Chondrocyte Cell Cultures Research Articles

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes.

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs) and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2) and decreased type I collagen (COL1) protein expression levels. SRY-box 9 (SOX9) mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

Release of adenosine from chitosan beads

Event Abstract Back to Event Release of adenosine from chitosan beads Allen Jed Mamaril1, Parwinder Singh1, Ravi Patel1, Joel D. Bumgardner1 and Jessica A. Jennings1 1 University of Memphis, Department of Biomedical Engineering, United States Introduction: As many as 27 million American suffer from the painful chronic condition of osteoarthritis[1]. Most treatments are geared toward symptom management, but recent advancements in tissue engineering aim to heal articular cartilage. Recent literature has shown that adenosine reduces inflammation, increases proliferation and matrix production, and promotes chondrocyte growth in osteoarthritic models[2],[3]. This study was conducted to demonstrate efficacy of chitosan microbeads as a delivery vehicle for adenosine. Methods: Chitosan (Chitopharm S) solutions (2% w/v) were created by dissolving 3 or 6 mg/mL adenosine base powder (MP Biomedicals) in 2% acetic acid, adding chitosan and stirring overnight. An emulsion was created by stirring the solution with an impeller at a setting of 2000 rpm in a 1:1 mixture of light to heavy chain mineral oil and 1% Span 80. Glyoxal was added at 0.5% to the emulsion and the mixture was heated at 70°C while impelling for 24 hours. Microbeads were recovered by centrifugation and washing with hexanes, methanol, and then acetone. Four separate samples of dried beads weighing 50 mg were added to 1 mL sterile DMEM/High-glucose and vortexed. Eluate samples were taken every day for 7 days after vortexing and centrifugation. Adenosine concentration was measured with high performance liquid chromatography and normalized to known standard concentrations. Results: Higher concentrations of adenosine were observed in the elution samples collected over the first two days of the trial, with a peak elution of 1360 μg/ml on day two of elution for 6mg/ml concentration (Figure 1). The presence of adenosine in elution samples declined constantly over the next five days in a first-order release pattern for both loading concentrations. Discussion: Injectable chitosan microbeads deliver controlled amounts of adenosine over time, which offers advantages for drug delivery systems directed toward cartilage repair. By altering adenosine concentration in microbeads, elution profile and duration of the drug release can be controlled. Since previous studies have shown highest bioactivity of adenosine in the range of 50-200 µg/ml[2], loading with the lower amount may have improved therapeutic effect over the high-dose loaded microbeads. Evaluation of adherence of adenosine-loaded microbeads to damaged tissue as well as testing efficacy in promoting cell growth through in vitro chondrocyte cell culture and preclinical models will provide insight into the refinement of functional delivery systems. Conclusions: Adenosine-loaded microbeads may provide effective controlled delivery of bioactive adenosine for applications in promoting healing of cartilage, skin, bone, and soft tissues. Minimally invasive delivery, inexpensive cost, and biocompatibility may offer an advantage of adenosine-loaded chitosan microbeads over other treatment options for tissue repair, such as surgical procedures, joint replacement, and growth factor therapy. FedEx Institute of Technology Innovation Fund; Herff College of Engineering

An In Vitro Chondrocyte Electrical Stimulation Framework: A Methodology to Calculate Electric Fields and Modulate Proliferation, Cell Death and Glycosaminoglycan Synthesis

Electric fields (EFs) have been used as external biophysical stimuli to increase cell population and stimulate the molecular synthesis of chondrocytes. However, in the methodology reported by different researchers there is no evidence about the procedure to calculate EFs. This work presents a new framework to calculate EFs and modulate proliferation, cell death and glycosaminoglycan synthesis. For this study, we used a computational analysis, by a finite elements method for experimental setting, to find the dielectric constants of the cell culture medium and the experimental geometrical configuration. Chondrocytes were stimulated with EFs of 4 and 8 mV/cm, and for each of these EFs, three exposure times were tested (30 min, 1 and 5 h). We observed an increment in cell population when an electric field of 4 mV/cm was applied during 30 min. The glycosaminoglycan synthesis remained stable after an electric field of 8 mV/cm was applied during 5 h. No changes in cell population and molecular synthesis were perceived when EFs were applied during 1 h. This approach is a promising tool that could be used to enhance in vitro procedures in order to obtain well-characterized chondrocyte cell cultures either by increasing cell proliferation or stimulating molecular synthesis.

