ABSTRACT
OBJECTIVES: Bone morphogenetic protein (BMP-) and Wnt-signalling play crucial roles in cartilage homeostasis. Our objective was to investigate whether activation of the BMP-pathway or stimulation of Wnt-signalling cascades effectively enhances cartilage-specific extracellular matrix (ECM) accumulation and functional biomechanical parameters of chondrocyte-seeded tissue engineering (TE)-constructs. DESIGN: Articular chondrocytes were cultured in collagen-type-I/III-matrices over 6 weeks to create a biomechanical standard curve. Effects of stimulation with 100 ng/mL BMP-4/-7 heterodimer or 10 mM lithium chloride (LiCl) on ECM-deposition was quantified and characterized histologically. Biomechanical parameters were determined by the Very Low Rubber Hardness (VLRH) method and under confined compression stress relaxation. RESULTS: BMP-4/-7 treatment resulted in stronger collagen type-II staining and significantly enhanced glycosaminoglycan (GAG) deposition (3.2-fold; *P < 0.01) correlating with improved hardness (â¼1.7-fold; *P = 0.001) reaching 83% of native cartilage values after 28 days, a value not reached before 9 weeks without stimulation. LiCl treatment enhanced VLRH slightly, but significantly (â¼1.3-fold; *P = 0.016) with a trend to more ECM-deposition. BMP-4/-7 treatment significantly enhanced the E Modulus (105.7 ± 34.1 kPa; *P = 0.000001) compared to controls (8.0 ± 4.2 kPa). Poisson's ratio was significantly improved by BMP-4/-7 treatment (0.0703 ± 0.0409; *P = 0.013) vs controls (0.0432 ± 0.0284) and a significantly lower permeability (5.8 ± 2.1056 × 10(-14) m4/N.s; *P = 0.00001) was detected compared to untreated scaffolds (4.4 ± 3.1289 × 10(-13) m4/N.s). CONCLUSIONS: While Wnt-activation is less effective, BMP-4/-7 heterodimer stimulation approximated native cartilage features in less than 50% of standard culture time representing a promising strategy for functional cartilage TE to improve biomechanical parameters of engineered cartilage.
Subject(s)
Bone Morphogenetic Proteins/physiology , Cartilage, Articular/physiology , Tissue Engineering/methods , Wnt Signaling Pathway/physiology , Animals , Biomechanical Phenomena , Bone Morphogenetic Protein 4/pharmacology , Bone Morphogenetic Protein 7/pharmacology , Cartilage, Articular/cytology , Cartilage, Articular/metabolism , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/metabolism , Collagen Type II/metabolism , Extracellular Matrix/physiology , Glycosaminoglycans/metabolism , Hardness , Lithium Chloride/pharmacology , Sus scrofaABSTRACT
Specific biomechanical properties represent important quality markers of cartilage tissue engineering (TE) constructs. The aim of the study was to identify a sensitive biomechanical test to assess mechanical properties of cartilage TE constructs. Biomechanical testing of in vitro cultivated constructs following the very low rubber hardness (VLRH) principle illustrated significant differences between constructs cultured under chondrogenic conditions over various periods of time. An increase in proteoglycan and collagen type II deposition corresponded to increasing VLRH hardness values. Although a decrease in proteoglycan was detected after ectopic implantation of constructs into SCID mice, no reduction in biomechanical hardness values was observed. A functional estimation of TE constructs requires determination of biomechanical and biochemical parameters as quality features.
Subject(s)
Biocompatible Materials/chemistry , Fractures, Cartilage/physiopathology , Fractures, Cartilage/surgery , Guided Tissue Regeneration/instrumentation , Regeneration/physiology , Tissue Scaffolds , Animals , Biomechanical Phenomena , Equipment Design , Equipment Failure Analysis/methods , Fractures, Cartilage/pathology , Humans , Materials Testing/methods , Swine , Treatment OutcomeABSTRACT
Human mesenchymal stem cells (MSC) have attracted much attention for tissue regeneration including repair of non-healing bone defects. Heterogeneity of MSC cultures and considerable donor variability however, still preclude standardised production of MSC and point on functional deficits for some human MSC populations. We aimed to identify functional correlates of donor-dependency of bone formation in order to develop a potency assay predicting the therapeutic capacity of human MSC before clinical transplantation. MSC from 29 donors were characterised in vitro and results were correlated to bone formation potency in a beta-tricalcium-phosphate (ß-TCP)-scaffold after subcutaneous implantation into immunocompromised mice. In contrast to osteogenic in vitro differentiation parameters, a doubling time below 43.23 hours allowed to predict ectopic bone formation at high sensitivity (81.8%) and specificity (100%). Enriched conditions adapted from embryonic stem cell expansion rescued bone formation of inferior MSC populations while growth arrest of potent MSC by mitomycin C abolished bone formation, establishing a causal relationship between neo-bone formation and growth. Gene expression profiling confirmed a key role for proliferation status for the bone forming ability suggesting that a rate limiting anabolism and open chromatin determined and predicted the therapeutic potency of culture-expanded MSC. Proliferation-based potency testing and switch to enriched expansion conditions may pave the way for standardised production of MSC for bone repair.
