Quantitatively measuring the cytotoxicity of viscous hydrogels with direct cell sampling in a micro scale format "MicroDrop" and its comparison to CCK8.

Tissue engineering holds promise for developing therapeutic applications using viscous materials e.g. hydrogels. However, assessing the cytotoxicity of such materials with conventional assays can be challenging due to non-specific interactions. To address this, we optimized a live/dead staining method for quantitative evaluation and compared it with the conventional CCK8 assay. Our MicroDrop method involved seeding droplets containing 5000 cells in 10 µl medium on 12-well plates. After allowing them to adhere for 4 h, various viscous samples were applied to the cells and measurements were conducted using a fluorescence microscope immediately and at daily intervals up to 72 h. A sodium dodecyl sulfate (SDS) dilution series compared the MicroDrop with the CCK8 assay. The findings revealed a cell-type specific pattern for 10 mg/ml hyaluronic acid (HA), wherein MC3T3-E1 cells maintained 95% viability until 72 h, while L929 cells experienced a gradual decline to 17%. 2 mg/ml HA exhibited consistent viability above 90% across all time points and cell lines. Similarly, fibrin demonstrated 90% viability across dilutions and time points, except for undiluted samples showing a decrease from 85% to 20%. Gelatin-methacrylol sustained viability above 70% across all time points at both 5% and 10% concentrations. The comparison of the SDS dilution series between viability (MicroDrop) and metabolic activity (CCK8) assay showed a correlation coefficient of 0.95. The study validates the feasibility of the established assay, providing researchers with an efficient tool for assessing cytotoxicity in viscous materials. Additionally, it holds the potential to yield more precise data on well-known hydrogels.

Amino Acid-Based Polyphosphorodiamidates with Hydrolytically Labile Bonds for Degradation-Tuned Photopolymers.

Photochemical additive manufacturing technologies can produce complex geometries in short production times and thus have considerable potential as a tool to fabricate medical devices such as individualized patient-specific implants, prosthetics and tissue engineering scaffolds. However, most photopolymer resins degrade only slowly under the mild conditions required for many biomedical applications. Herein we report a novel platform consisting of amino acid-based polyphosphorodiamidate (APdA) monomers with hydrolytically cleavable bonds. The substituent on the α-amino acid can be used as a handle for facile control of hydrolysis rates of the monomers into their endogenous components, namely phosphate and the corresponding amino acid. Furthermore, monomer hydrolysis is considerably accelerated at lower pH values. The monomers underwent thiol-yne photopolymerization and could be 3D structured via multiphoton lithography. Copolymerization with commonly used hydrophobic thiols demonstrates not only their ability to regulate the ambient degradation rate of thiol-yne polyester photopolymer resins, but also desirable surface erosion behavior. Such degradation profiles, in the appropriate time frames, in suitably mild conditions, combined with their low cytotoxicity and 3D printability, render these novel photomonomers of significant interest for a wide range of biomaterial applications.

Sleeping Beauty transposon system for GDNF overexpression of entrapped stem cells in fibrin hydrogel in a rat model of Parkinson's disease.

There is currently no causal treatment available for Parkinson's disease (PD). However, the use of glial cell line-derived neurotrophic factor (GDNF) to provide regenerative effects for neurons is promising. Such approaches require translational delivery systems that are functional in diseased tissue. To do so, we used a non-viral Sleeping Beauty (SB) transposon system to overexpress GDNF in adipose tissue-derived mesenchymal stromal cells (adMSCs). Entrapment of cells in fibrin hydrogel was used to boost potential neurorestorative effects. Functional GDNF-adMSCs were able to secrete 1066.8 ± 169.4 ng GDNF/120,000 cells in vitro. The GDNF-adMSCs were detectable for up to 1 month after transplantation in a mild 6-hydroxydopamine (6-OHDA) hemiparkinson male rat model. Entrapment of GDNF-adMSCs enabled GDNF secretion in surrounding tissue in a more concentrated manner, also tending to prolong GDNF secretion relatively. GDNF-adMSCs entrapped in hydrogel also led to positive immunomodulatory effects via an 83% reduction of regional IL-1ß levels compared to the non-entrapped GDNF-adMSC group after 1 month. Furthermore, GDNF-adMSC-treated groups showed higher recovery of tyrosine hydroxylase (TH)-expressing cells, indicating a neuroprotective function, although this was not strong enough to show significant improvement in motor performance. Our findings establish a promising GDNF treatment system in a PD model. Entrapment of GDNF-adMSCs mediated positive immunomodulatory effects. Although the durability of the hydrogel needs to be extended to unlock its full potential for motor improvements, the neuroprotective effects of GDNF were evident and safe. Further motor behavioral tests and other disease models are necessary to evaluate this treatment option adequately.