A mathematical model and computational framework for three-dimensional chondrocyte cell growth in a porous tissue scaffold placed inside a bi-directional flow perfusion bioreactor.

The in vitro chondrocyte cell culture for cartilage tissue regeneration in a perfusion bioreactor is a complex process. Mathematical modeling and computational simulation can provide important insights into the culture process, which would be helpful for selecting culture conditions to improve the quality of the developed tissue constructs. However, simulation of the cell culture process is a challenging task due to the complicated interaction between the cells and local fluid flow and nutrient transport inside the complex porous scaffolds. In this study, a mathematical model and computational framework has been developed to simulate the three-dimensional (3D) cell growth in a porous scaffold placed inside a bi-directional flow perfusion bioreactor. The model was developed by taking into account the two-way coupling between the cell growth and local flow field and associated glucose concentration, and then used to perform a resolved-scale simulation based on the lattice Boltzmann method (LBM). The simulation predicts the local shear stress, glucose concentration, and 3D cell growth inside the porous scaffold for a period of 30 days of cell culture. The predicted cell growth rate was in good overall agreement with the experimental results available in the literature. This study demonstrates that the bi-directional flow perfusion culture system can enhance the homogeneity of the cell growth inside the scaffold. The model and computational framework developed is capable of providing significant insight into the culture process, thus providing a powerful tool for the design and optimization of the cell culture process.

Gene expression of cultured human chondrocytes as a model for assessing neutralization efficacy of soluble TNFα by TNFα antagonists

Tumor necrosis factor-alpha (TNFα) antagonists are efficacious in the treatment of various immune-mediated inflammatory diseases. Because of rapidly growing demand for developing new or biosimilar versions of these biologicals, the need to create in vitro testing models that best represent physiological conditions is increasing.Primary human chondrocytes were used for potency evaluation and comparison between the molecular effects of anti-TNFα biologicals. Infliximab and etanercept were chosen to assess the suitability of chondrocyte cell culture for determination of anti-TNFα neutralization efficacy employing quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technology.Use of both anti-TNFα biologics resulted in decrease of TNFα-stimulated expression of various matrix metalloproteinases, interleukins and other inflammation-related genes in our cell model. Significant differences in inhibition efficacy of etanercept and infliximab were observed, which were confirmed also on protein level. To evaluate the potency of anti-TNFα biologicals, a selection of TNFα-responsive target genes was made from the gene array data. The selected genes were employed in development of statistical model, which enables comparability of anti-TNFα biologicals.The presented analytical approach is suitable for assessment of the neutralization efficacy of various anti-TNFα biologicals. As such, it can be used for additional comprehensive characterization and comparability of TNF antagonists in preclinical drug testing.

Computational modelling of the scaffold-free chondrocyte regeneration: a two-way coupling between the cell growth and local fluid flow and nutrient concentration

The in vitro chondrocyte cell culture process in a perfusion bioreactor provides enhanced nutrient supply as well as the flow-induced shear stress that may have a positive influence on the cell growth. Mathematical and computational modelling of such a culture process, by solving the coupled flow, mass transfer and cell growth equations simultaneously, can provide important insight into the biomechanical environment of a bioreactor and the related cell growth process. To do this, a two-way coupling between the local flow field and cell growth is required. Notably, most of the computational and mathematical models to date have not taken into account the influence of the cell growth on the local flow field and nutrient concentration. The present research aimed at developing a mathematical model and performing a numerical simulation using the lattice Boltzmann method to predict the chondrocyte cell growth without a scaffold on a flat plate placed inside a perfusion bioreactor. The model considers the two-way coupling between the cell growth and local flow field, and the simulation has been performed for 174 culture days. To incorporate the cell growth into the model, a control-volume-based surface growth modelling approach has been adopted. The simulation results show the variation of local fluid velocity, shear stress and concentration distribution during the culture period due to the growth of the cell phase and also illustrate that the shear stress can increase the cell volume fraction to a certain extent.

In vitro chondrocyte behavior on porous biodegradable poly (e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation

In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.

