Smoking delays chondrogenesis in a mouse model of closed tibial fracture healing.

Smoking delays the healing process and increases morbidity associated with many common musculoskeletal disorders, including long bone fracture. In the current study, a murine model of tibial fracture healing was used to test the hypothesis that smoking delays chondrogenesis after fracture. Mice were divided into two groups, a nonsmoking control group and a group exposed to cigarette smoke for 1 month prior to surgical tibial fracture. Mice were euthanized at 7, 14, and 28 days after surgery. The outcomes measured were immunohistochemical staining for type II collagen protein expression as a marker of cartilage matrix and proliferating cell nuclear antigen (PCNA) staining to measure proliferation at the site of injury. Toluidine blue staining and histomorphometry were used to quantify areas of cartilaginous and noncartilaginous fracture callus. Radiographs were analyzed for evidence of remodeling after injury. At day 7 after injury, mice exposed to cigarette smoke had a smaller fracture callus with less cartilage matrix compared to controls. Proliferation was present at high levels in both groups at this time point, but proliferating cells had a more immature morphology in the smoking group. At day 14, chondrogenesis was more active in smokers compared to controls, while a higher percentage of bone was present in the control animals. At day 28, X-ray analysis revealed a larger fracture callus remaining in the smoking animals. Together, these findings show that the chondrogenic phase of tibial fracture healing is delayed by smoking. This study represents, to our knowledge, the first analysis of molecular and cellular mechanisms of healing in a smoking mouse fracture model.     

Chondromyxoid fibroma resembles in vitro chondrogenesis, but differs in expression of signalling molecules

Chondromyxoid fibroma is a rare benign cartilaginous bone tumour characterized by morphological features that resemble different steps of chondrogenesis in terms of both cellular morphology, ranging from spindled to rounded cells, and the extracellular matrix formed, which ranges from fibrous to cartilaginous. The presence in chondromyxoid fibroma of signalling molecules that regulate the spatial expression of proteins involved in normal cartilage proliferation and differentiation was investigated in samples from 20 patients and compared with articular chondrocytes from 11 normal donors cultivated in 3D pellet culture. Sections were stained with safranin-O and H&E, and immunohistochemistry was performed for p16, cyclin D1, FGFR3, BCL2, p21, PTHLH, PTHR1 and N-cadherin. Expression patterns were analysed using hierarchical clustering. In chondromyxoid fibroma, specific morphological features correlated with a distinct pattern of expression. Comparison with normal chondrocytes in pellet culture showed a striking morphological resemblance, but with an unmistakably different pattern of expression. N-cadherin, PTHLH, and PTHR1 were expressed to a significantly higher level (p < 0.01) in articular chondrocyte pellets but, conversely, there was significantly lower expression of cyclin D1, p16 and BCL2 (p < 0.05) in these cells. Morphological similarities reflect common steps in cartilage differentiation, albeit driven by different molecular mechanisms. The proteins we have found to be differentially expressed seem crucial for neoplastic chondrogenesis.     

Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells

Two FDA-approved agents, ferumoxides (Feridex), a suspension of superparamagnetic iron oxide (SPIO) nanoparticles and protamine sulfate, a drug used to reverse heparin anticoagulation, can be complexed and used to label cells magnetically ex vivo. Labeling stem cells with ferumoxides-protamine sulfate (FePro) complexes allows for non-invasive monitoring by MRI. However, in order for stem cell trials or therapies to be effective, this labeling technique must not inhibit the ability of cells to differentiate. In this study, we examined the effect of FePro labeling on stem cell differentiation. Viability, phenotypic expression and differential capacity of FePro labeled CD34 + hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) were compared with unlabeled control cells. Colony-forming unit (CFU) assays showed that the capacity to differentiate was equivalent for labeled and unlabeled HSC. Furthermore, labeling did not alter expression of surface phenotypic markers (CD34, CD31, CXCR4, CD20, CD3 and CD14) on HSC, as measured by flow cytometry. SDF-1-induced HSC migration and HSC differentiation to dendritic cells were also unaffected by FePro labeling. Both FePro-labeled and unlabeled MSC were cultured in chondrogenesis-inducing conditions. Alcian blue staining for proteoglycans revealed similar chondrogenic differentiation for both FePro-labeled and unlabeled cells. Furthermore, collagen X proteins, indicators of cartilage formation, were detected at similar levels in both labeled and unlabeled cell pellets. Prussian blue staining confirmed that cells in labeled pellets contained iron oxide, whereas cells in unlabeled pellets did not. It is concluded that FePro labeling does not alter the function or differentiation capacity of HSC and MSC. These data increase confidence that MRI studies of FePro-labeled HSC or MSC will provide an accurate representation of in vivo trafficking of unlabeled cells.     

