A Combination of Surgical and Chemical Induction in a Rabbit Model for Osteoarthritis of the Knee.

BACKGROUND: Appropriate animal models of osteoarthritis (OA) are essential to develop new treatment modalities for OA. A combination of surgical and chemical induction could be appropriate for OA models. METHODS: Rabbit knee OA models developed by surgical induction (anterior cruciate ligament transection [ACLT]), chemical induction (monosodium iodoacetate [MIA] injection), and a combination of both were compared to assess compositional and structural destruction of the knee joint. Twenty-one New Zealand white rabbits were randomly divided into 3 groups to induce OA (group 1: ACLT, n = 3; group 2: MIA [3, 6, 9 mg] injection, n = 9; group 3: ACLT + MIA [3, 6, 9 mg] injection, n = 9). RESULTS: In all groups, the Modified Mankin score was significantly higher in the osteoarthritis-induced knee than in the control. Modified Mankin scores were compared by category. The ACLT group was observed to score high in cartilage structure. In the MIA group, chondrocytes and matrix staining showed higher scores, and the ACLT+MIA group scored higher in all categories for cartilage structure, chondrocytes, matrix staining, and tidemark integrity. The ACLT + 3 mg MIA showed definite OA characteristics such as cartilage surface destruction and degeneration of cartilage layers, and the ACLT + 6 mg MIA and ACLT + 9 mg MIA showed more prominent OA characteristics such as cartilage surface destruction, matrix disorganization, and osteophyte formation. CONCLUSION: The combination of MIA injection and ACLT could be an appropriate method for OA induction in rabbit models.

Soluble CCR2 gene therapy controls joint inflammation, cartilage damage, and the progression of osteoarthritis by targeting MCP-1 in a monosodium iodoacetate (MIA)-induced OA rat model.

BACKGROUND: Osteoarthritis (OA) is the most common type of degenerative arthritis and affects the entire joint, causing pain, joint inflammation, and cartilage damage. Various risk factors are implicated in causing OA, and in recent years, a lot of research and interest have been directed toward chronic low-grade inflammation in OA. Monocyte chemoattractant protein-1 (MCP-1; also called CCL2) acts through C-C chemokine receptor type 2 (CCR2) in monocytes and is a chemotactic factor of monocytes that plays an important role in the initiation of inflammation. The targeting of CCL2-CCR2 is being studied as part of various topics including the treatment of OA. METHODS: In this study, we evaluated the potential therapeutic effects the sCCR2 E3 gene may exert on OA. The effects of sCCR2 E3 were investigated in animal experiments consisting of intra-articular injection of sCCR2 E3 in a monosodium iodoacetate (MIA)-induced OA rat model. The effects after intra-articular injection of sCCR2 E3 (fusion protein encoding 20 amino acids of the E3 domain of the CCL2 receptor) in a monosodium iodoacetate-induced OA rat model were compared to those in rats treated with empty vector (mock treatment) and full-length sCCR2. RESULTS: Pain improved with expression of the sCCR2 gene. Improved bone resorption upon sCCR2 E3 gene activation was confirmed via bone analyses using micro-computed tomography. Histologic analyses showed that the sCCR2 E3 gene exerted protective effects against cartilage damage and anti-inflammatory effects on joints and the intestine. CONCLUSIONS: These results show that sCCR2 E3 therapy is effective in reducing pain severity, inhibiting cartilage destruction, and suppressing intestinal damage and inflammation. Thus, sCCR2 E3 may be a potential therapy for OA.

Characterization of wild-type and STAT3 signaling-suppressed mesenchymal stem cells obtained from hemovac blood concentrates.

BACKGROUND: Venous blood drained from the knee joint after total knee arthroplasty (TKA) using a hemovac line is a potential source of bone marrow components, including stem cells, from the cutting surface of cancellous bones of the knee joint. However, the function of mesenchymal stem cells (MSCs) in patients with osteoarthritis (OA-MSCs) can be disrupted by inflammation of the joint. Further, to override the invasive nature of the currently used methods to obtain stem cells, their functional modification is necessary for therapeutic applications. METHODS: The effects of signal transducer and activator of transcription 3 (STAT3) signaling suppression on MSCs (iSTAT3-MSCs) were evaluated by comparative analyses of the characteristics of OA-MSCs and iSTAT3-MSCs from 20 patients who underwent TKA. RESULTS: OA-MSCs and iSTAT3-MSCs were adherent, with fibroblast-like appearance and high rates of expression of MSC-specific markers, including CD73, CD90, and CD105 (>90%). Both OA-MSCs and iSTAT3-MSCs were able to differentiate into osteogenic, adipogenic, and chondrogenic cells; however, iSTAT3-MSCs showed higher levels of osteogenic and chondrogenic differentiation markers than OA-MSCs. Additionally, the anti-inflammatory and chondroprotective cytokine levels were higher in iSTAT3-MSCs than in OA-MSCs. CONCLUSIONS: These findings indicate that iSTAT3-MSCs after TKA are potentially effective for stem cell therapy in the context of bone and cartilage disorders.

