Rabbit Anterior Cruciate Ligament Research Articles

Bone marrow stromal and anterior cruciate ligament remnant cell co-culture-derived extracellular vesicles promote cell activity in both cell types.

The significance of anterior cruciate ligament (ACL) remnants during reconstruction remains unclear. Co-culturing ACL remnant cells and bone marrow stromal cells (BMSCs) may reduce apoptosis and enhance hamstring tendon activity. This study investigated whether extracellular vesicles (EVs), which facilitate cell-cell interactions, act as the active components, improving graft maturation in this co-culture. The effects of EVs on cell viability, proliferation, migration and gene expression in the rabbit ACL remnant cells and BMSCs were assessed using control (BMSC-only culture), co-culture (ACL remnant cells and BMSCs, CM) and co-culture without EVs (CM ∆ EVs) media. EVs were isolated from control (BMSC-EV) and co-culture (CM-EV) media and characterized. CM significantly enhanced the proliferation, migration and expression of transforming growth factor (TGF-β)-, vascular endothelial growth factor (VEGF)-, collagen synthesis- and tenogenesis-related genes. However, CM-induced effects were reversed by the CM ∆ EVs treatment. CM-EV treatment exhibited higher potential to enhance proliferation, migration and gene expression in the ACL remnant cells and BMSCs than BMSC-EV and non-EV treatments. In conclusion, EVs, secreted under the coexistence of ACL remnant cells and BMSCs, primarily increase the cell viability, proliferation, migration and gene expression of collagen synthesis-, TGF-β-, VEGF- and tenogenesis-related genes in both cell types.

Braided biomimetic PCL grafts for anterior cruciate ligament repair and regeneration

Anterior cruciate ligament (ACL) is a knee joint stabilizer with a limited regeneration capacity mainly because of low cellular content. State-of-the-art procedures are unable to restore the functions of the tissue as demonstrated by limited success rates. Regenerative engineering can offer a solution for restoring the functions of torn/ruptured ligaments provided that biomimetic grafts are available as grafts/scaffolds. However, a model construct to test behavior of cells to better understand the healing mechanism of ACL is still missing. This study, firstly, aimed at creating an injured rabbit ACL model. Then, the injured and healthy ACL tissues were characterized in terms of alignment and diameter distributions of collagen fibrils. Next, polycaprolactone (PCL) grafts were prepared from braided electrospun meshes and were characterized in terms of alignment and diameter distributions of fibers. Finally, biomechanical properties of ACL tissue and mechanical properties of PCL grafts were determined and compared. Findings demonstrated that distributions of the fiber diameters of PCL electrospun grafts were similar to diameter distribution of collagens of healthy and injured rabbit ACL. The novelty of this study relies on the determination of the diameter distribution of collagens of healthy and injured rabbit ACL tissues, and fabrication of PCL grafts with diameter distributions similar to that seen in healthy and injured ACLs. This study is significant because it addresses a worldwide clinical problem associated with millions of patients. The fibrous biomimetic graft designed in this study is different from the traditional grafts that exhibit unimodal distribution, and it is expected to have a significant contribution to ACL regeneration efforts.

Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts in vitro and promotion of tendon-bone healing in rabbits

To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.

Inhibition of CaMKK2 Decreases Progression of Post-traumatic Osteoarthritis in a Rabbit ACL Transection Model

Background and Hypothesis:Post-traumatic osteoarthritis (PTOA) is a multifactorial degenerative disease of the joint affecting 20-50% of all joint injuries with a total annual cost of $15 billion. There are no current disease-modifying therapies for PTOA. Mechanical stress due to ligament tear or impact injury triggers the release of inflammatory mediators in the joint. Resulting collagen damage, loss of proteoglycans, and cell death triggers further release of inflammatory mediators and reactive oxygen species. This cycle of inflammation leads to PTOA. We hypothesize that inhibition of Ca2+/CaM dependent protein kinase kinase 2 (CaMKK2), a kinase associated with the inflammatory effects in PTOA, will mitigate the disease-propagating mechanisms.&#x0D; Methods:We utilized a rabbit model of PTOA which involved surgical transection of the anterior cruciate ligament (ACL) to generate joint instability. Rabbits were then treated tri-weekly with either STO-609 (CaMKK2 inhibitor, 0.033 mg/kg) or saline (control) for 16 weeks. Rabbits were sacrificed at 16 weeks post-surgery. Tibiofemoral joints were harvested for staining with safranin O fast green (SO) and PTOA grading via Osteoarthritis Research Society International (OARSI) guidelines. Apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). RNA isolation of cartilage and subchondral bone tissue was conducted for qRT-PCR. Gene expression of MMP-13, IL-6, IL-1B, ACAN, COL2A1 was quantified and normalized to GAPDH.&#x0D; Results:Histology and gross morphology showed increased PTOA severity in saline controls compared to STO-609 treated rabbits. There was no significant difference in chondrocyte apoptosis in STO-609 treated rabbits compared to saline controls based on TUNEL staining. Gene expression analyses are in progress.&#x0D; Potential Impact:This study addresses the unmet clinical need for novel disease-modifying therapeutics for PTOA. Preliminary results show that inhibition of CaMKK2 has the potential to decrease cartilage degradation after joint injuries.