Mutant IDH is sufficient to initiate enchondromatosis in mice

Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

Activation of a7 Nicotinic Acetylcholine Receptors Prevents Monosodium Iodoacetate-Induced Osteoarthritis in Rats

Background/Aims: Although some evidence suggests that the prevalence of osteoarthritis (OA) is lower in smokers compared to nonsmokers, the mechanisms of nicotine-induced protection remain unclear. Stimulation of the a7 nicotinic acetylcholine receptor (a7-nAChR) appears to be a critical mechanism underlying the anti-inflammatory potential of cholinergic agonists in immune cells. The inhibition of secreted inflammatory molecules and the subsequent inflammatory processes have been proposed as a novel strategy for the treatment of OA. The objective of the present study was to determine whether nicotine-induced protection in a monosodium iodoacetate (MIA) rat model of OA occurs via a7-nAChR-mediated inhibition of chondrocytes. Methods: Both in vivo (MIA) and in vitro (MIA; Interleukin-1ß, IL-1ß) models of OA were used to investigate the roles and the possible mechanisms whereby a7-nAChRs protect against knee joint degradation. Multiple experimental approaches, including macroscopic, histological analysis, chondrocyte cell cultures, confocal microscopy, and western blotting, were employed to elucidate the mechanisms of a7-nAChR-mediated protection. Results: Systemic administration of nicotine alleviated MIA-induced joint degradation. The protective effects of nicotine were abolished by administration of the a7-nAChR-selective antagonist methyllycaconitine (MLA). In primary cultured rat chondrocytes, pretreatment with nicotine suppressed both p38, extracellular regulated kinase (Erk) 1/2 and c-Jun-N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) phosphorylation and phosphorylated nuclear factor-kappa B (NF-κB) p65 activation induced by MIA- or IL-1ß, and these effects were also reversed by MLA. Conclusion: Taken together, our results suggest that activation a7-nAChRs is an important mechanism underlying the protective effects of nicotine.

A simple two dimensional culture method to study the hypertrophic differentiation of rat articular chondrocytes

Chondrocytes hypertrophy is a physiological process observed in endochondral ossification during development until adolescence in human. It can also be observed during pathophysiological conditions such as osteoarthritis. Hypertrophic chondrocytes synthesise collagen X and express matrix metalloproteinase 13 and alkaline phosphatase. The cellular models available to study this process are either not convenient, they might lead to a rapid dedifferentiation of chondrocytes, or they are far from the physiological conditions. The objective of this study was to design an user-friendly 2D-primary cell culture of young articular chondrocytes of rat able to follow the terminal differentiation process. After confluence, chondrocytes were cultured according to 4 differentiation protocols. Protocol 1 contained DMEM/F12 supplemented with 10% foetal bovine serum (FBS) and 2 μg/ml insulin. Protocol 2 contained alpha-MEM supplemented with 5% FBS and 2 μg/ml insulin. Protocol 3 contained 2% FBS and 2 μg/ml insulin. Protocol 4 contained DMEM/F12 supplemented with 2% FBS in absence or in presence of 2 μg/ml insulin and 37.5 μg/ml ascorbate. The cell morphology was observed by phase-contrast microscopy and the expression of markers specific of mature and hypertrophic chondrocytes were assessed by RT-qPCR. The effect of a decrease in nutrient quality of the culture medium after confluence was tested using protocols 1, 2 and 3. Protocol 1 did not allow the maintenance of chondrocyte phenotype more than one week, because cells became fibroblastic. A decrease in Sox9 mRNA expression, in collagen II/collagen I and in aggrecan/versican mRNA ratios was also found with protocol 1. Protocol 3 was the best when compared with protocols 1 and 2. It allowed chondrocytes to adopt a hypertrophic morphology. Cells also expressed the collagen X specific hypertrophic marker, and presented an increase in collagen II/I and aggrecan/versican ratios after 15 days of culture post-confluence. The effect of the insulin/ascorbate supplementation was studied using protocol 4. The insulin/ascorbate supplementation allowed an earlier chondrocytes conversion to terminal differentiation with a prolonged effect till 3 weeks post-confluence, compared to control without insulin/ascorbate. Finally, the profile of chondrocyte differentiation was checked during 5 successive sub-cultures. Only the first passage could be used to study hypertrophy. A convenient protocol to study chondrocyte hypertrophy is proposed. Protocol 4 offers the possibility to study this differentiation phenotype which is crucial for the development of articular diseases such as osteoarthritis. Our model could also be used in tissue engineering for cartilage repair strategies in which hypertrophic differentiation of chondrocyte should be avoided.