Blends of gelatin and hyaluronic acid stratified by stereolithographic bioprinting approximate cartilaginous matrix gradients.

Stereolithographic bioprinting holds great promise in the quest for creating artificial, biomimetic cartilage-like tissue. To introduce a more biomimetic approach, we examined blending and stratifying methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) bioinks to mimic the zonal structure of articular cartilage. Bioinks were suspended with porcine chondrocytes before being printed in a digital light processing approach. Homogenous constructs made from hybrid bioinks of varying polymer ratios as well as stratified constructs combining different bioink blends were cultivated over 14 days and analyzed by histochemical staining for proteoglycans/collagen type II, cartilage marker expression analysis, and for cellular viability. The stiffness of blended bioinks increased gradually with HAMA content, from 2.41 ± 0.58 kPa (5% GelMA, 0% HAMA) to 8.84 ± 0.11 kPa (0% GelMA, 2% HAMA). Cell-laden constructs maintained vital chondrocytes and supported the formation of proteoglycans and collagen type II. Higher concentrations of GelMA resulted in increased formation of cartilaginous matrix proteins and a more premature phenotype. However, decreased matrix production in central areas of constructs was observed in higher GelMA content constructs. Biomimetically stratified constructs retained their gradient-like structure even after ECM formation, and exclusively exhibited a significant increase in COL2A1 gene expression (+178%). Concluding, we showed the feasibility of blending and stratifying photopolymerizable, natural biopolymers by SLA bioprinting to modulate chondrocyte attributes and to create zonally segmented ECM structures, contributing to improved modeling of cartilaginous tissue for regenerative therapies or in vitro models.

A Novel Method Facilitating the Simple and Low-Cost Preparation of Human Osteochondral Slice Explants for Large-Scale Native Tissue Analysis.

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-ß3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-ß3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.

Comparison of the Translational Potential of Human Mesenchymal Progenitor Cells from Different Bone Entities for Autologous 3D Bioprinted Bone Grafts.

Reconstruction of segmental bone defects by autologous bone grafting is still the standard of care but presents challenges including anatomical availability and potential donor site morbidity. The process of 3D bioprinting, the application of 3D printing for direct fabrication of living tissue, opens new possibilities for highly personalized tissue implants, making it an appealing alternative to autologous bone grafts. One of the most crucial hurdles for the clinical application of 3D bioprinting is the choice of a suitable cell source, which should be minimally invasive, with high osteogenic potential, with fast, easy expansion. In this study, mesenchymal progenitor cells were isolated from clinically relevant human bone biopsy sites (explant cultures from alveolar bone, iliac crest and fibula; bone marrow aspirates; and periosteal bone shaving from the mastoid) and 3D bioprinted using projection-based stereolithography. Printed constructs were cultivated for 28 days and analyzed regarding their osteogenic potential by assessing viability, mineralization, and gene expression. While viability levels of all cell sources were comparable over the course of the cultivation, cells obtained by periosteal bone shaving showed higher mineralization of the print matrix, with gene expression data suggesting advanced osteogenic differentiation. These results indicate that periosteum-derived cells represent a highly promising cell source for translational bioprinting of bone tissue given their superior osteogenic potential as well as their minimally invasive obtainability.

CCL25-Supplemented Hyaluronic Acid Attenuates Cartilage Degeneration in a Guinea Pig Model of Knee Osteoarthritis.

