Rabbit Knee Model Research Articles

Enoxaparin sodium bone cement displays local anti-inflammatory effects by regulating the expression of IL-6 and TNF-α

ObjectiveTo explore the roles of Enoxaparin Sodium-Polymethyl methacrylate bone cement on inflammatory factors Interleukin-6 and Tumour Necrosis Factor-α in a rabbit knee replacement model. As well as the mechanisms underlying its potential effects on lipopolysaccharide-induced endothelial cell injury. MethodsA knee replacement model was established using New Zealand rabbits. Forty rabbits were randomly divided into four groups: PMMA, ES-PMMA, sham-operated, and blank control groups (n = 10 in each group). Local tissues around the incision were taken at the 30th, 60th, and 90th minute after the surgical implantation of the corresponding bone cement. Immunohistochemistry in the surgical field was used to measure the expression of local inflammatory factors IL-6 and TNF-α. In the in vitro experiments, 1 cm3 of bone cement was immersed in 3 mL of the medium for 24 h. The bone cement was discarded and diluted to 25% with normal medium. Pre-experiments were screened for the best LPS-inducing concentration of 100 mg/mL, and the most compatible LPS concentration was used for subsequent experiments simulating the primary cultures of rats’ Inferior Vena Cava Endothelial Cells. The experiments were divided into four groups: blank control group, LPS induction group, PMMA + LPS group, and ES-PMMA + LPS group. The apoptosis rate was detected by flow cytometry, and the expression levels of TNF-α and IL-6 in the cells and supernatant were measured by ELISA, western blotting, and immunofluorescence. ResultsAccording to immunohistochemical results, IL-6-positive cells were concentrated in the tissue interstitial space. In the PMMA and sham-operated groups, the number of IL-6-positive cells gradually increased over time. At all time points, IL-6 expression in the ES-PMMA group was much lower than in the PMMA and sham-operated groups. At 30 min, TNF-α positive cells in the ES-PMMA group expressed less than those in the PMMA and sham-operated groups, with no discernible difference between the PMMA and ES-PMMA groups at 60 or 90 min. Using ELISA and flow cytometry, the expression levels of IL-6 and TNF-α were improved and the apoptosis rate was magnified in the LPS-induced group (***P < 0.001) in contrast with the blank control group. Additionally, the expression levels of IL-6 and TNF-α were reduced in the ES-PMMA + LPS group compared with the LPS-induced group (*P < 0.05) and the apoptosis rate was reduced (***P < 0.001), with statistically significant variations. Western blotting and immunofluorescence analysis confirmed that IL-6 and TNF-α protein expression in cells was upregulated in the LPS-induced group compared to the blank control group (***P < 0.001), and the mean fluorescence intensity was enlarged (***P < 0.001). Meanwhile, IL-6 and TNF-α expression in the ES-PMMA + LPS group were down-regulated (**P < 0.01 or *P < 0.05) compared with the LPS-induced group and PMMA + LPS crew protein expression, and the average fluorescence intensity of IL-6 and TNF-α was lowered in the ES-PMMA + LPS group compared to the LPS-induced group (***P < 0.001). ConclusionsES-PMMA bone cement reduced the expression levels of local inflammatory factors IL-6 and TNF-α in a rabbit knee model. ES-PMMA bone cement reduced the rate of LPS-induced endothelial cell apoptosis and diminished local inflammatory damage by regulating the secretion of inflammatory factors TNF-α and IL-6.

BMP2 and GDF5 for Compartmentalized Regeneration of the Scapholunate Ligament.

Background Chronic injuries to the scapholunate ligament (SLIL) alter carpal kinematics and may progress to early degenerative osteoarthritis. To date, there is no consensus for the best method for SLIL reconstruction. This study aims to assess the use of growth factors (bone morphogenetic protein [BMP]2 and growth and differentiation factor 5 [GDF5]) for compartmentalized regeneration of bone and ligament in this multiphasic scaffold in a rabbit knee model. Case Description A total of 100 µg of BMP2 and 30 µg of GDF5 were encapsulated into a heparinized gelatin-hyaluronic acid hydrogel and loaded into the appropriate compartment of the multiphasic scaffold. The multiphasic scaffold was implanted to replace the native rabbit medial collateral ligament ( n = 16). The rabbits were randomly assigned to two different treatment groups. The first group was immobilized postoperatively with the knee pinned in flexion with K-wires for 4 weeks ( n = 8) prior to sacrifice. The second group was immobilized for 4 weeks, had the K-wires removed followed by a further 4 weeks of mobilization prior to sample harvesting. Literature Review Heterotopic ossification as early as 4 weeks was noted on gross dissection and confirmed by microcomputed tomography and histological staining. This analysis revealed formation of a bony bridge located within and over the ligament compartment in the intra-articular region. Biomechanical testing showed increased ultimate force of the ligament compartment at 4 weeks postimplantation consistent with the presence of bone formation and higher numbers of scaffold failures at the bone-tendon junction. This study has demonstrated that the addition of BMP2 and GDF5 in the bone-ligament-bone (BLB) scaffold resulted in heterotopic bone formation and failure of the ligament compartment. Clinical Relevance The implantation of a three-dimensional-printed BLB scaffold alone demonstrated superior biomechanical and histological results, and further investigation is needed as a possible clinical reconstruction for the SLIL.