Reconstruction of Rabbit Anterior Cruciate Ligament by Bone Marrow-Derived Mesenchymal Stem Cell Implantation Through a Weft-Knitted Silk Mesh Scaffold Covering a Whip-Shaped Core

To investigate the feasibility of using whip core wrapped by silk weft knitted mesh sheath as a scaffold and bone marrow-derived mesenchymal stem cells (BMSCs) to reconstruct the rabbit anterior cruciate ligament (ACL), BMSC implantation using the mesh-whip scaffold was performed to construct a BMSC-scaffold complex. Then, the BMSC-scaffold complex was implanted into an animal model of an ACL deficient rabbit. Regenerated ACLs were then taken from the animal model three and six months after implantation, followed by hematoxylin-eosin and Masson staining, quantitative RT-PCR detection, as well as mechanical performance evaluation. The results showed that many Sharpey’s fibers had arranged regularly between the neo-ACL and the bone three months after surgery, and an interface structure formed six months after surgery. Regenerated ligaments contained silk fibers and suficient collagen. Type I collagen, type III collagen, and tenascin-C were all highly expressed in the experimental group compared to the control group (no BMSC implantation) in the regenerated ligaments. In addition, the maximum pullout force values of neo-ACL in the three- and six-month experimental groups were 70.6±17.8 N and 122.8±25.7 N, respectively. The findings suggest that BMSC implantation using the mesh-whip scaffold is a promising method to reconstruct rabbit ACL.

Gene Expression in Response to High Frequency, Low Magnitude Loading on the Anterior Cruciate Ligament

IntroductionPrevention of Anterior Cruciate Ligament (ACL) injuries is important, as ACL injuries are associated with the development of knee osteoarthritis, increased pain, functional limitations, and decreased quality of living. Despite the focus on numerous ACL injury prevention mechanisms, ACL injury rates are still on the rise. Investigating a way to strengthen the ACL may be an alternative method to prevent ACL tears. Traditionally, tissue strengthening would occur through a high magnitude load, applied a limited number of times. However, as ligaments are designed to restrict unhealthy joint movement, high magnitude loading of ligaments is likely non‐desirable, making high frequency, low magnitude loading an intriguing possibility. Previous studies have shown bone’s ability to adapt and strengthen in response to high frequency, low magnitude loading. Therefore, we hypothesized ligaments, the connective tissue connecting bones together, would also exhibit similar adaptive properties. However, there is limited research on a typical in vivo ligament response to mechanical loading.ObjectiveThe purpose of this study was to identify what genes were altered in expression in response to high frequency, low magnitude loading.MethodsSeven New Zealand white rabbits were loaded utilizing a custom device built to apply a high frequency, low magnitude load to the rabbit’s shank. While anesthetized, rabbits were placed into the loading device with the left leg secured in a cast and attached to the load cell with a non‐strain cord (Figure 1). To ensure the loading occurred along the longitudinal axis of the ACL, the hip was extended, knee positioned at 30° knee flexion, with the device pulling on the tibia at 35‐40° to apply tibial anterior translation. Loads were applied oscillating between 2‐12 N, at 15 Hz for 20 minutes. Four hours post loading, both of the rabbit’s ACLs were harvested (loaded ACL &amp; internal control). Three rabbits served as external controls and received no loading. Total RNA was extracted from the ACLs using a Qiagen RNAeasy kit following manufacturer’s instructions. Next Generation Sequencing RNAseq detected gene expression differences between the loaded ACL and internal control ACL, and the loaded ACL and the external control ACL using a Sleuth Wald test during data analysis.ResultsIn response to mechanical loading, there were three genes differentially expressed between the loaded and the internal control ACL, none of which were annotated within the rabbit genome. Between the loaded and external control ACL, there were 121 genes differentially expressed. Grouping these genes by function, there were 15 related to mechanotransduction, five to metabolic pathways, four to interleukin response and tissue repair, three to actin cytoskeleton regulation, two relating to protein transport, and one to collagen synthesis.ConclusionsThis is the first data demonstrating a ligament adaptive response to in vivohigh frequency, low magnitude loading. Additionally, these results shed light on the possible mechanotransductive response pathway in ligament. Future studies should characterize optimal loading parameters (i.e., loading frequency, magnitude, and duration), and investigate the long‐term effects of mechanical loading on a ligament.