There is evidence that the application of mesenchymal stromal cells (MSCs) counteracts osteoarthritis (OA) progression. However, the prospect of extracting and expanding these cells might be limited. The aim of this study was to investigate whether hyaluronic acid (HA) supplemented with MSC-recruiting chemokine C-C motif ligand 25 (CCL25) can influence the natural course of spontaneous OA in the guinea pig. CCL25 concentration in synovial fluid (SF) was quantified with enzyme-linked immunosorbent assay. Boyden chamber cell migration assay was used to test CCL25-mediated migration of guinea pig MSC. Forty-nine 11-month-old male guinea pigs were divided into seven groups. The main treatments consisted of five intra-articular injections of HA in pure form and in combination with three doses of CCL25 (63, 693, and 6,993 pg) given at a weekly interval. The severity of cartilage damage was assessed by using a modified Mankin score. The measured average physiological concentration of CCL25 in SF of animals is 85 ± 39 pg/ml. MSC showed a 3.2-fold increase in cell migration at 1,000 nM CCL25 in vitro demonstrating the biological migratory activity of CCL25 on these cells. In vivo, treatment with HA alone did not reduce OA progression. Similarly, OA scores were not found significantly reduced after treatment with 63 pg CCL25 + HA. However, when compared to pure HA, treatment with 693 pg CCL25 + HA and 6,993 pg CCL25 + HA significantly reduced the OA score from 10.1 to 7.4 (-28%) and 8.4 (-20%), respectively. These data suggest that intra-articular injections of HA supplemented with CCL25 attenuates OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1723-1729, 2019.

Photopolymerizable gelatin and hyaluronic acid for stereolithographic 3D bioprinting of tissue-engineered cartilage.

To create artificial cartilage in vitro, mimicking the function of native extracellular matrix (ECM) and morphological cartilage-like shape is essential. The interplay of cell patterning and matrix concentration has high impact on the phenotype and viability of the printed cells. To advance the capabilities of cartilage bioprinting, we investigated different ECMs to create an in vitro chondrocyte niche. Therefore, we used methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) in a stereolithographic bioprinting approach. Both materials have been shown to support cartilage ECM formation and recovery of chondrocyte phenotype. We used these materials as bioinks to create cartilage models with varying chondrocyte densities. The models maintained shape, viability, and homogenous cell distribution over 14 days in culture. Chondrogenic differentiation was demonstrated by cartilage-typical proteoglycan and type II collagen deposition and gene expression (COL2A1, ACAN) after 14 days of culture. The differentiation pattern was influenced by cell density. A high cell density print (25 × 106 cells/mL) led to enhanced cartilage-typical zonal segmentation compared to cultures with lower cell density (5 × 106 cells/mL). Compared to HAMA, GelMA resulted in a higher expression of COL1A1, typical for a more premature chondrocyte phenotype. Both bioinks are feasible for printing in vitro cartilage with varying differentiation patterns and ECM organization depending on starting cell density and chosen bioink. The presented technique could find application in the creation of cartilage models and in the treatment of articular cartilage defects using autologous material and adjusting the bioprinted constructs size and shape to the patient. © 2019 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2649-2657, 2019.

Biomimetic sulfated polyethylene glycol hydrogel inhibits proteoglycan loss and tumor necrosis factor-α-induced expression pattern in an osteoarthritis in vitro model.

This study aimed to evaluate the potential of an anti-inflammatory polyethylene glycol (PEG) hydrogel for osteoarthritis (OA) management in an OA in vitro model. Freshly isolated porcine chondrocytes were maintained in high-density cultures to form cartilage-like three-dimensional micromasses. Recombinant porcine tumor necrosis factor-alpha (TNF-α) was used to induce OA-like changes. Normal and OA-like micromasses were treated with dendritic polyglycerol sulfate-based PEG hydrogel. Live/dead staining showed that all micromasses remained vital and presented similar morphological characteristics. Safranin-O staining demonstrated a typical depletion of glycosaminoglycans in TNF-α-treated micromasses but not in the presence of the hydrogel. There was no distinct difference in immunohistochemical detection of type II collagen. Microarray data showed that rheumatoid arthritis and TNF signaling pathways were down regulated in hydrogel-treated OA-like micromasses compared to nontreated OA-like micromasses. The hydrogel alone did not affect genes related to OA such as ANPEP, COMP, CXCL12, PTGS2, and TNFSF10, but it prevented their regulation caused by TNF-α. This study provides valuable insights toward a fully synthetic hydrogel for the intra-articular treatment of OA. The findings proved the potential of this hydrogel to prevent the development of TNF-α-induced OA with regard to proteoglycan loss and TNF-α-induced expression pattern without additional signs of differentiation and inflammation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 490-500, 2019.