Expedited gene delivery for osteochondral defect repair in a rabbit knee model: A one-year investigation

ObjectiveTo evaluate a single-step, gene-based procedure for repairing osteochondral lesions. DesignOsteochondral lesions were created in the patellar groove of skeletally mature rabbits. Autologous bone marrow aspirates were mixed with adenovirus vectors carrying cDNA encoding green fluorescent protein (Ad.GFP) or transforming growth factor-β1 (Ad.TGF-β1) and allowed to clot. The clotted marrow was press-fit into the defects. Animals receiving Ad.GFP were euthanized at 2 weeks and intra-articular expression of GFP examined by fluorescence microscopy. Animals receiving Ad.TGF-β1 were euthanized at 3 months and 12 months; repair was compared to empty defects using histology and immunohistochemistry. Complementary in vitro experiments assessed transgene expression and chondrogenesis in marrow clots and fibrin gels. In a subsequent pilot study, repair at 3 months using a fibrin gel to encapsulate Ad.TGF-β1 was evaluated. ResultsAt 2 weeks, GFP expression was seen at variable levels within the cartilaginous lesion. At 3 months, there was no statistically significant improvement (p ​> ​0.05) in healing of lesions receiving Ad.TGF-β1 and variability was high. At 12 months, there were still no significant difference (p ​> ​0.05) between the empty defects and those receiving Ad.TGF-β1 in the overall, cartilage, and bone scores. Variability was still high. In vitro experiments suggested that variability reflected variable transduction efficiency and chondrogenic activity of the marrow clots; using fibrin gels instead of marrow may address this issue but more research is needed. ConclusionsThis approach to improving the repair of osteochondral lesions needs further refinement to reduce variability and provide a more robust outcome.

The Beneficial Effect of an Intra-articular Injection of Losartan on Microfracture-Mediated Cartilage Repair Is Dose Dependent

Background: A previous publication demonstrated that the oral intake of losartan promoted microfracture-mediated hyaline-like cartilage repair in osteochondral defects of a rabbit knee model. However, an intra-articular (IA) injection of losartan may have direct beneficial effects on cartilage repair and has not been studied. Purpose: To determine the dosage and beneficial effects of an IA injection of losartan on microfracture-mediated cartilage repair and normal cartilage homeostasis. Study Design: Controlled laboratory study. Methods: Rabbits were divided into 5 groups (n = 6 each): a microfracture group (MFX group) and 4 different losartan treatment groups that received varying doses of IA losartan (0.1, 1, 10, and 100 mg per knee). An osteochondral defect (5 mm) was created in the trochlear groove cartilage of 1 limb in each rabbit, and 5 microfracture perforations were made in the osteochondral defect. Both the injured and the contralateral knee joints were injected with IA losartan immediately after microfracture and at 2 and 4 weeks after surgery. Rabbits were sacrificed at 6 weeks after surgery for analysis including gross observation, micro–computed tomography, histology, and reverse transcription quantitative polymerase chain reaction. Results: Micro–computed tomography and gross observation demonstrated comparable subchondral bone healing and hyaline-like cartilage morphology in the 0.1-, 1-, and 10-mg losartan groups relative to the MFX group. Conversely, the 100-mg losartan group showed neither bony defect healing nor cartilage repair. Histology revealed higher O’Driscoll scores and hyaline-like cartilage regeneration in the 1-mg losartan group compared with the MFX group. In contrast, the 100-mg losartan group showed the lowest histology score and no cartilage repair. An IA injection of losartan at the doses of 0.1, 1, and 10 mg did not cause adverse effects on uninjured cartilage, while the 100-mg dose induced cartilage damage. Quantitative polymerase chain reaction results showed downregulation of the transforming growth factor β (TGF-β) signaling pathway after IA losartan injection. Conclusion: An IA injection of losartan at the dose of 1 mg was most effective for the enhancement of microfracture-mediated cartilage repair without adversely affecting uninjured cartilage. Conversely, a high dose (100 mg) IA injection of losartan inhibited cartilage repair in the osteochondral defect and was chondrotoxic to normal articular cartilage. Clinical Relevance: An IA injection of losartan at an optimal dosage represents a novel microfracture enhancement therapy and warrants a clinical trial for future clinical applications.

Comparison of chondrogenesis-related biological behaviors between human urine-derived stem cells and human bone marrow mesenchymal stem cells from the same individual