Embryotoxicity of chromium: Distribution in pregnant mice and effects on embryonic cells in vitro

The embryonic and fetal uptake of Na₂ ⁵¹Cr₂O₇(Cr VI) was about 10 times higher than that of ⁵¹CrCl₃(Cr III) when these two were given in the same doses i. v. to pregnant C57BL mice. On day 13 of gestation, embryonic concentrations were 12% (Cr VI) and 0.4% (Cr III) of the maternal serum concentration 1 h after injection to the mother. After injection of Cr(III) radioactivity was not detectable in embryonic structures in early gestation, when autoradiographic techniques were used. In late gestation, administration of both forms of Cr resulted in an accumulation in the calcified areas of the fetal skeleton. The radioactivity after administration of Cr(VI) may represent Cr(III) after reduction in the tissues. When added to chick limb bud mesenchymal cells in vitro, Cr(VI) inhibited chondrogenesis at a concentration of about 0.1 g/ml medium, which is around 1/10 of the embryonic concentration achieved after giving teratogenic doses to pregnant mice. Cr(III) on the other hand did not show any overt cytotoxicity even at 15 g/ml ( 500 times higher than the in vivo embryonic concentrations after teratogenic doses). Especially Cr(III) accumulated strongly in the visceral yolk sac, probably after binding to and transport by maternal serum proteins. The possibilities that Cr(III) excerts its teratogenic action by inhibiting embryotrophic nutrition is discussed.     

Progress of co-culture systems in cartilage regeneration

ABSTRACT

Introduction: Cartilage tissue engineering has rapidly developed in recent decades, exhibiting promising potential to regenerate and repair cartilage. However, the origin of a large amount of a suitable seed cell source is the major bottleneck for the further clinical application of cartilage tissue engineering. The use of a monoculture of passaged chondrocytes or mesenchymal stem cells results in undesired outcomes, such as fibrocartilage formation and hypertrophy. In the last two decades, co-cultures of chondrocytes and a variety of mesenchymal stem cells have been intensively investigated in vitro and in vivo, shedding light on the perspective of co-culture in cartilage tissue engineering.

Areas covered: We summarize the recent literature on the application of heterologous cell co-culture systems in cartilage tissue engineering and compare the differences between direct and indirect co-culture systems as well as discuss the underlying mechanisms.

Expert opinion: Co-culture system is proven to address many issues encountered by monocultures in cartilage tissue engineering, including reducing the number of chondrocytes needed and alleviating the dedifferentiation of chondrocytes. With the further development and knowledge of biomaterials, cartilage tissue engineering that combines the co-culture system and advanced biomaterials is expected to solve the difficult problem regarding the regeneration of functional cartilage.     

Investigating cellulose derived glycosaminoglycan mimetic scaffolds for cartilage tissue engineering applications

Articular cartilage has a limited capacity to heal and, currently, no treatment exists that can restore normal hyaline cartilage. Creating tissue engineering scaffolds that more closely mimic the native extracellular matrix may be an attractive approach. Glycosaminoglycans, which are present in native cartilage tissue, provide signalling and structural cues to cells. This study evaluated the use of a glycosaminoglycan mimetic, derived from cellulose, as a potential scaffold for cartilage repair applications. Fully sulfated sodium cellulose sulfate (NaCS) was initially evaluated in soluble form as an additive to cell culture media. Human mesenchymal stem cell (MSC) chondrogenesis in pellet culture was enhanced with 0.01% NaCS added to induction media as demonstrated by significantly higher gene expression for type II collagen and aggrecan. NaCS was combined with gelatine to form fibrous scaffolds using the electrospinning technique. Scaffolds were characterized for fibre morphology, overall hydrolytic stability, protein/growth factor interaction and for supporting MSC chondrogenesis in vitro. Scaffolds immersed in phosphate buffered saline for up to 56 days had no changes in swelling and no dissolution of NaCS as compared to day 0. Increasing concentrations of the model protein lysozyme and transforming growth factor‐β3 were detected on scaffolds with increasing concentrations of NaCS (p < 0.05). MSC chondrogenesis was enhanced on the scaffold with the lowest NaCS concentration as seen with the highest collagen type II production, collagen type II immunostaining, and expression of cartilage‐specific genes. These studies demonstrate the feasibility of cellulose sulfate as a scaffolding material for cartilage tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.     