Quantitative high-resolution 7T MRI to assess longitudinal changes in articular cartilage after anterior cruciate ligament injury in a rabbit model of post-traumatic osteoarthritis

ObjectiveTo demonstrate an ultra-high field (UHF) 7 ​T delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) protocol for quantitative post-traumatic osteoarthritis (PTOA) detection and monitoring in a rabbit anterior cruciate ligament transection (ACLT) model. DesignACL transections were performed unilaterally in 5 rabbits (33-weeks-old, 3.5 ​± ​0.5 ​kg) to induce PTOA. MRI exams were performed at 7 ​T prior to and 2, 4, 7 and 10-weeks after ACLT using a modified dGEMRIC protocol. Voxel-based T1 and T2 maps were created over manually drawn femoral cartilage ROIs from the center of the tibial plateau to the posterior meniscus. Femoral, tibial, and patellar epiphyses were harvested 10-weeks post-surgery and processed for μCT imaging and histology. ResultsQuantitative analysis revealed a 35% and 39% decrease in dGEMRIC index in the medial ACLT knee compartment 7- and 10-weeks post-surgery, respectively (p ​= ​0.009 and p ​= ​0.006) when compared to baseline. There was no significant change in the lateral ACLT compartment or in either compartment of the control knees. Visual inspection of histology confirmed PTOA in the ACLT knees. Osteophytes were found only in ACLT knees (osteophyte volume in femur: 94.53 ​± ​44.08 ​mm3, tibia: 29.35 ​± ​13.79 ​mm3, and patella: 3.84 ​± ​0.92 ​mm3) and were significantly larger in the medial compartments of the femur than lateral (p ​= ​0.0312). ConclusionThe dGEMRIC technique quantitatively applied at 7 ​T UHF-MRI demonstrates site-specific cartilage degeneration in a large animal PTOA model. This should encourage further investigation, with potential applications in drug and therapeutic animal trials as well as human studies.

Injectable recombinant block polymer gel for sustained delivery of therapeutic protein in post traumatic osteoarthritis

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. Here, we demonstrate that E5C, a recombinant protein block polymer, consisting of five repeats of elastin like polypeptide (E) and a coiled-coil domain of cartilage oligomeric matrix protein (C), is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin, provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C gels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the progranulin-derivative Atsttrin, for therapeutic application in OA.

Intra-Articular Adeno-Associated Virus-Mediated Proteoglycan 4 Gene Therapy for Preventing Posttraumatic Osteoarthritis.

Lubricin, a glycoprotein encoded by the proteoglycan 4 (PRG4) gene, is an essential boundary lubricant that reduces friction between articular cartilage surfaces. The loss of lubricin subsequent to joint injury plays a role in the pathogenesis of posttraumatic osteoarthritis. In this study, we describe the development and evaluation of an adeno-associated virus (AAV)-based PRG4 gene therapy intended to restore lubricin in injured joints. The green fluorescent protein (GFP) gene was inserted the PRG4 gene to facilitate tracing the distribution of the transgene product (AAV-PRG4-GFP) in vivo. Transduction efficiency of AAV-PRG4-GFP was evaluated in joint cells, and the conditioned medium containing secreted PRG4-GFP was used for shear loading/friction and viability tests. In vivo transduction of joint tissues following intra-articular injection of AAV-PRG4-GFP was confirmed in the mouse stifle joint in a surgical model of destabilization of the medial meniscus (DMM), and chondroprotective activity was tested in a rabbit anterior cruciate ligament transection (ACLT) model. In vitro studies showed that PRG4-GFP has lubricin-like cartilage-binding and antifriction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP before cyclic shear loading (n = 3). Polymerase chain reaction and confocal microscopy confirmed the presence of PRG4-GFP DNA and protein, respectively, in a mouse DMM (n = 3 per group). In the rabbit ACLT model, AAV-PRG4-GFP gene therapy enhanced lubricin expression (p = 0.001 vs. AAV-GFP: n = 7-14) and protected the cartilage from degeneration (p = 0.014 vs. AAV-GFP: n = 9-10) when treatments were administered immediately postoperation, but efficacy was lost when treatment was delayed for 2 weeks. AAV-PRG4-GFP gene therapy protected cartilage from degeneration in a rabbit ACLT model; however, data from the ACLT model suggest that early intervention is essential for efficacy.