Dose-Dependent Effect of Mesenchymal Stromal Cell Recruiting Chemokine CCL25 on Porcine Tissue-Engineered Healthy and Osteoarthritic Cartilage.

Thymus-expressed chemokine (CCL25) is a potent cell attractant for mesenchymal stromal cells, and therefore it is a candidate for in situ cartilage repair approaches focusing on the recruitment of endogenous repair cells. However, the influence of CCL25 on cartilage is unknown. Accordingly, in this study, we investigated the effect of CCL25 on tissue-engineered healthy and osteoarthritic cartilage. Porcine chondrocytes were cultured in a three-dimensional (3D) micromass model that has been proven to mimic key-aspects of human cartilage and osteoarthritic alterations upon stimulation with tumor necrosis factor-α (TNF-α). Micromass cultures were stimulated with CCL25 (0, 0.05, 0.5, 5, 50, 500 nmol/L) alone or in combination with 0.6 nmol/L TNF-α for seven days. Effects were evaluated by life/dead staining, safranin O staining, histomorphometrical analysis of glycosaminoglycans (GAGs), collagen type II (COL2A1) real-time RT-PCR and Porcine Genome Array analysis. 500 nmol/L CCL25 led to a significant reduction of GAGs and COL2A1 expression and induced the expression of matrix metallopeptidases (MMP) 1, MMP3, early growth response protein 1 (EGR1), and superoxide dismutase 2 (SOD2). In concentrations lower than 500 nmol/L, CCL25 seems to be a candidate for in situ cartilage repair therapy approaches.

Hyaluronic Acid Influence on Normal and Osteoarthritic Tissue-Engineered Cartilage.

The aim of this study is to identify gene expression profiles associated with hyaluronic acid (HA) treatment of normal and osteoarthritis (OA)-like tissue-engineered cartilage. 3D cartilage micromasses were treated with tumour necrosis factor-α (TNF-α) (OA-inducer) and/or HA for 7 days. Viability was examined by PI/FDA staining. To document extracellular matrix (ECM) formation, glycosaminoglycans (GAG) were stained with Safranin-O and cartilage-specific type II collagen was detected immunohistochemically. Genome-wide gene expression was determined using microarray analysis. Normal and OA-like micromasses remained vital and showed a spherical morphology and homogenous cell distribution regardless of the treatment. There was no distinct difference in immunolabeling for type II collagen. Safranin-O staining demonstrated a typical depletion of GAG in TNF-α-treated micromasses (-73%), although the extent was limited in the presence of HA (-39%). The microarray data showed that HA can influence the cartilage metabolism via upregulation of TIMP3 in OA-like condition. The upregulation of VEGFA and ANKRD37 genes implies a supportive role of HA in cartilage maturation and survival. The results of this study validate the feasibility of the in vitro OA model for the investigation of HA. On the cellular level, no inhibiting or activating effect of HA was shown. Microarray data demonstrated a minor impact of HA on gene expression level.

Injectable hydrogels for treatment of osteoarthritis - A rheological study.

Osteoarthritis (OA) is a disabling condition especially in the elderly population. The current therapeutic approaches do not halt the OA progression or reverse joint damage. In order to overcome the problem of rapid clearance of hyaluronic acid (HA), a standard viscosupplement for OA, we investigated the rheological properties of a relatively non-degradable dendritic polyglycerol sulfate (dPGS) hydrogel to determine a suitable concentration for intra articular injections that mimics HA in terms of its viscoelastic and mechanical properties. To do so, the concentration range from 3.6 to 4.8wt% of dPGS and, as a reference, blends of commercially available HAs (Ostenil®, GO-ON®, Synocrom® Forte and Synvisc®), were investigated by means of oscillating and flow rheology, thereby yielding storage (G') and loss modulus (G"), as well as yield stress and shear viscosity. In our rheological experiments we observe a pronounced coupling of the molecular weight and the rheological properties for the HAs. Furthermore, we find the dPGS hydrogel to form more compact networks with increasing concentration. From a broader comparison the current findings suggest that an overall polymer concentration of 4.0wt% dPGS has viscoelastic properties that are comparable to hyaluronic acid in the medically relevant frequency range, where for medical application the dPGS hydrogel has the advantage of being much less easily displaced from its injection place than HA.