BackgroundStem cells are the main choice for seed cells in tissue engineering, but using most traditional stem cells requires invasive and complicated procedures. Human urine-derived stem cells (hUSCs) are an alternative stem cell source with the advantages of being isolated noninvasively and repetitively from the same individual. The aim of this study was to compare chondrogenesis-related biological behaviors between hUSCs and human bone marrow mesenchymal stem cells (hBMSCs) from the same individual.MethodshUSCs and hBMSCs were isolated from six patients who underwent iliac bone grafting. Cell morphology, proliferation, colony-forming, migration, and multidifferentiation analyses were performed in vitro. Then, acellular cartilage extracellular matrix (ACM) scaffolds were fabricated for in vivo implantation. The comparisons of cell viability, morphology, proliferation, and chondrogenesis between hUSCs and hBMSCs cultured on scaffolds were performed before implantation. The scaffolds loaded with hUSCs or hBMSCs were implanted into a rabbit knee model to repair cartilage defects. Magnetic resonance imaging (MRI) and micro-computed tomography (μCT) Analyses, inflammation and toxicity assays, gross observation, and histological evaluation were performed to evaluate the cartilage repair effects.ResultsIn in vitro experiments, hUSCs had better capacity for proliferation, colony-forming, and migration compared to hBMSCs in the same passage, while hBMSCs had greater osteogenic, adipogenic, and chondrogenic abilities compared to hUSCs in the same passage. Both hUSCs and hBMSCs at passage 3 had the strongest potential for proliferation, colony-forming, and multilineage differentiation compared to cells in other passages. The ACM scaffolds loaded with hUSCs or hBMSCs both significantly promoted the repair of cartilage defects in the rabbit knee model at 12 weeks’ postimplantation, and the new tissue was mainly hyaline cartilage. However, there was no significant difference in cartilage repair effects between hUSCs and hBMSCs.ConclusionsIn in vitro experiments, hUSCs presented better capacity for proliferation, while hBMSCs had greater chondrogenic ability. However, hUSCs and hBMSCs had similar cartilage repair effects in vivo. Results indicated that hUSCs can be a stem cell alternative for cartilage regeneration and provide a powerful platform for cartilage tissue engineering and clinical transformation.Graphical abstract

Ultrapurified Alginate Gel Containing Bone Marrow Aspirate Concentrate Enhances Cartilage and Bone Regeneration on Osteochondral Defects in a Rabbit Model

Background: Ultrapurified alginate (UPAL) gel implantation has been demonstrated as effective in cartilage repair for osteochondral defects; however, cell transplantation within UPAL gels would be required to treat larger defects. Hypothesis: The combination of UPAL gel and bone marrow aspirate concentrate (BMAC) would enhance cartilage repair and subchondral bone repair for large osteochondral defects. Study Design: Controlled laboratory study. Methods: A total of 104 osteochondral defects (1 defect per knee) of 52 rabbits were randomly divided into 4 groups (26 defects per group): defects without any treatment (Defect group), defects treated using UPAL gel alone (UPAL group), defects treated using UPAL gel containing allogenic bone marrow mesenchymal stromal cells (UPAL-MSC group), and defects treated using UPAL gel containing BMAC (UPAL-BMAC group). At 4 and 16 weeks postoperatively, macroscopic and histologic evaluations and measurements of repaired subchondral bone volumes of reparative tissues were performed. Collagen orientation and mechanical properties of the reparative tissue were assessed at 16 weeks. Results: The defects in the UPAL-BMAC group were repaired with hyaline-like cartilage with well-organized collagen structures. The histologic scores at 4 weeks were significantly higher in the UPAL-BMAC group (16.9 ± 2.0) than in the Defect group (4.7 ± 1.9; P < .05), the UPAL group (10.0 ± 3.3; P < .05), and the UPAL-MSC group (12.2 ± 2.9; P < .05). At 16 weeks, the score in the UPAL-BMAC group (24.4 ± 1.7) was significantly higher than those in the Defect group (9.0 ± 3.7; P < .05), the UPAL group (14.2 ± 3.9; P < .05), and the UPAL-MSC group (16.3 ± 3.6; P < .05). At 4 and 16 weeks, the macroscopic evaluations were significantly superior in the UPAL-BMAC group compared with the other groups, and the values of repaired subchondral bone volumes in the UPAL-BMAC group were significantly higher than those in the Defect and UPAL groups. The mechanical properties of the reparative tissues were significantly better in the UPAL-BMAC group than in the other groups. Conclusion: The implantation of UPAL gel containing BMAC-enhanced hyaline-like cartilage repair and subchondral bone repair of osteochondral defects in a rabbit knee model. Clinical Relevance: These data support the potential clinical application of 1-step treatment for large osteochondral defects using biomaterial implantation with cell transplantation.

Articular chondroprogenitors in platelet rich plasma for treatment of osteoarthritis and osteochondral defects in a rabbit knee model

BackgroundArticular chondroprogenitors are a promising contender for cartilage repair due to their inherent nature which stands primed for chondrogenesis and minimal hypertrophic preponderance. Platelet rich plasma (PRP) has been extensively used for treating cartilage defects and osteoarthritis (OA), due to its chondro-inductive properties and abundant pool of growth factors. The aim of this study was to assess the efficacy of chondroprogenitors injected with PRP versus PRP alone in the healing of experimentally created early OA and osteochondral defects (OCD) in a rabbit model. MethodsAdult New Zealand White male rabbits were used for cell and PRP isolation. Chondroprogenitors were isolated by fibronectin adhesion assay, labelled with iron oxide, characterized for surface markers, differential potential and expanded. PRP was isolated by double spin centrifugation using a TriCell kit. Study groups included (a) Monosodium iodoacetate induced early OA and (b) critical OCD. Following intervention (test arm: PRP+ chondroprogenitors and control arm: PRP), assessment was performed at 6- and 12-weeks which included histopathological examination and scoring (OARSI and Modified Wakitani score), immunohistochemistry analysis (Collagen type II and X) and synovial fluid S100A12 levels. Results and conclusionComparable, evident healing was noticed in both test and control arms when the OA group samples were assessed at both time points. In the OCD group, PRP alone exhibited significantly better results than the test arm, although repair was notable in both interventions. Further evaluation of chondroprogenitors is required to assess their role as a standalone therapy and in combination with PRP to further cartilage regeneration.

