M1 macrophages. In vitro studies revealed that appropriate dosing of MFG-E8 could significantly promote the efficiency of macrophage efferocytosis and subsequently increase M2 polarisation. More importantly, significantly increased peri-tunnel new bone formation, tighter connected interface and better mechanical properties were observed after ACLR when treated with MFG-E8 in vivo. We further demonstrated that MFG-E8 remarkably facilitated the clearance of apoptotic cells and increased the number of M2 macrophages at the interface between the tendon graft and bone tunnel in the early postoperative stage.ConclusionMFG-E8 promoted tendon-bone healing histologically and biomechanically, probably by the regulation of inflammatory processes via macrophage efferocytosis and M2 polarisation.The translational potential of this article: Regulation of macrophage efferocytosis and M2 polarization by MFG-E8 is expected to be a therapeutic strategy for promoting tendon-bone healing in patients undergoing ACLR.

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Enhancement of tendon-to-bone healing after anterior cruciate ligament reconstruction using bone marrow-derived mesenchymal stem cells genetically modified with bFGF/BMP2.

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