Preparation and Characterization of Nanofibrous Polymer Scaffolds for Cartilage Tissue Engineering

Polymer substrates obtained from poly(lactic acid) (PLA) nanofibres modified with carbon nanotubes (CNTs) and gelatin (GEL) for cartilage tissue engineering are studied. The work presents the results of physical, mechanical, and biological assessment. The hybrid structure of PLA and gelatine nanofibres, carbon nanotubes‐ (CNTs‐) modified PLA nanofibres, and pure PLA‐based nanofibres was manufactured in the form of fibrous membranes. The fibrous samples with different microstructures were obtained by electrospinning method. Microstructure, physical and mechanical properties of samples made from pure PLA nanofibres, CNTs‐, and gelatin‐modified PLA‐nanofibres were studied. The scaffolds were also tested in vitro in cell culture of human chondrocytes collected from patients. To assess the influence of the nanofibrous scaffolds upon chondrocytes, tests for cytotoxicity and genotoxicity were performed. The work reveals that the nanofibrous structures studied were neither genotoxic nor cytotoxic, and their microstructure, physical and mechanical properties create promising scaffolds for potential use in cartilage repairing.

Variation in growth pattern and morphological appearance of primary monolayer cultures of chondrocytes and neural cells isolated from the chick embryo at different stages

Aim: This study was an attempt at investigating the variation in growth and morphology of primary avian chondrocyte and neural cell cultures established under standard laboratory conditions. Methods: For establishing chondrocyte cultures, the tibia were dissected from chick embryos that were 12 (E12), 14 (E14) and 15 (E15) days old. For the neural cell culture initiation, the two lobes of the forebrain were dissected from chick embryos that were 4 (E4), 8 (E8) and 12 (E12) days old. The final characterization of the cells was performed by H&E and CFV staining. Results: In the chondrocyte cultures, two forms of morphology were observed which is reversible in process.The primary variation evaluated in chondrocytes cultures was the effect of the media on the state of the cells. Based on whether the cells were cultured in DMEM or MEM, there was a difference in the transformation of the attached cells from the differentiated to de-differentiated forms. The primary variation examined in neuron cultures was the embryonic age of the tissue and the effect that it had on the proliferation and neurite formation of the cells. E8 embryo neural cells cultures result in well-developed cells with neurite formation along with axonal and dendritic outgrowths with highly complex interconnections between them. Conclusion: Ultimately, this study demonstrated the composition of culture media had an effect on the morphological appearance of the chondrocytes, as well as, confirmed that the culture of primary neurons is best performed using cells from an E8 embryo.

329 CHONDROGENIC POTENTIAL OF PORCINE ADIPOSE-DERIVED STEM CELLS, CHONDROCYTES, PERIOSTEAL CELLS, AND FIBROBLASTS IN A PELLET CULTURE SYSTEM