DLX5 and HOXC8 enhance the chondrogenic differentiation potential of stem cells from apical papilla via LINC01013

BackgroundMesenchymal stem cell (MSC)-based cartilage tissue regeneration is a treatment with great potential. How to enhance the MSC chondrogenic differentiation is a key issue involved in cartilage formation. In the present study, we seek to expound the phenotypes and mechanisms of DLX5 in chondrogenic differentiation function in MSCs.MethodsStem cells from apical papilla (SCAPs) were used. The Alcian Blue staining, pellet culture system, and cell transplantation in rabbit knee cartilage defect were used to evaluate the chondrogenic differentiation function of MSCs. Western blot, real-time RT-PCR, and ChIP assays were used to evaluate the molecular mechanisms.ResultsDLX5 and HOXC8 expressions were upregulated during chondrogenic differentiation. In vitro results showed that DLX5 and HOXC8 enhanced the expression of chondrogenic markers including collagen II (COL2), collagen V (COL5), and sex-determining region Y box protein 9 (SOX9) and promoted the chondrogenic differentiation and the formation of cartilage clumps in the pellet culture system. Mechanically, DLX5 and HOXC8 formed protein complexes and negatively regulated the LncRNA, LINC01013, via directly binding its promoter. In vivo transplantation experiment showed that DLX5 and HOXC8 could restore the cartilage defect in the rabbit knee model. In addition, knock-down of LINC01013 enhanced the chondrogenic differentiation of SCAPs.ConclusionsIn conclusion, DLX5 and HOXC8 enhance the chondrogenic differentiation abilities of SCAPs by negatively regulating LINC01013 in SCAPs, and provided the potential target for promoting cartilage tissue regeneration.

A cell-free approach with a supporting biomaterial in the form of dispersed microspheres induces hyaline cartilage formation in a rabbit knee model.

The objective of this study was to test a regenerative medicine strategy for the regeneration of articular cartilage. This approach combines microfracture of the subchondral bone with the implant at the site of the cartilage defect of a supporting biomaterial in the form of microspheres aimed at creating an adequate biomechanical environment for the differentiation of the mesenchymal stem cells that migrate from the bone marrow. The possible inflammatory response to these biomaterials was previously studied by means of the culture of RAW264.7 macrophages. The microspheres were implanted in a 3 mm-diameter defect in the trochlea of the femoral condyle of New Zealand rabbits, covering them with a poly(l-lactic acid) (PLLA) membrane manufactured by electrospinning. Experimental groups included a group where exclusively PLLA microspheres were implanted, another group where a mixture of 50/50 microspheres of PLLA (hydrophobic and rigid) and others of chitosan (a hydrogel) were used, and a third group used as a control where no material was used and only the membrane was covering the defect. The histological characteristics of the regenerated tissue have been evaluated 3 months after the operation. We found that during the regeneration process the microspheres, and the membrane covering them, are displaced by the neoformed tissue in the regeneration space toward the subchondral bone region, leaving room for the formation of a tissue with the characteristics of hyaline cartilage.

Biomimetic proteoglycans can molecularly engineer early osteoarthritic cartilage in vivo.

Biomimetic proteoglycans (BPGs) have the potential to treat osteoarthritis (OA) given that these molecules mimic the structure and properties of natural proteoglycans, which are significantly reduced in OA. We examined the effects of BPGs injected into the intra-articular space in an in vivo OA rabbit knee model and evaluated the effect on histological response, joint friction, and BPG distribution and retention. Rabbits underwent ACL transection to create an arthritic state after 5 weeks. OA rabbits were treated with BPGs or Euflexxa® (hyaluronic acid) intra-articular injections. Non-OA rabbits were injected similarly with BPGs; contralateral joints served as controls. The progression of OA and response to injections were evaluated using Mankin and gross grading systems indicating that mild OA was achieved in operated joints. The coefficient of friction (COF) of the intact knee joints were measured using a custom pendulum friction apparatus, showing that OA joints and OA + Euflexxa® joints demonstrated increased COF than non-operated controls, while BPG-injected non-OA and OA + BPGs were not significantly different from non-OA controls. Injected fluorescently labeled BPGs demonstrated that BPGs diffused into cartilage with localization in the pericellular region. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:403-411, 2019.

Better understanding of acute gouty attack using CT perfusion in a rabbit model.

To assess hemodynamic changes related to acute gouty knee arthritis in a rabbit with CT perfusion (CTP) METHODS: Forty-two rabbits were randomly separated into two groups: the treated group of 30 and the control group of 12. The right knee was injected with monosodium urate solution and polymyxin in the treated group and saline and polymyxin in the control group. At 2, 16, 32, 48, 60, and 72h after injection, five rabbits from the treated group and two rabbits from the control group were selected for CTP. At each time point, blood flow (BF), blood volume (BV), and clearance rate (CL) were measured, and microvessel density (MVD) was evaluated with a microscope. In the treated group, BF, BV, CL, and MVD were significantly higher than in the control group (p < 0.001). Differences within paired comparison of BV, BF, CL, and MVD were all significant (all p < 0.001). Peak time of BV, BF, and MVD was 32h and 48h for CL. After multivariate stepwise linear regression analysis, BV was linearly associated with MVD and vice versa, which also applied to BF with MVD and BF with CL, separately. The ascending rate of MVD was the highest among that of all parameters; so was the descending rate of CL. CTP in this rabbit knee model accurately detected hemodynamic changes during a gouty attack. • Acute gouty arthritis can be evaluated with CTP in a rabbit knee model. • Following injection of MSU crystals, producing an acute gouty attack, CTP successfully assessed hemodynamic changes. • The ascending rate of MVD was the highest among that of all parameters; so was the descending rate of CL.