Improved Healing of Rabbit Patellar Tendon Defects After an Atelocollagen Injection.

BACKGROUND: Patellar tendinopathy is a common cause of limitations in daily life activities in young and/or active people. The patellar tendon consists of a complex of collagen fibers; therefore, collagen could be used as a scaffold in the treatment of patellar tendinopathy. PURPOSE: To evaluate the healing capacity of injected atelocollagen as a treatment scaffold for patellar tendon defect and, hence, its potential for the treatment of patellar tendinopathy. STUDY DESIGN: Controlled laboratory study. METHODS: After receiving a full-thickness patellar tendon defect, 24 New Zealand White rabbits were divided into a control group (without treatment) and an experimental group that received an atelocollagen injection into the defect. Six rabbits from each group were subsequently used for either histologic scoring or biomechanical testing. The Mann-Whitney U test was used to compare histologic evaluation scores and load to failure between the 2 groups. Statistical significance was set at P < .05. RESULTS: The experimental group showed excellent repair of the damaged patellar tendon and good remodeling of the defective area. In contrast, the control group showed defective healing with loose, irregular matrix fibers and adipose tissue formation. A statistically significant difference was found between the 2 groups in both histologic scores and biomechanical tests at postoperative week 12. CONCLUSION: Injection of atelocollagen significantly improved the regeneration of damaged patellar tendons. CLINICAL RELEVANCE: Atelocollagen gel injections could be used to treat patellar tendinopathy in outpatient clinic settings.

Hemovac blood after total knee arthroplasty as a source of stem cells.

BACKGROUND: With increasing life expectancy, stem cell therapy is receiving increasing attention. However, its application is restricted by ethical concerns. Hence a need exists for design of safe procedures for stem cell procurement. Here, we investigated whether hemovac blood (HVB) is an appropriate stem cell source. METHODS: HVB concentrates (HVBCs) from 20 total knee arthroplasty (TKA) patients and bone marrow aspirate (BMA) concentrates (BMACs) from 15 patients who underwent knee cartilage repair were comparatively evaluated. A bone marrow aspiration needle was inserted into the anterior superior iliac spine. Aspiration was performed using a 50-mL syringe, including 4 mL of anticoagulant, followed by centrifugation to obtain BMACs. To obtain HVBCs, blood was aspirated from the hemovac immediately after TKA surgery. Different cell types were enumerated. Isolation of BMA and HVB mononuclear cells was performed using density gradient centrifugation. Non-hematopoietic fibroblast colonies were quantified by colony forming unit-fibroblast assay surface marker analysis of HVB, HVBC, BMA, and BMAC was performed via flow cytometry. Mesenchymal stem cells (MSCs) isolated from HVBCs and BMACs were examined for osteogenic, adipogenic, and chondrogenic differentiation potential. Gene expression analysis was performed by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The number of cells from HVB and HVBC was significantly lower than from BMA and BMAC; however, the number of colonies in HVBC and BMAC did not differ significantly (P>0.05). Isolated cells from both sources had a fibroblast-like appearance, adhered to culture flasks, and formed colonies. Under different culture conditions, MSC-specific surface markers (CD29, CD44, CD90, CD105), osteogenic markers [RUNX2, osteopontin, osteocalcin, and alkaline phosphatase (ALP)] and adipogenic markers (PPARγ and C/EBPα) were expressed. Moreover, SOX9, type II collagen, and aggrecan were significantly upregulated upon chondrogenic differentiation. CONCLUSIONS: HVB from TKA patients is a useful source of stem cells for research.

Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells.