Regenerative medicine has long sought to develop therapies for articular cartilage repair and for enhancing endochondral ossification to address complications of long bone healing. The objective of this study was to determine the chondrogenic potential of porcine primary cell cultures for possible utility in orthopedic tissue engineering applications. Adipose-derived mesenchymal stem cell (ASC), chondrocyte (positive control), periosteal cell, and fibroblast (negative control) primary cell cultures from 8- to 12-month-old Yorkshire pigs were plated at 5000 to 10 000 cells cm–2 in 75-cm2 cell culture flasks using high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with NaHCO3, 10% fetal bovine serum (FBS), and antimicrobials (penicillin-streptomycin, gentamicin sulfate, and amphotericin B), then incubated at 37°C, 5% CO2, and 18% O2. Cells were trypsinized at ~80% confluency and transferred into 15-mL conical tubes at 500 000 cells tube–1. Suspensions were washed twice by centrifugation in DPBS then condensed via a final centrifugation in 1.0 mL of negative control media (DMEM + 10% FBS) or chondrogenic base media consisting of high-glucose DMEM with 40 µg mL–1 of proline, 50 µM ascorbic acid-2-phosphate, 100 nM dexamethasone, antimicrobials, and 1× insulin-transferrin-selenium solution added. Chondrogenic additives tested were 2% FBS, Kartogenin (200 nM, 400 nM, or 4 µM), 10 ng mL–1 of BMP-4, or a combination of 10 ng mL–1 of BMP-6 + 10 ng mL–1 of TGFβ-3. Pellets were incubated with media changed every 2 to 3 days for a period of 2 to 4 weeks, fixed with 4% paraformaldehyde in DPBS, and then frozen at –80°C in Neg 50 Frozen Section Medium. Eight-micrometer sections were cut using a cryostat onto charged slides. Histochemical staining was performed using hematoxylin and eosin (H&E) for cell morphology and Safranin O and Alcian Blue for cartilage matrix markers. Immunofluorescent staining was done to detect collagen II and aggrecan with the nuclear marker lamin-C used to assess cell viability. Chondrocyte pellets grown in chondrogenic media, regardless of additive, exhibited cartilage matrix formation with H&E and stained strongly positive with Safranin O, Alcian Blue, and collagen II. The ASC pellets grown in chondrogenic media showed mixed cell morphology and areas of early cartilage matrix formation with H&E and stained faintly positive with Safranin O, Alcian Blue, and collagen II at 3 weeks in culture. Periosteal cell pellets had similar morphology in all conditions and did not stain positive for cartilage matrix markers. Fibroblast pellets did not survive any condition for processing. In conclusion, porcine chondrocytes and ASC were able to form cartilage matrix in a pellet culture system with chondrogenic media regardless of additive, while porcine fibroblasts and periosteal cells showed limited to no chondrogenic potential in the conditions tested.

Primary murine growth plate and articular chondrocyte isolation and cell culture.

The ability to isolate primary chondrocytes from wild-type and genetically altered mice has provided tremendous advances in the understanding of signaling networks that regulate chondrocytes in health and disease. Isolation of chondrocytes from both growth plate tissues and articular cartilage has been challenging due to the cells being embedded within a highly organized tissue matrix. Here we describe highly reproducible methods for the isolation of pure populations of growth plate chondrocytes from the murine sternum and ribs and articular chondrocytes from the knee joint.

Ketamine is toxic to chondrocyte cell cultures.

Ketamine has been used in combination with a variety of other agents for intra-articular analgesia, with promising results. However, although it has been shown to be toxic to various types of cell, there is no available information on the effects of ketamine on chondrocytes. We conducted a prospective randomised controlled study to evaluate the effects of ketamine on cultured chondrocytes isolated from rat articular cartilage. The cultured cells were treated with 0.125 mM, 0.250 mM, 0.5 mM, 1 mM and 2 mM of ketamine respectively for 6 h, 24 hours and 48 hours, and compared with controls. Changes of apoptosis were evaluated using fluorescence microscopy with a 490 nm excitation wavelength. Apoptosis and eventual necrosis were seen at each concentration. The percentage viability of the cells was inversely proportional to both the duration and dose of treatment (p = 0.002 and p = 0.009). Doses of 0.5 mM, 1 mM and 2mM were absolutely toxic. We concluded that in the absence of solid data to support the efficacy of intra-articular ketamine for the control of pain, and the toxic effects of ketamine on cultured chondrocytes shown by this study, intra-articular ketamine, either alone or in combination with other agents, should not be used to control pain. Cite this article: Bone Joint J 2014; 96-B:989-94.

Discriminating multiplexed GFP reporters in primary articular chondrocyte cultures using image cytometry.

Flow cytometry has become a standard tool for defining a heterogeneous cell population based on surface expressed epitopes or GFP reporters that reflect cell types or cellular differentiation. The introduction of image cytometry raised the possibility of adaptation to discriminate GFP reporters used to appreciate cell heterogeneity within the skeletal lineages. The optical filters and LEDs were optimized for the reporters used in transgenic mice expressing various fluorescent proteins. In addition, the need for compensation between eGFP and surrounding reporters due to optical cross-talk was eliminated by selecting the appropriate excitation and emission filters. Bone marrow or articular cartilage cell cultures from GFP and RFP reporter mouse lines were established to demonstrate the equivalency in functionalities of image to flow cytometry analysis. To examine the ability for monitoring primary cell differentiation, articular chondrocyte cell cultures were established from mice that were single or doubly transgenic (Dkk3eGFP and Col2A1GFPcyan), which identify the progression of superficial small articular cell to a mature chondrocyte. The instrument was able to rapidly and accurately discriminate cells that were Dkk3eGFP only, Dkk3eGFP/Col2A1GFPcyan, and Col2A1GFP, which provides a useful tool for studying the impact of culture conditions on lineage expansion and differentiation.