Treatment of post-traumatic joint contracture in a rabbit model using pulsed, high intensity laser and ultrasound

Post-traumatic joint contracture induced by scar tissues following a surgery or injury can leave patients in a permanent state of pain and disability, which is difficult to resolve by current treatments. This randomized controlled trial examines the therapeutic effect of pulsed high-intensity laser (PHIL) and pulsed high-intensity focused ultrasound (PHIFU) for post-traumatic joint contracture due to arthrofibrosis. The peak power levels of both PHIL and PHIFU are much higher than that of laser or ultrasound currently used in physical therapy, while short pulses are utilized to prevent damage. To test the effectiveness of these treatments, a rabbit knee model for joint contracture was established. Twenty-one rabbits were split into four groups: untreated control (n = 5), PHIL (n = 5), PHIFU (n = 5), and a PHIL + PHIFU group (n = 6). Maximum extension of the surgically modified rabbit knee was compared to that of the contralateral control knee over the course of 16 weeks. The rabbits in the untreated control group maintained a relatively consistent level of joint contracture, while every rabbit in each of the treatment groups had improved range of motion, eventually leading to a restoration of normal joint extension. Average recovery time was 7.6 ± 1.5 weeks for the PHIL treatment group, 9.8 ± 3.7 weeks for the PHIFU group, and 8.0 ± 2.2 weeks for the combined treatment group. Histopathology demonstrated reduced density and accelerated resorption of scar tissues in the treated knee joints. This study provides evidence that both PHIL and PHIFU are effective in treating post-traumatic joint contracture in rabbits and warrant further investigation into the underlying mechanisms to optimize PHIL and PHIFU based treatments in a larger number of animals.

Synthetic Poly(Vinylalcohol)-Based Membranes for Cartilage Surgery and Repair.

Cell-based therapies for cartilage repair are continually being developed to treat osteoarthritis. The cells are either introduced directly by intra-articular injection or via a cell-seeded matrix scaffold. Here, poly(vinylalcohol)-based membranes are developed to be used for mesenchymal stem cell implantation in cartilage repair procedures, having controllable physicochemical properties such as porosity, mechanical strength, and permeability, and a unique self-sealing property. The membranes possess a bilayer structure with a less porous layer providing mechanical strength and selective permeability, exhibit an elastic modulus of between 0.3 and 0.9 MPa, and are permeable to molecules <40 kDa, which is in the range of cartilage permeability. Three different peptide ligands with the sequences Ac-GCGYGRGDSPG, Ac-GCG(OPG)4REGOFG(OPG)4, and Ac-GCG(OPG)7, respectively, are conjugated to the membranes and subject to in vitro cell adhesion and differentiation assays. Col I/Col II gene expression ratios indicated that the collagen-mimetic peptide, Ac-GCG(OPG)7, best supported mesenchymal stem cell differentiation into the chondrogenic lineage. Although low retention of the membrane is observed in vivo in a rabbit knee model, results suggest that the membrane was able to facilitate mesenchymal stem cell implantation and differentiation to chondrocytes. These PVA-based membranes provide a feasible, synthetic, off-the-shelf material for the delivery of stem cells, and can be modified for other surgical applications.

Randomized, Placebo-controlled Clinical Trial Evaluating Ketotifen Fumarate in Reduction of Post-traumatic Elbow Joint Contracture

Grant support received from: American Foundation for Surgery of the Hand There is no financial information to disclose. Post-traumatic joint contractures (PTJC) are a significant complication following intra-articular injuries. Our research has established that a myofibroblast-mast cell-neuropeptide axis underlies the joint capsule fibrosis that limits motion of the human elbow and a rabbit knee model of PTJC.1-3 In our rabbit model, we have established that Ketotifen Fumarate (KF), a mast cell stabilizer, prevents growth factor release and decreased contracture severity by 50% concomitant with decreased measures of fibrosis.2-4 We hypothesized that oral KF would decrease elbow PTJC severity, compared with placebo, in patients with intra-articular elbow fractures and/or dislocations. A randomized, placebo-controlled clinical trial comparing 6-weeks of oral KF 5 mg twice daily to a lactose placebo in patients sustaining distal humerus (AO/OTA 13) and proximal radius +/- ulna (AO/OTA 21) fractures. The primary outcome measure was flexion-extension range of motion (ROM) arc at 12-weeks post-injury. Secondary outcomes included the Disabilities of the Arm, Hand and Shoulder (DASH) score, complication rate, and fracture healing. Statistical analysis consisted of chi-square for categorical variables and ANOVA for continuous variables. Multiple linear regression based on robust estimators was used to assess the adjusted effect of KF. 145 participants were randomized (76 KF, 69 placebo). There were no significant differences between the two groups for baseline characteristics including age, sex, side of injury, hand dominance, pre-injury work status, time to randomization, injury types, and operation; all P > 0.05 (Table 75-1). The 12-week follow up rate was 88% in the KF group and 89% in the placebo group. There was no significant difference between the treatment groups in the primary outcome measure, serious adverse event rates, or (re) operation rates; P > 0.05. But there was a significant difference between operative and non-operative groups on extension-flexion arc of motion (P = 0.03). Fracture healing at the time of analysis found 48% of participants in each group had achieved union. •Patients requiring surgical treatment, as a surrogate for increased injury severity, demonstrated increased PTJC.•The greater loss of motion at 12 weeks in the operative sub-group will inform future Phase III multi-centre injury inclusion criteria to elbow fractures or dislocations requiring surgical treatment.•Several measures of safety were compiled that confirmed the safety of Ketotifen in elbow fractures or dislocations.Table 75-1Baseline CharacteristicsCharacteristicKetotifenPlaceboAge – years, mean (SD)46.7 (18.3)44.4 (13)Sex – female (%)41 (54)29 (42)Side of Injury – right (%)32 (42)26 (38)Hand dominance – right (%)63 (83)56 (81)Work status – employed (%)56 (74)52 (75)Injury to randomization – days, mean (SD)3.7 (2.1)3.7 (2.1)Injury Fracture Type 13 (distal humerus): Type 21 (proximal radius +/- ulna)11:517:48Injury Dislocation Posterior1212Operation – yes (%)34 (45)31 (45) Open table in a new tab This research was supported by a 2012 Clinical Research Grant from the American Foundation for Surgery of the Hand.