A P19 and P19CL6 cell-based complementary approach to determine paracrine effects in cardiac tissue engineering.

The negligible self-repair potential of the myocardium has led to cell-based tissue engineering approaches to restore heart function. There is more and more consensus that, in addition to cell development, paracrine effects in particular play a pivotal role in the repair of heart tissue. Here, we present two complementary murine P19 and P19CL6 embryonic carcinoma cell-based in vitro test approaches to study the potential of repair cells and the factors secreted by these cells to induce cardiomyogenesis. P19 cells were 3-dimensionally cultured in hanging drops and P19CL6 cells in a monolayer. Both systems, capable of inducible differentiation towards the cardiomyogenic lineage shown by the appearance of beating cells, the expression of connexin 43 and cardiac troponins T and I, were used to test the cardiomyogenesis-inducing potential of human cardiac-derived adherent proliferating (CardAP) cells, which are candidates for heart repair. CardAP cells in coculture as well as CardAP cell-conditioned medium initiated beating in P19 cells, depending on the cell composition and concentration of the medium. CardAP cell-dependent beating was not observed in P19CL6 cultures, but connexin 43 and cardiac troponin formation as well as expression of GATA-binding protein 4 indicated the dose-dependent stimulatory cardiomyogenic effect of human CardAP cells. In summary, in different ways, P19 and P19CL6 cells have shown their capability to detect paracrine effects of human CardAP cells. In a complementary approach, they could be beneficial for determining the stimulatory cardiomyogenic potential of candidate cardiac-repair cells in vitro.

Suitability of porcine chondrocyte micromass culture to model osteoarthritis in vitro.

In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (r = 0.90), human cartilage (r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (-75%) and collagen depletion (-50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development (GPNMB, FRZB) including transcription factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone formation (COL1A1, COL10A1) was predominantly found decreased. These findings indicating significant parallels between human articular cartilage and the presented porcine micromass model and vice versa confirm the applicability of known cartilage marker and their characteristics in the porcine micromass model. TNF-α treatment enabled the initiation of typical OA reaction patterns in terms of extensive ECM loss, cell death, formation of an inflammatory environment through the induction of genes coding for chemokines and enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.

A method to screen and evaluate tissue adhesives for joint repair applications.

BACKGROUND: Tissue adhesives are useful means for various medical procedures. Since varying requirements cause that a single adhesive cannot meet all needs, bond strength testing remains one of the key applications used to screen for new products and study the influence of experimental variables. This study was conducted to develop an easy to use method to screen and evaluate tissue adhesives for tissue engineering applications. METHOD: Tissue grips were designed to facilitate the reproducible production of substrate tissue and adhesive strength measurements in universal testing machines. Porcine femoral condyles were used to generate osteochondral test tissue cylinders (substrates) of different shapes. Viability of substrates was tested using PI/FDA staining. Self-bonding properties were determined to examine reusability of substrates (n = 3). Serial measurements (n = 5) in different operation modes (OM) were performed to analyze the bonding strength of tissue adhesives in bone (OM-1) and cartilage tissue either in isolation (OM-2) or under specific requirements in joint repair such as filling cartilage defects with clinical applied fibrin/PLGA-cell-transplants (OM-3) or tissues (OM-4). The efficiency of the method was determined on the basis of adhesive properties of fibrin glue for different assembly times (30 s, 60 s). Seven randomly generated collagen formulations were analyzed to examine the potential of method to identify new tissue adhesives. RESULTS: Viability analysis of test tissue cylinders revealed vital cells (>80%) in cartilage components even 48 h post preparation. Reuse (n = 10) of test substrate did not significantly change adhesive characteristics. Adhesive strength of fibrin varied in different test settings (OM-1: 7.1 kPa, OM-2: 2.6 kPa, OM-3: 32.7 kPa, OM-4: 30.1 kPa) and was increasing with assembly time on average (2.4-fold). The screening of the different collagen formulations revealed a substance with significant higher adhesive strength on cartilage (14.8 kPa) and bone tissue (11.8 kPa) compared to fibrin and also considerable adhesive properties when filling defects with cartilage tissue (23.2 kPa). CONCLUSION: The method confirmed adhesive properties of fibrin and demonstrated the dependence of adhesive properties and applied settings. Furthermore the method was suitable to screen for potential adhesives and to identify a promising candidate for cartilage and bone applications. The method can offer simple, replicable and efficient evaluation of adhesive properties in ex vivo specimens and may be a useful supplement to existing methods in clinical relevant settings.