Low-Molecular-Weight Collagen Peptide Ameliorates Osteoarthritis Progression through Promoting Extracellular Matrix Synthesis by Chondrocytes in a Rabbit Anterior Cruciate Ligament Transection Model.

This study examined whether the oral administration of low-molecular-weight collagen peptide (LMCP) containing 3% Gly-Pro-Hyp with >15% tripeptide (Gly-X-Y) content could ameliorate osteoarthritis (OA) progression using a rabbit anterior cruciate ligament transection (ACLT) model of induced OA and chondrocytes isolated from a patient with OA. Oral LMCP administration (100 or 200 mg/kg/day) for 12 weeks ameliorated cartilage damage and reduced the loss of proteoglycan compared to the findings in the ACLT control group, resulting in dose-dependent (p < 0.05) improvements of the OARSI score in hematoxylin & eosin (H&E) and Safranin O staining. In microcomputed tomography analysis, LMCP also significantly (p < 0.05) suppressed the deterioration of the microstructure in tibial subchondral bone during OA progression. The elevation of IL-1βand IL-6 concentrations in synovial fluid following OA induction was dose-dependently (p < 0.05) reduced by LMCP treatment. Furthermore, immunohistochemistry illustrated that LMCP significantly (p < 0.05) upregulated type II collagen and downregulated matrix metalloproteinase-13 in cartilage tissue. Consistent with the in vivo results, LMCP significantly (p < 0.05) increased the mRNA expression of COL2A1 and ACAN in chondrocytes isolated from a patient with OA regardless of the conditions for IL-1βinduction. These findings suggest that LMCP has potential as a therapeutic treatment for OA that stimulates cartilage regeneration.

Sustained delivery of pgrn-derivative atsttrin via e5c hydrogel protects cartilage and bone quality in a rabbit model of post-traumatic osteoarthritis

Purpose: We have developed a protein-engineered thermo-responsive hydrogel, comprised of elastin (E) and cartilage oligomeric matrix protein coiled-coil (C), capable of providing sustained delivery of our chondroprotective engineered progranulin (PGRN)-derivative, Atsttrin (Tang et al., Science, 2011). Here, we characterize the impact of hydrogel delivered Atsttrin on chondrocyte viability and metabolism in vitro and describe the protective effects of injectable E5C hydrogel and Atsttrin-loaded E5C hydrogel in a rabbit anterior cruciate ligament transection (ACLT) induced model of post-traumatic osteoarthritis.

Periodic injections of adipose-derived stem cell sheets attenuate osteoarthritis progression in an experimental rabbit model

BackgroundSubcutaneous adipose tissue represents an abundant source of multipotent adult stem cells named as Adipose-derived stem cells (ADSCs). With a cell sheet approach, ADSCs survive longer, and can be delivered in large quantities. We investigated whether intra-articular ADSC sheets attenuated osteoarthritis (OA) progression in a rabbit anterior cruciate ligament transection (ACLT) model.MethodsFabricating medium containing ascorbate-2-phosphate was used to enhance collagen protein secretion by the ADSCs to make ADSC sheets. At 4 weeks after ACLT, autologous ADSC sheets were injected intra-articularly into the right knee (ADSC sheets group), and autologous cell death sheets treated by liquid nitrogen were injected into the left knee (control group). Subsequent injections were administered once weekly. Femoral condyles were compared macroscopically and histologically.ResultsMacroscopically, OA progression was significantly milder in the ADSC sheets than in the control groups. Histologically, control knees showed obvious erosions in the medial and lateral condyles, while cartilage was retained predominantly in the ADSC sheets group. Immunohistochemically, MMP-1, MMP-13, ADAMTS-4 were less expressive in the ADSC sheets than in the control groups.ConclusionsPeriodic ADSC sheets injections inhibited articular cartilage degeneration without inducing any adverse effects. A large quantity of autologous ADSCs delivered by cell sheets homed to the synovium and protected chondrocytes.

