Platelet-rich fibrin matrix (PRFM) has been proved to enhance tenocyte proliferation but has mixed results when used during rotator cuff repair. The optimal PRFM preparation protocol should be determined before clinical application. To screen the best PRFM to each individual’s tenocytes effectively, small-diameter culture wells should be used to increase variables. The gelling effect of PRFM will occur when small-diameter culture wells are used. A co-culture device should be designed to avoid this effect. Tenocytes harvested during rotator cuff repair and blood from a healthy volunteer were used. Tenocytes were seeded in 96-, 24-, 12-, and six-well plates and co-culture devices. Appropriate volumes of PRFM, according to the surface area of each culture well, were treated with tenocytes for seven days. The co-culture device was designed to avoid the gelling effect that occurred in the small-diameter culture well. Cell proliferation was analyzed by water soluble tetrazolium-1 (WST-1) bioassay.Objectives
Methods
This study aimed to explore the role of miR-320a in the pathogenesis of osteoarthritis (OA). Human cartilage cells (C28/I2) were transfected with miR-320a or antisense oligonucleotides (ASO)-miR-320a, and treated with IL-1β. Subsequently the expression of collagen type II alpha 1 (Col2α1) and aggrecan (ACAN), and the concentrations of sulfated glycosaminoglycans (sGAG) and matrix metallopeptidase 13 (MMP-13), were assessed. Luciferase reporter assay, qRT-PCR, and Western blot were performed to explore whether pre-B-cell leukemia Homeobox 3 (PBX3) was a target of miR-320a. Furthermore, cells were co-transfected with miR-320a and PBX3 expressing vector, or cells were transfected with miR-320a and treated with a nuclear factor kappa B (NF-κB) antagonist MG132. The changes in Col2α1 and ACAN expression, and in sGAG and MMP-13 concentrations, were measured again. Statistical comparisons were made between two groups by using the two-tailed paired Objectives
Methods
Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1.Objectives
Materials and Methods
The patellofemoral joint is an important source of symptoms in osteoarthritis of the knee. We have used a newly designed surgical model of patellar strengthening to induce osteoarthritis in BALB/c mice and to establish markers by investigating the relationship between osteoarthritis and synovial levels of matrix metalloproteinases (MMPs). Osteoarthritis was induced by using this microsurgical technique under direct vision without involving the cavity of the knee. Degeneration of cartilage was assessed by the Mankin score and synovial tissue was used to determine the mRNA expression levels of MMPs. Irrigation fluid from the knee was used to measure the concentrations of MMP-3 and MMP-9. Analysis of cartilage degeneration was correlated with the levels of expression of MMP. After operation the patellofemoral joint showed evidence of mild osteoarthritis at eight weeks and further degenerative changes by 12 weeks. The level of synovial MMP-9 mRNA correlated with the Mankin score at eight weeks, but not at 12 weeks. The levels of MMP-2, MMP-3 and MMP-14 mRNA correlated with the Mankin score at 12 weeks. An increase in MMP-3 was observed from four weeks up to 16 weeks. MMP-9 was notably increased at eight weeks, but the concentration at 16 weeks had decreased to the level observed at four weeks. Our observations suggest that MMP-2, MMP-3 and MMP-14 could be used as markers of the progression of osteoarthritic change.
We investigated the antibiotic concentration in fresh-frozen femoral head allografts harvested from two groups of living donors. Ten samples were collected from patients with osteoarthritis of the hip and ten from those with a fracture of the neck of the femur scheduled for primary arthroplasty. Cefazolin (1 g) was administered as a pre-operative prophylactic antibiotic. After storage at −80°C for two weeks the pattern of release of cefazolin from morsellised femoral heads was evaluated by an We concluded that allografts of morsellised bone from the femoral head harvested from patients undergoing arthroplasty of the hip contained cefazolin, which had been administered pre-operatively and they exhibited inhibitory effects against bacteria
In a rabbit model we investigated the efficacy of a silk fibroin/hydroxyapatite (SF/HA) composite on the repair of a segmental bone defect. Four types of porous SF/HA composites (SF/HA-1, SF/HA-2, SF/HA-3, SF/HA-4) with different material ratios, pore sizes, porosity and additives were implanted subcutaneously into Sprague-Dawley rats to observe biodegradation. SF/HA-3, which had characteristics more suitable for a bone substitite based on strength and resorption was selected as a scaffold and co-cultured with rabbit bone-marrow stromal cells (BMSCs). A segmental bone defect was created in the rabbit radius. The animals were randomised into group 1 (SF/HA-3 combined with BMSCs implanted into the bone defect), group 2 (SF/HA implanted alone) and group 3 (nothing implanted). They were killed at four, eight and 12 weeks for visual, radiological and histological study. The bone defects had complete union for group 1 and partial union in group 2, 12 weeks after operation. There was no formation of new bone in group 3. We conclude that SF/HA-3 combined with BMSCs supports bone healing and offers potential as a bone-graft substitute.