Intra-articular implantation of collagen scaffold carriers is safe in both native and arthrofibrotic rabbit knee joints.

ObjectivesSustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated in vivo animal model of knee arthrofibrosis.Materials and MethodsA total of 96 rabbits were randomly and equally assigned to four different groups: arthrotomy alone; arthrotomy and collagen scaffold placement; contracture surgery; and contracture surgery and collagen scaffold placement. Animals were killed in equal numbers at 72 hours, two weeks, eight weeks, and 24 weeks. Joint contracture was measured, and cartilage and synovial samples underwent histological analysis.ResultsAnimals that underwent arthrotomy had equivalent joint contractures regardless of scaffold implantation (-13.9° versus -10.9°, equivalence limit 15°). Animals that underwent surgery to induce contracture did not demonstrate equivalent joint contractures with (41.8°) or without (53.9°) collagen scaffold implantation. Chondral damage occurred in similar rates with (11 of 48) and without (nine of 48) scaffold implantation. No significant difference in synovitis was noted between groups. Absorption of the collagen scaffold occurred within eight weeks in all animalsConclusionOur data suggest that intra-articular implantation of a collagen sponge does not induce synovitis or cartilage damage. Implantation in a native joint does not seem to induce contracture. Implantation of the collagen sponge in a rabbit knee model of contracture may decrease the severity of the contracture.Cite this article: J. A. Walker, T. J. Ewald, E. Lewallen, A. Van Wijnen, A. D. Hanssen, B. F. Morrey, M. E. Morrey, M. P. Abdel, J. Sanchez-Sotelo. Intra-articular implantation of collagen scaffold carriers is safe in both native and arthrofibrotic rabbit knee joints. Bone Joint Res 2016;6:162–171. DOI: 10.1302/2046-3758.63.BJR-2016-0193.

Changes in genetic expression in a rabbit knee model of posttraumatic extrinsic contracture

Objective To investigate the changes in genetic expression in a rabbit knee model of posttraumatic extrinsic contracture. Methods A model of posttraumatic extrinsic contracture was created in rabbit knees to study the differentially expressed genes. The left knees of 3 mature New Zealand rabbits (12 to 18 months old) were operated on to result in stable intra-articular fractures which were then immobilized as the trauma group while the right knees without any operation served as the control group. The gene expression profiles of joint capsule were studied with Agilent rabbit whole genome microarray technology. Differentially expressed genes were screened by comparing gene expression profiles between the trauma and control groups. Among those differentially expressed genes, matrix metalloproteinase 2 (MMP2), Actin alpha 2 (Acta2), and Keratinocyte growth factor (KGF), related to fibrosis, were selected for verification by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) analysis. Results When compared to normal capsules, 90 differentially expressed genes were screened out in the contracted capsules. Of them, 21 were up-regulated and 69 down-regulated. These genes were associated with regulation of response to external stimulus, regulation of lipopolysaccharide-mediated signaling pathway, regulation of response to biotic stimu-lus, et al. The real-time RT-PCR testing showed the MMP2, Acta2 and KGF genes were all significantly up-regulated compared to the control group (P< 0.05), consistent with the results of the microarray. Conclusions There exist differentially expressed genes in the joint capsules in a rabbit knee model of posttraumatic joint extrinsic contracture compared with normal ones. Posttraumatic extrinsic contracture may be associated with MMP2 and Acta2. The function of KGF genes in human joint contracture needs further re-search. Key words: Gene expression; Stifle; Arthrogryposis; Rabbits

Development &amp; performance assessment of a new ATMP for cartilage tissue engineering