Effective engineering approaches for cartilage regeneration involve a combination of cells and biomaterial scaffolds. Multipotent mesenchymal stem cells (MSCs) are important sources for cartilage regeneration. Atelocollagen provides a suitable substrate for MSC attachment and enhancing chondrogenic differentiation. Here, we assessed the chondrogenic potential of adipose tissue derived human MSCs (hMSCs) mixed with atelocollagen gel. We observed cell attachment, viability, and microstructures by electron microscopy over 21 days. The levels of Sox9, type II collagen, aggrecan, type I collagen, Runx2, type X collagen, ALP, Osterix, and MMP13 were measured by RT-qPCR. Cartilage matrix-related proteins were assessed by enzyme-linked immunosorbent assay (ELISA), histology, and immunohistochemistry. hMSCs of all groups exhibited well-maintained cell survival, distribution and morphology. Abundant type II collagen fibers developed on day 21; while Sox9, type II collagen, and aggrecan expression increased over time in the atelocollagen group. However, type I collagen, RUNX2, type X collagen (CoL10A1), Osterix, and ALP were not expressed. These results corroborated the protein expression detected by ELISA. Further, histological analysis revealed lacunae-like structures, while staining demonstrated glycosaminoglycan accumulation. Cumulatively, these results indicate that atelocollagen scaffolds improve hMSC chondrogenic differentiation and are a potential approach for cartilage regeneration.

Effect of java citronella essential oil addition on the physicochemical properties of Gelidium corneum-chitosan composite films.

A new composite film was developed by combining Gelidium corneum (GC) with chitosan to enhance the physicochemical characteristics of GC film. In addition, to confer new functional property on the GC-chitosan composite film, various amounts (0.5%, 1.0%, and 1.5%) of java citronella essential oil (JCEO) were incorporated into the film. As the concentration of JCEO increased, the extensibility of the GC-chitosan film improved. In addition, the film became more opaque due to decreased light transmission. Especially, ultraviolet light was completely blocked in the composite films containing JCEO. Radical scavenging activities of the films also increased with increasing JCEO content, indicating that the films have antioxidant activity. Therefore, GC-chitosan composite film containing JCEO is applicable in food packaging to preserve food quality by retarding lipid oxidation.

The Therapeutic Effect of STAT3 Signaling-Suppressed MSC on Pain and Articular Cartilage Damage in a Rat Model of Monosodium Iodoacetate-Induced Osteoarthritis.

Osteoarthritis (OA) is a degenerative disease that induces pain, cartilage deformation, and joint inflammation. Mesenchymal stem cells (MSCs) are potential therapeutic agents for treatment of OA. However, MSC therapy can cause excessive inflammation. Signal transducer and activator of transcription 3 (STAT3) modulates secretion of many proinflammatory cytokines. Experimental OA was induced by intra-articular (IA) injection of monosodium iodoacetate (MIA) to the right knee of rats. MSCs from OA patients (OA-MSCs) were treated with STA21, a small molecule that blocks STAT3 signaling, by IA or intravenous (IV) injection after MIA injection. Pain severity was quantified by assessment of secondary tactile allodynia using the von Frey assessment test. Cartilage degradation was measured by microcomputed tomography image analysis, histological analysis, and the Mankin score. Protein and gene expression was evaluated by enzyme-linked immunosorbent assay, immunohistochemistry, and real-time polymerase chain reaction. MSCs increased production of proinflammatory cytokines under inflammatory conditions. STA21 significantly decreased expression of these proinflammatory molecules via inhibition of STAT3 activity but increased gene expression of molecules related to migration potential and immunomodulation in OA-MSCs. STAT3-inhibited OA-MSCs administrated by IV or IA injection decreased pain severity and cartilage damage in rats with MIA-induced OA rats by decreasing proinflammatory cytokines in the joints. Combined IA and IV-injected STAT3-inhibited OA-MSCs had an additive effect of pain relief in MIA-induced OA rats. STAT3 inhibition may optimize the therapeutic activities of MSCs for treating OA by attenuating pain and progression of MIA by inhibiting inflammation and cartilage damage.

In vitro cellular uptake of fibroin microspheres and its dependency on the cell cycle stage.

Cellular uptake of a protein microsphere was investigated in vitro. Fibroin microspheres with an average diameter of approximately 1 µm were prepared with Fluorescein isothiocyanate-dextran for the fluorescent core. Tomography, performed using confocal laser scanning microscopy, confirmed microsphere uptake into the cytoplasmic area of 3T3 cells. Flow cytometry showed that cellular uptake was proportional to the co-incubation time and the microsphere concentration. It also revealed that fibroin microspheres were internalised by 3T3 cells at different rates depending on the cell cycle stage. The following cell stages had higher concentrations of internalised microspheres: G(2)/M > S > G(0)/G(1), when using 0.1 mg of microspheres per 10(6) cells. The internalised microspheres per cell also increased in the same order of cell cycle stages when co-incubating cells with 1 mg of microspheres. These results provide information that can be used to develop fibroin microspheres for intracellular delivery of large cargos.