Electrodeposition of calcium phosphate onto polyethylene terephthalate artificial ligament enhances graft-bone integration after anterior cruciate ligament reconstruction

It is a big challenge to develop a polyethylene terephthalate (PET) artificial ligament with excellent osteogenetic activity to enhance graft-bone integration for ligament reconstruction. Herein, we evaluated the effect of biomineralization (BM) and electrodeposition (ED) method for depositing calcium-phosphate (CaP) on the PET artificial ligament in vitro and in vivo. Scanning electron microscopy and energy-dispersive X-Ray spectrometer mapping analysis revealed that the ED-CaP had more uniform particles and element distribution (Ca, P and O), and thermogravimetric analysis showed there were more CaP on the PET/ED-CaP than the PET/BM-CaP scaffold. Moreover, the hydrophilicity of PET scaffolds was significantly improved after CaP deposition. In vitro study showed that CaP coating via BM or ED method could improve the attachment and proliferation of MC3T3-E1 cells, and ED-CaP coating significantly increased osteogenic differentiation of the cells, in which the Wnt/β-catenin signaling pathway might be involved. In addition, radiological, histological and immunohistochemical results of in vivo study in a rabbit anterior cruciate ligament (ACL) reconstruction model demonstrated that the PET/BM-CaP and PET/ED-CaP scaffolds significantly improved graft-bone integration process compared to the PET scaffold. More importantly, larger areas of new bone ingrowth and the formation of fibrocartilage tissue were observed at 12 weeks in the PET/ED-CaP group, and the biomechanical tests showed increased ultimate failure load and stiffness in PET/ED-CaP group compared to PET/BM-CaP and PET group. Therefore, ED of CaP is an effective strategy for the modification of PET artificial ligament and can enhance graft-bone integration both in vitro and in vivo.

Disturbances in Metabolic Pathways and the Identification of a Potential Biomarker Panel for Early Cartilage Degeneration in a Rabbit Anterior Cruciate Ligament Transection Model.

This study aimed to assess the association between synovial fluid (SF) metabolites and magnetic resonance imaging (MRI) measurements of cartilage biochemical composition to identify potential SF biomarkers for detecting the early onset of cartilage degeneration in a rabbit model. Both knees of 12 New Zealand White rabbits were used. The anterior cruciate ligament transection (ACLT) model was performed on right knees, and the sham surgery on left knees. MRI UTE-T2* scanning and SF sample collection were performed on ACLT knees at 4 and 8 weeks postsurgery and on sham surgery knees at 4 weeks postsurgery. Ultra-performance liquid chromatography-mass spectrometry and multivariate statistical analysis were used to distinguish samples in three groups. Pathway and receiver operating characteristic analyses were utilized to identify potential metabolite biomarkers. There were 12 knees in sham surgery models, 11 in ACLT models at 4 weeks postsurgery, and 10 in ACLT models at 8 weeks postsurgery. UTE-T2* values for the lateral tibia cartilage showed significant decreases over the study period. Levels of 103 identified metabolites in SF were markedly different among three groups. Furthermore, 24 metabolites were inversely correlated with UTE-T2* values of the lateral tibia cartilage, while hippuric acid was positively correlated with UTE-T2* values of the lateral tibia cartilage. Among 25 potential markers, N1-acetylspermidine, 2-amino-1,3,4-octadecanetriol, l-phenylalanine, 5-hydroxy-l-tryptophan, and l-tryptophan were identified as potential biomarkers with high area under the curve values and Pearson correlation coefficients. Five differential metabolites in SF were found as potential biomarkers for the early detection of cartilage degeneration in the rabbit ACLT model.

Biomineralizaion of hydroxyapatite on polyethylene terephthalate artificial ligaments promotes graft-bone healing after anterior cruciate ligament reconstruction: An in vitro and in vivo study.