Treatment of osteochondritis dissecans of the patella in adolescents with nanofractured autologous matrix-induced chondrogenesis (NAMIC): A report of five cases

PurposeOsteochondritis dissecans (OCD) of the patella is a very rare affliction. The aim of this case series is to evaluate the effectiveness of the Nanofractured Autologous Matrix-Induced Chondrogenesis (NAMIC) technique for the treatment of OCD in young adults.MethodsFive consecutive patients with patellar osteochondral lesions treated with NAMIC were prospectively studied. There were 4 males and 1 female with a mean age of 15.2 years, ranging from 12 to 18 years. Clinically, they presented pain when going up and down stairs or squatting, effusion, swelling and functional limitation. The International Cartilage Repair Society (ICRS) grade was III in 4 patients and IV in 1 patient. Patients were functionally evaluated with the Lysholm knee score, the Tegner Activity Score (TAS) and the Knee Injury and Osteoarthritis Outcome Score (KOOS) and radiologically using the Magnetic resonance Observation of Cartilage Repair Tissue (MOCART) score.ResultsAll the scores significantly improved two years after surgery with respect to pre-operative values (Lysholm score from 63.8 ± 3.9 before surgery to 91 ± 3.2 at 2 years; Tegner activity score from 3.6 ± 0.5 to 8.2 ± 0.8 and the KOOS score from 45.2 ± 2.6 to 91.2 ± 2.4). Both the X-rays and the Magnetic Resonance Imaging (MRI) showed good joint regularity with an average MOCART of 80 ± 7.1/100.ConclusionsTreatment of juvenile patellar osteochondritis dissecans with the NAMIC technique seems to be a reliable technique in the short term to restore the patella joint surface and obtain good functional results.     

NELL-1促成骨和成软骨的作用机制及应用前景

NELL-1（neural epidermal growth factor-like 1）是一种对骨软骨细胞系具有高度特异性的生长因子,因其局部诱导骨形成的效应而受到密切关注;另外,它还具有作为全身治疗药物的成骨潜能以及促软骨形成的功能。促成骨方面,NELL-1主要通过Runx、MAPK信号通路、经典Wnt信号通路起作用,和经典的成骨因子BMPs存在协同作用。最近几年,由于生物信息学和高通量RNA测序的不断进步,研究者也发现了NELL-1促成骨分化时RNA网络的潜在调控作用。而NELL-1的促软骨形成活性主要依赖于Runx3介导的Ihh信号转导。本文主要就NELL-1促进成骨和成软骨的相关机制,以及其在骨组织再生、软骨组织再生领域的应用作一综述。     

CD146 as a new marker for an increased chondroprogenitor cell sub‐population in the later stages of osteoarthritis

Cartilage‐derived mesenchymal stem cells (MSCs) have been isolated with different methods. In this study lateral and medial femoral condyles were respectively collected from patients with late‐stage osteoarthritis during the total knee arthroplasty. After digestion of the cartilage tissues with type II collagenase and analysis by fluorescence‐activated cell sorting (FACS) with CD146, a chondroprogenitor cell sub‐population were isolated and purified. The expression of other MSC‐associated markers in the CD146⁺ chondroprogenitors was analyzed by flow cytometry. Multi‐lineage differentiation capacity of CD146⁺ chondroprogenitors was compared with that of unsorted chondrocytes and adipose‐derived MSCs (ADMSCs). Higher percentage of CD146⁺ chondroprogenitors isolated from the medial femoral condyles was observed than that from the lateral. CD146⁺ chondroprogenitors expressed high levels of MSC‐specific surface antigens, and showed higher chondrogenesis capacity than ADMSCs and unsorted chondrocytes in a 3D cell pellet culture model. Thus CD146 might be a new cell surface marker for cartilage progenitor cell population in the late‐stage osteoarthritis. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:84–91, 2015.