Insulin-Like Growth Factor I (IGF-1) Ec/Mechano Growth Factor – A Splice Variant of IGF-1 within the Growth Plate

Human insulin-like growth factor 1 Ec (IGF-1Ec), also called mechano growth factor (MGF), is a splice variant of insulin-like growth factor 1 (IGF-1), which has been shown in vitro as well as in vivo to induce growth and hypertrophy in mechanically stimulated or damaged muscle. Growth, hypertrophy and responses to mechanical stimulation are important reactions of cartilaginous tissues, especially those in growth plates. Therefore, we wanted to ascertain if MGF is expressed in growth plate cartilage and if it influences proliferation of chondrocytes, as it does in musculoskeletal tissues. MGF expression was analyzed in growth plate and control tissue samples from piglets aged 3 to 6 weeks. Furthermore, growth plate chondrocyte cell culture was used to evaluate the effects of the MGF peptide on proliferation. We showed that MGF is expressed in considerable amounts in the tissues evaluated. We found the MGF peptide to be primarily located in the cytoplasm, and in some instances, it was also found in the nucleus of the cells. Addition of MGF peptides was not associated with growth plate chondrocyte proliferation.

Opiates do not violate the viability and proliferative activity of human articular chondrocytes

Articular cartilage injuries present a challenge for the clinician. Autologous chondrocyte implantation embedded in scaffolds are used to treat cartilage defects with favorable outcomes. Autologous serum is often used as a medium for chondrocyte cell culture during the proliferation phase of the process of such products. A previous report showed that opiate analgesics (fentanyl, alfentanil and diamorphine) in the sera have a significant inhibitory effect on chondrocyte proliferation. In order to determine if opiates in serum inhibit chondrocyte proliferation, twenty two patients who underwent knee arthroscopy and were anesthetized with either fentanyl or remifentanil were studied. Blood was drawn before and during opiate administration and up to 2 h after its discontinuation. The sera were used as medium for in vitro proliferation of both cryopreserved and freshly isolated chondrocytes, and the number and viability of cells were measured. There was no difference in the yield or cell viability between the serum samples of patients anesthetized with fentanyl when either fresh or cryopreserved human articular chondrocytes (hACs) were used. Some non-significant reduction in the yield of cells was observed in the serum samples of patients anesthetized with remifentanil when fresh hAC were used. We conclude that Fentanyl in human autologous serum does not inhibit in vitro hAC proliferation. Remifentanil may show minimal inhibitory effect on in vitro fresh hAC proliferation.

Bupivacaine and levobupivacaine induce apoptosis in rat chondrocyte cell cultures at ultra-low doses

Osteoarthritis (OA) is characterized by chondrocyte apoptosis and necrosis which play a key role during the progression of OA. Intra-articular administration of bupivacaine is a practical and effective way of postoperative pain control following various joint surgeries. 0.25% bupivacaine showed to be safe in terms of chondrocyte toxicity. Around 200 nM of bupivacaine was shown to be effective for peripheral nerve block. This study aims to observe the possible cytotoxic effects of bupivacaine and its enantiomer levobupivacaine on chondrocyte cell culture at 7.69, 76.9, and 384.5μM or at 0.0125, 0.0025, and 0.00025% concentrations, respectively. Chondrocytes were isolated from rat articular cartilage after incubating with collagenase in RPMI-1640 medium. Cells were treated with bupivacaine and levobupivacaine at 7.69, 76.9, and 384.5μM concentrations for 6, 24, and 48h. Treated chondrocytes were stained with acridine orange and ethidium bromide and examined under a fluorescence microscope at a 490nm excitation wavelength for apoptotic changes. Study results suggest that both bupivacaine and levobupivacaine have dose-dependent chondrocyte toxicity, and this is significantly lesser at 7.69μM dose. There was no significant difference in terms of chondrocyte apoptosis, (p>0.05). Clinicians should be skeptic for the serious long-term side effects of bupivacaine and its analogs, even at ultra-low doses.

A Combined Approach for the Assessment of Cell Viability and Cell Functionality of Human Fibrochondrocytes for Use in Tissue Engineering

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5–P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5–P6 for cell therapy protocols.