The purpose of the present study is to modify the polyethylene terephthalate ligament with hydroxyapatite via biomineralization and to investigate its effect on graft-bone healing. After biomineralization of hydroxyapatite, the surface characterization of polyethylene terephthalate ligament was examined by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and water contact angle measurements. The compatibility and osteoinduction, along with the underlying signaling pathway involved of hydroxyapatite-polyethylene terephthalate ligament, were evaluated in vitro. Moreover, a rabbit anterior cruciate ligament reconstruction model was established, and the polyethylene terephthalate or hydroxyapatite-polyethylene terephthalate artificial ligament was implanted into the knee. The micro-computed tomography analysis, histological, and immunohistochemical examination as well as biomechanical test were performed to investigate the effect of hydroxyapatite coating in vivo. The results of scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction showed that the hydroxyapatite was successfully deposited on the polyethylene terephthalate ligament. Water contact angle of the hydroxyapatite-polyethylene terephthalate group was significantly smaller than that of the polyethylene terephthalate group. The in vitro study showed that hydroxyapatite coating significantly improved adhesion and proliferation of MC3T3-E1 cells. The osteogenic differentiation of cells was also enhanced through the activation of ERK1/2 pathway. The micro-computed tomography, histological, and immunohistochemical results showed that biomineralization of hydroxyapatite significantly promoted new bone and fibrocartilage tissue formation at 12 weeks postoperatively. Moreover, the failure load and stiffness in the hydroxyapatite-polyethylene terephthalate group were higher than that in the polyethylene terephthalate group. Therefore, biomineralizaion of hydroxyapatite enhances the biocompatibility and osseointegration of the polyethylene terephthalate artificial ligament, thus promoting graft-bone healing for anterior cruciate ligament reconstruction through the activation of ERK1/2 pathway.

Deep learning-based segmentation from histology allows for automated quantification of calcified cartilage morphology in a rabbit model of post-traumatic osteoarthritis

Purpose: Calcified cartilage (CC) undergoes constant remodeling due to two competing events: 1) mineralization of the deep non-calcified cartilage that advances the tidemark, and 2) new bone formation through endochondral ossification that advances the cement line. However, CC modifications in the presence of osteoarthritis (OA) progression are not well understood. In this study, we investigate CC morphology during OA progression and aging in the rabbit anterior cruciate ligament transection (ACLT) model.

Evaluation of a bioengineered ACL matrix's osteointegration with BMP-2 supplementation.

A poly (l-lactic) acid bioengineered anterior cruciate ligament (ACL) matrix has previously demonstrated the ability to support tissue regeneration in a rabbit ACL reconstruction model. The matrix was designed for optimal bone and ligament regeneration by developing a matrix with differential pore sizes in its bone and ligament compartments. Building upon past success, we designed a new bioengineered ACL matrix that is easier to install and can be used with endobutton fixation during ACL reconstruction. To achieve this, a new braiding procedure was developed to allow the matrix to be folded in half, making two-limbs, while maintaining its bone and ligament compartments. The osteointegration of the matrix with and without bone morphogenetic protein 2 (BMP-2) supplementation was evaluated in a rabbit ACL reconstruction model. Two doses of BMP-2 were evaluated, 1 and 10 μg, and delivered by saline injection into the bone tunnel at the end of surgery. A fibrous matrix-to-bone interface with occasional Sharpey’s fibers was the primary mode of osteointegration observed. The matrix was also found to support a fibrocartilage matrix-to-bone interface. In some cases, the presence of chondrocyte-like cells was observed at the aperture of the bone tunnel and the center of the matrix within the bone tunnel. Treatment with BMP-2 was associated with a trend towards smaller bone tunnel cross-sectional areas, and 1 μg of BMP-2 was found to significantly enhance osteoid seam width in comparison with no BMP-2 or 10 μg of BMP-2 treatment. Regenerated tissue was well organized within the bioengineered ACL matrix and aligned with the poly (l-lactic) acid fibers. Disorganized tissue was found between the two-limbs of the bioengineered ACL matrix and hypothesized to be due to a lack of structural scaffolding. This study suggests that the bioengineered ACL matrix can undergo similar modes of osteointegration as current autografts and allografts, and that BMP-2 treatment may enhance osteoblastic activity within the bone tunnels.

A LbL-Assembled Bioactive Coating Modified Nanofibrous Membrane for Rapid Tendon-Bone Healing in ACL Reconstruction.

