Fang Wan

Enhancement of osseointegration of polyethylene terephthalate artificial ligament by coating of silk fibroin and depositing of hydroxyapatite

Background Application of artificial ligament in anterior cruciate ligament reconstruction is one of the research focuses of sports medicine but the biological tendon–bone healing still remains a problem. The preliminary study of hydroxyapatite (HAP) coating on the polyethylene terephthalate (PET) surface could effectively induce the osteoblast differentiation, but the tendon–bone healing was still not stable. As a green synthesis process, the biomimetic mineralization can simulate the natural bone growth in vitro and in vivo. Methods HAP crystals were grown under the guide of silk fibroin (SF) PET surface by biomimetic route. Several techniques including scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy were utilized for proving the introduction of both SF and HAP. The viability and osseointegration of bone marrow stromal cells on the surface of three kinds of ligament, including PET group (non-coating group), PET+SF group (SF-coating group), and PET+SF+HAP group (combined HAP- and SF-coating group), were analyzed by CCK-8 assays and alkaline phosphatase (ALP) detection. Seventy-two mature male New Zealand rabbits were randomly divided into three groups. Among them, 36 rabbits were sacrificed for mechanical testing, and histological examination for the others. Results The SF and SF+HAP were successfully coated on the surface of PET fiber. The CCK-8 assay showed that the cell proliferation on PET+SF+HAP group was better than the other two groups from 24 to 120 hours. After 14 days of culture, the cells in the PET+SF+HAP group delivered higher levels of ALP than the other two groups. After 3 days of culture, the expression level of integrin β1 in the PET+SF+HAP group and PET+SF group were higher than in the PET group. The mean load to failure and the stiffness value of the PET+SF+HAP group were both higher than the other two groups. Hematoxylin and eosin staining showed that new bone tissue formation was only found in the PET+SF+HAP group 8 weeks postoperatively. Masson staining showed that in the PET+SF+HAP group 8 weeks postoperatively, the PET fibers were almost completely encircled by collagen. Histomorphometric analysis showed that the width of the graft–bone interface in the PET+SF+HAP group was narrower than that in the other two groups 4 and 8 weeks postoperatively. The mRNA level of BMP-7 in the PET+SF+HAP groups was significantly higher than those in the other two groups 4 and 8 weeks postoperatively. Conclusion The study showed that the combined SF and HAP coating by biomimetic route on the surface of PET artificial ligament could induce graft osseointegration in the bone tunnel, providing theoretical and experimental foundation for manufacturing novel artificial ligaments meeting the clinical needs.

Enhance the biocompatibility and osseointegration of polyethylene terephthalate ligament by plasma spraying with hydroxyapatite in vitro and in vivo

Purpose This study was designed to evaluate the biocompatibility and osseointegration of polyethylene terephthalate ligament after coating with hydroxyapatite (PET/HA) by using the plasma spraying technique in vitro and in vivo. Methods In this study, PET/HA sheets were prepared by using the plasma spraying technique. The characterization, the viability of bone marrow stromal cells (BMSCs), and the mRNA expression of bone formation-related genes were evaluated in vitro. The osseointegration in vivo was investigated in the rabbit anterior cruciate ligament (ACL) reconstruction model by micro-computed tomography (micro-CT) analysis, histological evaluation, and biomechanical tests. Results Scanning electron microscopy (SEM) results showed that the surface of polyethylene terephthalate (PET) becomes rough after spraying with hydroxyapatite (HA) nanoparticles, and the water contact angle was 75.4°±10.4° in the PET/HA-plasma group compared to 105.3°±10.9° in the control group (p<0.05). The cell counting kit-8 counting results showed that the number of BMSCs significantly increased in the PET/HA-plasma group (p<0.05). Reverse transcription polymerase chain reaction (RT-PCR) results showed that there was an upregulated mRNA expression of bone formation-related genes in the PET/HA-plasma group (p<0.05). Micro-CT results showed that the transactional area of tibial tunnels and femoral tunnels was smaller in the PET/HA-plasma group (p<0.05). The histological evaluation scores of the PET/HA-plasma group were significantly superior to those of the PET control group at 8 and 12 weeks (p<0.05). The biomechanical tests showed an increased maximum load to failure and stiffness in the PET/HA-plasma group compared to those in the control group at 8 and 12 weeks. Conclusion Both in vitro and in vivo results demonstrated in this study suggest that the biocompatibility and osseointegration of PET/HA ligament were significantly improved by increasing the proliferation of cells and upregulating the expression of bone formation-related genes. In a word, the PET/HA-plasma ligament is a promising candidate for ACL reconstruction in future.

Silk fibroin and hydroxyapatite segmented coating enhances graft ligamentization and osseointegration processes of the polyethylene terephthalate artificial ligament in vitro and in vivo

The inferior biocompatibility of the polyethylene terephthalate (PET) artificial ligament may lead to poor healing in both the intra-articular part (IAP) and the intraosseous part (IOP) after anterior cruciate ligament (ACL) reconstruction. This study aimed to systematically investigate the effect of silk fibroin (SF) and hydroxyapatite (HA) segmented coating on graft ligamentization and osseointegration processes of the PET ligament. Several techniques including scanning electron microscopy (SEM) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray diffraction (XRD) and water contact angle (WCA) measurements were carried out to validate the introduction of SF and HA. The segmented coating ligament was assessed both in vitro and in vivo. The results of SEM and cell counting kit-8 (CCK-8) assay revealed that the L929 fibroblasts and MC3T3-E1 osteoblasts exhibited better adhesion and proliferation performance on the PET-SF and PET-HA fibers, respectively, compared to those on the uncoated PET fibers. HA promoted osteogenic differentiation of MC3T3-E1 in terms of the levels of alkaline phosphatase (ALP) activity and calcium deposition. Furthermore, the in vivo study in a beagle ACL reconstruction model demonstrated that the segmented coating could enhance the graft ligamentization and osseointegration processes as indicated by the better tissue infiltration in the IAP and more bone ingrowth in the IOP of the ligament than the control group according to the results of micro-computed tomography (micro-CT), histology, real-time polymerase chain reactions (RT-PCRs) and biomechanical tests. Therefore, the SF and HA segmented coating ligaments may display a great potential application for the clinical augmentation of graft healing in ACL reconstruction surgery.