In an osteological collection of 3100 specimens, 70 were found with unilateral clavicular fractures which were matched with 70 randomly selected normal specimens. This formed the basis of a study of the incidence of arthritis of the acromioclavicular joint and the effect of clavicular fracture on the development of arthritis in the ipsilateral acromioclavicular joint. This was graded visually on a severity scale of 0 to 3. The incidence of moderate to severe arthritis of the acromioclavicular joint in normal specimens was 77% (100 specimens). In those with a clavicular fracture, 66 of 70 (94%) had arthritis of the acromioclavicular joint, compared to 63 of 70 (90%) on the non-injured contralateral side (p = 0.35). Clavicles with shortening of 15 mm or less had no difference in the incidence of arthritis compared to those with shortening greater than 15 mm (p = 0.25). The location of the fracture had no effect on the development of arthritis.
Several experimental models have been used to produce intravascular fat embolism. We have developed a simple technique to induce fat embolism using corn oil emulsified with distilled water to form fatty micelles. Fat embolism was produced by intravenous administration of these fatty micelles in anaesthetised rats, causing alveolar oedema, haemorrhage and increased lung weight. Histopathological examination revealed fatty droplets and fibrin thrombi in the lung, kidney and brain. The arteriolar lumen was filled with fatty deposits. Following fat embolism, hypoxia and hypercapnia occurred. The plasma phospholipase A2, nitrate/nitrite, methylguidanidine and proinflammatory cytokines were significantly increased. Mass spectrometry showed that the main ingredient of corn oil was oleic acid. This simple technique may be applied as a new animal model for the investigation of the mechanisms involved in the fat embolism syndrome.
Ovine articular chondrocytes were isolated from cartilage biopsy and culture expanded All defects were assessed using the International Cartilage Repair Society (ICRS) classification. Those treated with ACFC, ACI and AF exhibited median scores which correspond to a nearly-normal appearance. On the basis of the modified O’Driscoll histological scoring scale, ACFC implantation significantly enhanced cartilage repair compared to ACI and AF. Using scanning electron microscopy, ACFC and ACI showed characteristic organisation of chondrocytes and matrices, which were relatively similar to the surrounding adjacent cartilage. Implantation of ACFC resulted in superior hyaline-like cartilage regeneration when compared with ACI. If this result is applicable to humans, a better outcome would be obtained than by using conventional ACI.
To clarify the pathomechanisms of discogenic low back pain, the sympathetic afferent discharge originating from the L5-L6 disc via the L2 root were investigated neurophysiologically in 31 Lewis rats. Sympathetic afferent units were recorded from the L2 root connected to the lumbar sympathetic trunk by rami communicantes. The L5-L6 discs were mechanically probed, stimulated electrically to evoke action potentials and, finally, treated with chemicals to produce an inflammatory reaction. We could not obtain a response from any units in the L5-L6 discs using mechanical stimulation, but with electrical stimulation we identified 42 units consisting mostly of A-delta fibres. In some experiments a response to mechanical probing of the L5-L6 disc was recognised after producing an inflammatory reaction. This study suggests that mechanical stimulation of the lumbar discs may not always produce pain, whereas inflammatory changes may cause the disc to become sensitive to mechanical stimuli, resulting in nociceptive information being transmitted as discogenic low back pain to the spinal cord through the lumbar sympathetic trunk. This may partly explain the variation in human symptoms of degenerate discs.
We have compared the concentrations of stromal-cell-derived factor-1 (SDF-1), matrix metalloproteinase-1 (MMP-1), MMP-9 and MMP-13 in serum before and after synovectomy or total knee replacement (TKR). We confirmed the presence of SDF-1 and its receptor CXCR4 in the synovium and articular cartilage by immunohistochemistry. We established chondrocytes by using mutant CXCR4 to block the release of MMPs. The level of SDF-1 was decreased 5.1- and 6.7-fold in the serum of patients with OA and RA respectively, after synovectomy compared with that before surgery. MMP-9 and MMP-13 were decreased in patients with OA and RA after synovectomy. We detected SDF-1 in the synovium and the bone marrow but not in cartilage. CXCR4 was detected in articular cartilage. SDF-1 increased the release of MMP-9 and MMP-13 from chondrocytes in a dose-dependent manner. The mutant CXCR4 blocked the release of MMP-9 and MMP-13 from chondrocytes by retrovirus vector. Synovectomy is effective in patients with OA or RA because SDF-1, which can regulate the release of MMP-9 and MMP-13 from articular chondrocytes for breakdown of cartilage, is removed by the operation.
Extracorporeal shock-wave (ESW) treatment hasbeen shown to be effective in promoting the healing of fractures. We aimed to determine whether ESW could enhance the growth of bone-marrow osteoprogenitor cells. We applied ESW to the left femur of rats 10 mm above the knee at 0.16 mJ/mm2 in a range of between 250 and 2000 impulses. Bone-marrow cells were harvested after ESW for one day and subjected to assessment of colony-forming unit (CFU) granulocytes, monocytes, erythocytes, megakaryocytes (CFU-Mix), CFU-stromal cells (CFU-S) and CFU-osteoprogenitors (CFU-O). We found that the mean value for the CFU-O colonies after treatment with 500 impulses of ESW was 168.2 CFU-O/well ( Our findings suggest that optimal treatment with ESW could enhance rat bone-marrow stromal growth and differentiation towards osteoprogenitors presumably by induction of TGF-β1.