In vivo effect of bone marrow-derived mesenchymal stem cells in a rat kidney transplantation model with prolonged cold ischemia.

Brain death and prolonged cold ischemia are major contributors to the poorer long-term outcome of transplants from deceased donor kidney transplants, with an even higher impact if expanded criteria donors ('marginal organs') are used. Targeting ischemia-reperfusion injury-related intragraft inflammation is an attractive concept to improve the outcome of those grafts. As mesenchymal stem cells (MSCs) express both immunomodulatory and tissue repair properties, we evaluated their therapeutic efficacy in a rat kidney transplant model of prolonged cold ischemia. The in vitro immunomodulatory capacity of bone marrow-derived rat MSCs was tested in co-cultures with rat lymph node cells. For in vivo studies, Dark Agouti rat kidneys were cold preserved and transplanted into Lewis rats. Syngeneic Lewis MSCs were administered intravenously. Transplants were harvested on day 3, and inflammation was examined by quantitative RT-PCR and histology. Similarly to MSCs from other species, rat MSCs in vitro also showed a dose-dependent immunomodulatory capacity. Most importantly, in vivo administration of MSCs reduced the intragraft gene expression of different pro-inflammatory cytokines, chemokines, and intercellular adhesion molecule-1. In addition, fewer antigen-presenting cells were recruited into the renal allograft. In conclusion, rat MSCs ameliorate inflammation induced by prolonged cold ischemia in kidney transplantation.

Gene expression profiling of primary human articular chondrocytes in high-density micromasses reveals patterns of recovery, maintenance, re- and dedifferentiation.

The high-density micromass culture has been widely applied to study chondrocyte cell physiology and pathophysiological mechanisms. Since an integrated image has not been established so far, we analyzed the phenotypic alterations of human articular chondrocytes in this model on the broad molecular level. Freshly isolated chondrocytes were assembled as micromasses and maintained up to 6 weeks in medium containing human serum. Formation of cartilaginous extracellular matrix (ECM) was evaluated by histological and immunohistochemical staining. At 0, 3 and 6 weeks, chondrocyte micromasses were subjected to gene expression analysis using oligonucleotide microarrays and real-time RT-PCR. Micromasses developed a cartilaginous ECM rich in proteoglycans and type II collagen. On gene expression level, time-dependent expression patterns was observed. The induction of genes associated with cartilage-specific ECM (COL2A1 and COL11A1) and developmental signaling (GDF5, GDF10, ID1, ID4 and FGFR1-3) indicated redifferentiation within the first 3 weeks. The repression of genes related to stress response (HSPA1A and HSPA4), apoptotic events (HYOU1, NFKBIA and TRAF1), and degradation (MMP1, MMP10 and MMP12) suggested a recovery of chondrocytes. Constant expression of other chondrogenic (ACAN, FN1 and MGP) and hypertrophic markers (COL10A1, ALPL, PTHR1 and PTHR2) indicated a pattern of phenotypic maintenance. Simultaneously, the expression of chondrogenic growth (BMP6, TGFA, FGF1 and FGF2) and transcription factors (SOX9, EGR1, HES1 and TGIF1), and other cartilage ECM-related genes (COMP and PRG4) was consistently repressed and expression of collagens related to dedifferentiation (COL1A1 and COL3A1) was steadily induced indicating a progressing loss of cartilage phenotype. Likewise, a steady increase of genes associated with proliferation (GAS6, SERPINF1, VEGFB and VEGFC) and apoptosis (DRAM, DPAK1, HSPB, GPX1, NGFRAP1 and TIA1) was observed. Sequence and interplay of identified expression patterns suggest that chondrocyte micromass cultures maintain a differentiated phenotype up to 3 weeks in vitro and might be useful for studying chondrocyte biology, pathophysiology and differentiation. Cultivation longer than 6 weeks leads to progressing dedifferentiation of chondrocytes that should be considered on long-term evaluations.

Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation.

INTRODUCTION: Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. METHODS: Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score > 3, Ahlbäck Score > 2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using real-time PCR. RESULTS: Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (ACAN, COL2A1, COMP, CRTL1, SOX9) and genes involved in matrix synthesis (BGN, CILP2, COL9A2, COL11A1, TIMP4) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (ALPL, COL1A1, COL3A1, COL10A1, MMP13, POSTN, PTH1R, RUNX2) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, was differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. CONCLUSIONS: Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA, and OA chondrocytes fulfill the requirements for matrix-associated ACT.

Chondrogenic differentiation capacity of human mesenchymal progenitor cells derived from subchondral cortico-spongious bone.

Microfracture is frequently used to repair articular cartilage defects and allows mesenchymal progenitors to migrate from subchondral bone into the defect and form cartilaginous repair tissue. The aim of our study was to analyze the cell surface antigen pattern and the differentiation capacity of cells derived from human subchondral bone. Human progenitor cells were derived from subchondral cortico-spongious bone and grown in the presence of human serum. Stem cell-related cell surface antigens were analyzed by flowcytometry. Cortico-spongious progenitor (CSP) cells showed presence of CD73, CD90, CD105, and STRO-1. Multilineage differentiation potential of CSP cells was documented by histological staining and by gene expression analysis of osteogenic, adipogenic, and chondrogenic marker genes. CSP cells formed a mineralized matrix as demonstrated by von Kossa staining and showed induction of osteocalcin, independent of osteogenic stimulation. During adipogenic differentiation, the adipogenic marker genes fatty acid binding protein 4 and peroxisome proliferative activated receptor gamma were induced. Immunohistochemical staining of cartilage-specific type II collagen and induction of the chondrocytic marker genes cartilage oligomeric matrix protein, aggrecan, and types II and IX collagen confirmed TGF beta 3-mediated chondrogenic lineage development. CSP cells from subchondral bone, as known from microfracture, are multipotent stem cell-like mesenchymal progenitors with a high chondrogenic differentiation potential.

Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes. METHODS: Human chondrocytes were cultured three-dimensionally for 14 days in alginate beads and subsequently stimulated for 48 hours with supernatants from SV40 T-antigen immortalized human synovial fibroblasts (SF) derived from a normal donor (NDSF) and from a patient with RA (RASF), respectively. To identify RA-related factors released from SF, supernatants of RASF and NDSF were analyzed with antibody-based protein membrane arrays. Stimulated cartilage-like cultures were used for subsequent gene expression profiling with oligonucleotide microarrays. Affymetrix GeneChip Operating Software and Robust Multi-array Analysis (RMA) were used to identify differentially expressed genes. Expression of selected genes was verified by real-time RT-PCR. RESULTS: Antibody-based protein membrane arrays of synovial fibroblast supernatants identified RA-related soluble mediators (IL-6, CCL2, CXCL1-3, CXCL8) released from RASF. Genome-wide microarray analysis of RASF-stimulated chondrocytes disclosed a distinct expression profile related to cartilage destruction involving marker genes of inflammation (adenosine A2A receptor, cyclooxygenase-2), the NF-kappaB signaling pathway (toll-like receptor 2, spermine synthase, receptor-interacting serine-threonine kinase 2), cytokines/chemokines and receptors (CXCL1-3, CXCL8, CCL20, CXCR4, IL-1beta, IL-6), cartilage degradation (matrix metalloproteinase (MMP)-10, MMP-12) and suppressed matrix synthesis (cartilage oligomeric matrix protein, chondroitin sulfate proteoglycan 2). CONCLUSION: Differential transcriptome profiling of stimulated human chondrocytes revealed a disturbed catabolic-anabolic homeostasis of chondrocyte function and disclosed relevant pharmacological target genes of cartilage destruction. This study provides comprehensive insight into molecular regulatory processes induced in human chondrocytes during RA-related destruction of cartilage. The established model may serve as a human in vitro disease model of RA-related destruction of cartilage and may help to elucidate the molecular effects of anti-rheumatic drugs on human chondrocyte gene expression.