Micro‑scale assessment of the postoperative effect of anterior cruciate ligament reconstruction preclinical study using a 7.1T micro‑magnetic resonance imaging

High-field micro-magnetic resonance imaging (MRI) scanning may provide additional information for quantitative analysis of graft bone healing processes, thus serving as a promising supplementary method in graft and bone healing evaluation following anterior cruciate ligament reconstruction (ACLR) surgery during preclinical studies. The present study included 12 New Zealand white rabbits that underwent ACLR with polyethylene terephthalate (PET) ligament. At 4, 8, and 16 weeks following surgery, 4 rabbits were euthanized and knee joint samples were harvested for a 7.1T micro-magnetic resonance imaging (MRI) scan. The graft bone tunnel diameter and signal noise ratio (SNR) at the region of interest (ROI) were measured. Hematoxylin-eosin staining was performed at each time point to verify the graft bone healing process in histology. The bone tunnel diameter at the graft tunnel interface decreased over time in both femoral and tibial parts. Notably, the tunnel size was smaller than the diameter of the drilling Kirschner wire that was used to observe the femoral part and proximal site of the tibial part at 16 weeks following surgery. SNR research demonstrated that both the femoral and tibial part PET ligaments selected in the ROI exhibited a marked increase in SNR from the initial 4-week results. The micro-MRI result was consistent with that of histological analysis. Micro-MRI scanning was applied in an animal model that underwent ACL reconstruction surgery with PET ligament, and it was determined that micro-MRI is promising in quantitatively observing graft bone healing processes directly with a focus on graft tunnel distances and SNRs.

Advantages of an Attached Semitendinosus Tendon Graft in Anterior Cruciate Ligament Reconstruction in a Rabbit Model

Background: The semitendinosus tendon graft with an intact tibial insertion has a sustainable blood supply and might be beneficial for graft maturation after anterior cruciate ligament reconstruction (ACLR); however, its potential advantages for graft tendon-bone healing is still unclear. Hypothesis: Intact tibial insertion of the hamstring tendon can preserve enough blood supply to keep the harvested tendon alive, which can improve tendon-bone healing and the biomechanical strength of the graft. Study Design: Controlled laboratory study. Methods: Sixty-four healthy New Zealand White rabbits underwent unilateral ACLR with a semitendinosus tendon autograft after random enrollment into 2 groups (study group, n = 32 rabbits with semitendinosus tendon–preserved tibial insertions; control group, n = 32 rabbits with free semitendinosus tendons). At weeks 3, 6, 12, and 24, 8 rabbits in each group were sacrificed to evaluate tendon-bone healing by histologic staining, micro–computed tomography (micro-CT) examination, and biomechanical test. Results: The grafts in the study group maintained a similar cell count with no signs of necrosis or hypocellularity across all time points, but the grafts in the control group underwent a characteristic stage of necrosis at weeks 3 and 6. Sharpey-like fibers were observed from postoperative 3 weeks at the tendon-bone interface in the study group, and a normal insertion-like structure was formed at week 12, which became more mature at week 24. In the control group, however, Sharpey-like fibers could not be observed until week 12, and a normal transition through cartilage from bone to tendon was not observed at any time point. Histologic scores of the tendon-bone interface in the study group were significantly higher than those in the control group at week 6 (P = .04), week 12 (P < .001), and week 24 (P = .04). As compared with the control group via micro-CT, the study group had a significantly smaller bone tunnel area at week 6 (P = .01) and larger bone volume/total volume at week 3 (P = .0026) and week 6 (P = .01). Also, the study group had a significantly higher failure load at weeks 12 and 24 (both P = .03) and a significantly higher stiffness at week 24 (P < .001) versus the control group. Conclusion: The semitendinosus tendon graft with an intact tibial insertion in ACLR would bypass the graft avascular necrosis stage, which improves tendon-bone healing and biomechanical strength. Clinical Relevance: An alive graft in ACLR could improve tendon-bone healing and the biomechanical strength of the graft, which might be beneficial to early and intensive rehabilitation after ACLR.

Synthetic Ligaments for ACL Reconstruction

The early experience with synthetic ligaments for ACL reconstruction was disappointing. A variety of ligaments made from various substances including carbon fiber, polyester, polytetrafluoroethylene, and polypropylene all resulted in similar problems – synovitis, foreign body reaction, graft rupture, and osteolysis. As a result, they were all ultimately withdrawn from the market. The Ligament Augmentation Reinforcement System (LARS) is made from polyethylene terephthalate and has been used sporadically over the past two decades. It has been reported to have less problems with synovitis but clinical outcomes remain variable. The LARS device has been used extensively in China with apparent success, particularly in older patients. Areas of research interest include the use of alternative materials, various biological coatings, and alternative fixation methods.