IntroductionIn anterior cruciate ligament (ACL) reconstruction, hamstring tendon autograft is a well-accepted surgical choice as an alternative ACL graft. But the main disadvantage of autograft is its inefficient healing with host bone-tunnel which will leading to surgery failure.MethodsA biomimetic nanofibrous membrane for tendon-bone integration is fabricated in this work, which is composed of polycaprolactone (PCL) electrospinning membrane and chitosan/hyaluronic acid (CS/HA) multilayers film.ResultsBy using layer-by-layer (LbL) self-assembly this functional CS/HA multilayer films are deposited on the surface of PCL nanofiber to enable the local delivery of stromal cell-derived factor-1 α (SDF-1α) and bone morphogenetic protein-2 (BMP-2) in tendon-bone interface. This membrane can promote cell proliferation and recruitment, as well as inducing the osteogenic differentiation and recruitment of BMSCs.ConclusionFurther in vivo studies demonstrate that to wrap the tendon autograft using the membrane may afford superior tendon-bone integration and inhibit scar tissue formation in a rabbit ACL reconstruction model. More importantly, the biomechanical properties of the tendon-bone interface have been improved. This study shows that this biomimetic nanofibrous membrane is effective for improving tendon-bone healing after ACL reconstruction surgery.

Acute molecular biological responses during spontaneous anterior cruciate ligament healing in a rat model

Anterior cruciate ligament (ACL) injury is one of the most common injuries of the knee joint and is becoming more prevalent. While ACL reconstruction is considered the “gold standard” of treatment, some studies have demonstrated natural spontaneous healing of the ACL in humans, rabbits, and rats. At this time, the mechanism of ACL healing is poorly understood. The purpose of this study was to determine the process of ACL healing by examining the molecular and histological changes in the acute phases, using an ACL-Transection model and a spontaneous ACL healing model. Sixty adult male Wistar rats were randomly assigned to two groups: the ACL transection (ACLT) group and the controlled abnormal movement (CAM) group. Thirty rats were randomly assigned to three groups (day 1, day 3, and day 5). Then, all rats underwent an ACL transection procedure. The CAM group rats underwent controlled abnormal extra-articular tibial translation. Samples were harvested from rats and used for histological and biochemical analyses. Both, the ACLT and the CAM groups exhibited ruptured ACLs. However, in the CAM group, the ends of the proximal remnants were not retracted. Expressions of MMP-3 and PDGF-α increased, and expression of TGF-β1 decreased in the CAM group on day 5 (p < 0.01); PDGF-β expression in the CAM group increased significantly at each time point (p < 0.01). Our results suggested that controlling abnormal movements changed intra-articular responses positively during the acute phase both histologically and biochemically.

Early degeneration of the meniscus revealed by microbiomechanical alteration in a rabbit anterior cruciate ligament transection model

BackgroundThe microbiomechanical properties of the meniscus influence the cell response to the surrounding biomechanical environment ​and are beneficial to understand meniscus repairing and healing. To date, however, this information remains ambiguous. This study aims to characterise the microbiomechanical properties of the meniscus after degeneration in a rabbit anterior cruciate ligament transection (ACLT) model and to analyse the corresponding histology at the macroscale and chemical composition. MethodsTwenty New Zealand white rabbits were used. Menisci were collected from the knee joints 4 and 8 weeks after the ACLT and from those of the corresponding control groups. The central portions of both medial and lateral menisci were investigated using atomic force microscopy, histological study, and an energy-dispersive spectrometer. The evaluation was conducted regionally within the inner, middle, and outer sites from the top layer (facing the femoral surface) to the bottom layer (facing the tibial surface) in both the lateral and medial menisci to obtain the site-dependent properties. ResultsAt 4 weeks after surgery, the dynamic elastic modulus at the microlevel increased significantly at both the top and bottom layers compared with the intact meniscus (P ​= ​0.021). At 8 weeks after surgery, the stiffening occurred in all regions (P ​= ​0.030). The medial meniscus showed greater change than the lateral meniscus. All these microbiomechanical alterations occurred before the histological findings at the macroscale. ConclusionThe microbiomechanical properties in the meniscus changed significantly after ACLT and were site dependent. Their alterations occurred before the histological changes of degeneration were observed. The Translational Potential of this ArticleThe results of our study indicated that degeneration promoted meniscus stiffening. Thus, they provide a better understanding of the disease process affecting the meniscus. Our results might be beneficial to understand how mechanical forces distribute throughout the healthy and pathologic joint. They indicate the possibility of early diagnosis using a minimally invasive arthroscopic tool, as well as they might guide treatment to the healthy and pathologic meniscus and joint.