Event Abstract Back to Event Development & performance assessment of a new ATMP for cartilage tissue engineering Alain Morais1, 2, Cristina Correia3, Carlos A. Vilela1, 2, Ana C. Gertrudes3, David Learmonth3, Joaquim M. Oliveira1, 2, Rui A. Sousa3 and Rui L. Reis1, 2, 3 1 University of Minho, 3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, Portugal 2 University of Minho, ICVS/3B’s—PT Government Associate Laboratory, Portugal 3 Stemmatters SA, Portugal Introduction: Joint disorders are responsible of reduced quality of life and high socio-economic burden in our progressively ageing and physically active population. Even though huge efforts on the development of cartilage repair strategies, presently no approach has demonstrate full efficiency. The present study aimed to create an innovative advanced-therapy medicinal product (ATMP) where human adipose-derived stem/stromal cells (hASCs)[1] encapsulated within a novel hydrogel based on methacrylated gellan gum (mimsys G)[2] will develop cartilage-like tissue. These complete xeno-free 3D approach was developed and in vitro experiment were conducted under static and dynamic conditions, the latest using a rotational dual chamber bioreactor[3]. Preliminary in vivo assay using rabbit model of cartilage defect was based on autologous and allogenic approaches. Both in vitro and in vivo experiments were established to evaluate the performance of this system on focal cartilage tissue engineering. Materials and Methods: Human ASC (hASC xeno-free, irisbiosciences), isolated in GMP conditions, were expanded in xeno-free media until sub-confluency. mimsys G hydrogel (mimsys®G, irisbiosciences) was prepared to yield a 2% w/V solution. Cells were encapsulated at 10x106 cells/mL, ionically crosslinked and cultured with or without chondrogenic induction for 21 days. For dynamic growth, a rotational dual chamber bioreactor was used to control the perfusion and flow gradient within the cellular scaffolds. Cell viability was determined by live/dead assay, and cell metabolic activity by MTS reduction. A in vivo study evaluated regeneration of focal cartilage lesions on a rabbit knee model for 8 weeks, using autologous and allogenic approaches. Rabbit ASCs (rASCs), isolated from fat pad, and hASCs were encapsulated into mimsys G prior administration into the cartilage defect. Chondrogenesis was assessed on both in vitro and in vivo samples by histological staining of cartilage matrix using safranin O and immunolocalization of collagen type II. Expression of chondrogenic-related genes (aggrecan, collagen type II and sox-9) were also determined. Results: In vitro analysis of the new ATMP demonstrated high viability of the cells and progressive metabolic activity along static and dynamic culture. Additionally, intense cartilage matrix deposition was detected through up-regulation of chondrogenic maker Collagen II gene and protein (Immunohistochemistry) by end of culture (Fig.1). For the in vivo study, both autologous and allogenic approaches demonstrated the establishment of lateral and subchondral integration (Fig.2). Fig. 1 Fig. 2 Fig.1. In vitro Chondrogenesis – Collagen II gene (A) and protein (B) expression at day 0 and day 21, after chondrogenic induction in static culture conditions. Fig 2. In vivo study – Safranin-O staining after regeneration of cartilage lesion. Discussion and Conclusions: The proposed innovative ATMP demonstrated intense cartilage development in vitro through evaluation of specific markers. Preliminary in vivo data demonstrates promising repair performance in focal cartilage lesions, suggesting a valuable approach for further therapeutic exploitation. The authors thank the financial support under NORTE-07-0202-FEDER-023189 / ARTICULATE - Desenvolvimento de novos produtos e terapias regenerativas para o tratamento de patologias articulares / ADI

Synovial Stem Cells in Musculoskeletal Regeneration

The synovium is a thin layer of connective tissue that lines the joint surface, tendon sheaths, and bursae at freely moving articulations in the body. Embryologic origin of the synovial tissue is the mesenchymal layer, which also gives rise to bone, cartilage, ligament, and muscle tissue. The synovium has several functions, including lubrication of the articulating surfaces, nutrition of articular cartilage, and regulation of immune response within the joint. Synovial Cells The synovium contains two main cell types: type A and type B synoviocytes. Type A synoviocytes are tissue macrophages and have phagocytic functions. Type B synoviocytes are fibroblast-like cells and function in the formation of synovial fluid.1 In 2001, De Bari et al2 isolated mesenchymal stem cells (MSCs) from the synovium (Figure 1). Type A synoviocytes can be characterized and eliminated from mixed synovial cell populations through a selective culturing process. However, type B synoviocytes and synovial-MSCs have similar phenotypic features, and specific characteristics to clearly differentiate these two cell types from each other have not been determined yet.1Figure 1: Histologic appearance of human synovial mesenchymal stem cells under the phase contrast microscope 14 days after initial plating.In terms of immune phenotype, there are many similarities between synovial-MSCs and MSCs of other origins. Both cell types are positive for surface markers such as CD44, CD90, and CD105. However, cells derived from synovium, including synovial-MSCs, have higher expression of CD44 (a hyaluronan receptor) and can express uridine diphosphoglucose dehydrogenase, which is a vital enzyme involved in hyaluronan synthesis.3 In vitro studies have shown that synovial-MSCs have superior potential to differentiate into chondrocytes and to produce cartilage compared with MSCs of other origins.4 Moreover, synovial-MSCs have greater proliferation and colony-forming capacity than do other stem cell sources.4 Synovial-MSCs and Cartilage Regeneration Based on the promising results from in vitro studies, investigators have launched animal model studies to evaluate the effects of synovial-MSCs in vivo. In a rabbit model with full-thickness articular cartilage defect, Koga et al5 demonstrated that local transplantation of synovial-MSCs results in extensive cartilage matrix formation at the defect site. These authors also observed that in the deeper zone of the defect, synovial-MSCs differentiated into bone cells, whereas synovial-MSCs at the superficial zones differentiated into chondrocytes. This observation supported the multilineage differentiation potential of synovial-MSCs according to local microenvironments in vivo. In a pig model, transplantation of synovial-MSCs into a full-thickness articular cartilage defect promoted cartilage regeneration based on arthroscopic, MRI, and histologic analysis as early as 3 months after the procedure.6 Bilge et al7 used a rabbit knee model as an in vivo culture medium to evaluate the effects of synovium on chondrocyte growth. These authors observed that cartilage grafts that are in direct contact with the synovium produce more chondrocytes compared with cartilage grafts that are not. Studies of animal meniscal defect models demonstrated that transplanted synovial-MSCs adhere to sites of meniscal injury, differentiate into cells resembling meniscal fibrochondrocytes, and enhance meniscal regeneration.8,9 Sporadic human studies have reported that the number of synovial-MSCs in synovial fluid increases in knees with degenerated cartilage and osteoarthritis and following intraarticular ligament injury.10,11 This observation raises the question whether the number of synovial-MSCs that are mobilized from synovium into synovial fluid increases according to the degree of cartilage degeneration as part of the reparative process. Human trials investigating the effects of intra-articular synovial-MSC transplantation to promote cartilage regeneration and/or to prevent osteoarthritis should answer this question. Application of Synovial-MSCs for Bone, Tendon, and Muscle Regeneration Synovial-MSCs may offer an alternative cell-based treatment strategy for bone, tendon, and muscle regeneration. In a rabbit bone defect model, Matsusaki et al12 demonstrated that using tissue-engineered construct derived from synovial-MSCs with hydroxyapatite accelerates osteoinduction. In a rat Achilles tendon graft model, synovial-MSC implantation into bone tunnel accelerated early remodeling of tendon-to-bone healing.13 Another rat model study showed that synovial-MSCs have myogenic potential and contribute to skeletal muscle regeneration in vivo.14 In spite of these reports, there is not sufficient evidence regarding osteogenic and myogenic potential of synovial-MSCs compared with bone marrow- and muscle-derived MSCs. Future Perspectives In vitro and animal model studies support the use of synovial-MSCs in cartilage regeneration as an important, arguably superior, cell-based treatment alternative. Further investigations in the near future should help in our understanding the complexity of synovial-MSC biology in terms of isolation, characterization, culturing, distinguishing from, and interacting with other cell types before this promising cell-based therapy can be translated into clinical practice.

Carrageenan-induced transient inflammation in a rabbit knee model: molecular changes consistent with an early osteoarthritis phenotype

Inflammation following a knee injury is one of the factors associated with initiation of cartilage degeneration leading to osteoarthritis (OA). The hypothesis tested was that inflammation results in elevated expression of proteinases implicated in OA. Mature female rabbits received a single carrageenan injection to the right hind knee and the left knee served as the control. Five animals were killed at time points of 1, 2 and 4 weeks. The synovium and cartilage from both knees were collected and analysed for specific mRNA levels. Interleukin (IL)-1β and IL-6 mRNA levels peaked at 2 weeks and returned to normal levels in tissues by 4 weeks post-carrageenan treatment. Matrix metalloproteinase (MMP)-13, MMP-1, MMP-3 and cathepsin K followed the trend set by the inflammatory cytokines. Both synovium and cartilage tissues exhibited similar patterns of molecular expression, with cartilage from the tibial plateau responding more strongly than the femoral condyles. The acute inflammatory milieu controls the transient expression of many degradative proteinases in the knee. However, a single acute exposure to inflammation in the rabbit knee is insufficient to create a chronic inflammatory environment and other complementary factors, such as persistent mechanical instability and/or injury, may contribute to the establishment of OA.

Modulation of the intramedullary pressure responses by calcium dobesilate in a rabbit knee model of osteoarthritis

Background and purpose The presence of bone marrow edema in patients with osteoarthritis is associated with pain and disease progression. Management of bone edema with the synthetic prostacyclin iloprost may be complicated by side effects. Calcium dobesilate, a treatment for chronic venous disease, shares some pharmacological actions with iloprost but appears to be better tolerated. Anecdotal reports have suggested that calcium dobesilate may be useful for medical management of osteoarthritis, possibly by reducing bone marrow edema, and this study was performed to investigate possible benefits of treatment.Methods The effects of a 6-week period of oral calcium dobesilate administration on tibial intramedullary pressure dynamics and physical joint characteristics were evaluated in 20 rabbits with unilaterally induced knee osteoarthritis that were randomly allocated to either a treatment group or a placebo control group. Treatment or placebo started 8 weeks after induction of osteoarthritis, and was followed by a 4-week washout period.Results Calcium dobesilate did not affect joint thickness or range of motion, nor individual pressure measurements, compared to placebo. Pressure ranges in the operated limb were greater than in the intact limb after 8 weeks, and approached those of the intact limb after 6 weeks of treatment with calcium dobesilate but not with placebo. Inter-limb differences were lower (p = 0.02) in the dobesilate group following the washout period.Interpretation Calcium dobesilate had a detectable effect on pressure dynamics in the subchondral bone of osteoarthritic joints in this model. The significance of these effects for pain and function should be established.