Subject(s)
Bone Marrow Cells/cytology , Mesenchymal Stem Cells/cytology , Osteogenesis , Adolescent , Adult , Aged , Aged, 80 and over , Alkaline Phosphatase/metabolism , Animals , Bone Marrow Cells/physiology , Bone Regeneration , Calcification, Physiologic , Calcium Phosphates/therapeutic use , Cell Differentiation , Cell Proliferation/drug effects , Cells, Cultured , Child , Cluster Analysis , Enzyme Assays , Female , Gene Expression Profiling , Humans , Male , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Mice , Middle Aged , Mitomycin/pharmacology , Tissue Scaffolds , Transplantation, Heterologous , Young AdultABSTRACT
In order to evaluate the biomedical potential of three-dimensional chitinous scaffolds of poriferan origin, chondrocyte culturing experiments were performed. It was shown for the first time that freshly isolated chondrocytes attached well to the chitin scaffold and synthesized an extracellular matrix similar to that found in other cartilage tissue engineering constructs. Chitin scaffolds also supported deposition of a proteoglycan-rich extracellular matrix of chondrocytes seeded bioconstructs in an in vivo environment. We suggest that chitin sponge scaffolds, apart from the demonstrated biomedical applications, are highly optimized structures for use as filtering systems, templates for biomineralization as well as metallization in order to produce catalysts.
Subject(s)
Biomimetics/methods , Chitin/chemistry , Chitin/pharmacology , Molecular Conformation , Porifera/chemistry , Tissue Engineering/methods , Tissue Scaffolds , Animals , Cartilage/drug effects , Cartilage/physiology , Chitin/isolation & purification , Chondrocytes/cytology , Chondrocytes/drug effects , Humans , Regenerative Medicine , Tissue Scaffolds/chemistryABSTRACT
Marine invertebrate organisms including sponges (Porifera) not only provide an abundant source of biologically active secondary metabolites but also inspire investigations to develop biomimetic composites, scaffolds and templates for practical use in materials science, biomedicine and tissue engineering. Here, we presented a detailed study of the structural and physico-chemical properties of three-dimensional skeletal scaffolds of the marine sponges Aiolochroia crassa, Aplysina aerophoba, A. cauliformis, A. cavernicola, and A. fulva (Verongida: Demospongiae). We show that these fibrous scaffolds have a multilayered design and are made of chitin. (13)C solid-state NMR spectroscopy, NEXAFS, and IR spectroscopy as well as chitinase digestion and test were applied in order to unequivocally prove the existence of alpha-chitin in all investigated species.
Subject(s)
Chitin/analysis , Chitin/isolation & purification , Molecular Conformation , Porifera/chemistry , Animals , Chitin/chemistry , Chitin/metabolism , Chitinases/metabolism , Minerals/metabolism , Porifera/anatomy & histology , Spectrum Analysis , Trichoderma/enzymologyABSTRACT
Common in vitro protocols for chondrogenesis of mesenchymal stem cells (MSCs) induce an inadequate, hypertrophic differentiation cascade reminiscent of endochondral bone formation. We aimed to modify chondrogenic protocols in order to identify potent inducers, promotors, and inhibitors to achieve better chondrogenesis. Nine factors suspected to stimulate or inhibit chondrogenesis were used for chondrogenic in vitro induction of MSC. Differentiation was assessed by immunohistochemistry, alcian-blue staining, qRT-PCR, and quantification of alkaline phosphatase (ALP) activity. Pre-differentiated pellets were transplanted subcutaneously into SCID mice to investigate stable cartilage formation. Transforming growth factor (TGF)-beta was always required for chondrogenic differentiation and deposition of a collagen-type-II-positive extracellular matrix, while bone morphogenetic protein (BMP)-2, -4, -6, -7, aFGF, and IGF-I (10 ng/ml) were alone not sufficiently inductive. Each of these factors allowed differentiation in combination with TGF-beta, however, without preventing collagen type X expression. bFGF or parathyroid hormone-like peptide (PTHrP) inhibited the TGF-beta-responsive COL2A1 and COL10A1 expression and ALP induction when added from day 0 or 21. In line with a reversible ALP inhibition, in vivo calcification of pellets was not prevented. Late up-regulation of PTH1R mRNA suggests that early PTHrP effects may be mediated by a receptor-independent pathway. While TGF-beta was a full inducer, bFGF and PTHrP were potent inhibitors for early and late chondrogenesis, seemed to induce a shift from matrix anabolism to catabolism, but did not selectively suppress COL10A1 expression. Within a developmental window of collagen type II(+)/collagen type X(-) cells, bFGF and PTHrP may allow inhibition of further differentiation toward hypertrophy to obtain stable chondrocytes for transplantation purposes.
Subject(s)
Cell Differentiation , Chondrocytes/metabolism , Chondrogenesis , Intercellular Signaling Peptides and Proteins/metabolism , Mesenchymal Stem Cells/metabolism , Parathyroid Hormone-Related Protein/metabolism , Peptide Fragments/metabolism , Alcian Blue , Alkaline Phosphatase/metabolism , Animals , Biomarkers/metabolism , Bone Morphogenetic Proteins/metabolism , Cell Shape , Cells, Cultured , Chondrocytes/pathology , Chondrocytes/transplantation , Collagen Type II/genetics , Collagen Type X/genetics , Coloring Agents , Fibroblast Growth Factor 1/metabolism , Humans , Hypertrophy , Immunohistochemistry , Insulin-Like Growth Factor I/metabolism , Matrix Metalloproteinase 13/genetics , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/pathology , Mice , Mice, SCID , Phenotype , RNA, Messenger/metabolism , Receptor, Parathyroid Hormone, Type 1/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Staining and Labeling/methods , Time Factors , Transforming Growth Factor beta/metabolismABSTRACT
OBJECTIVE: Mesenchymal stem cells (MSCs) are a population of cells broadly discussed to support cartilage repair. The differentiation of MSCs into articular chondrocytes is, however, still poorly understood on the molecular level. The aim of this study was to perform an almost genome-wide screen for genes differentially expressed between cartilage and MSCs and to extract new markers useful to define chondrocyte differentiation stages. METHODS: Gene expression profiles of MSCs (n=8) and articular cartilage from OA patients (n=7) were compared on a 30,000 cDNA-fragment array and differentially expressed genes were extracted by subtraction. Expression of selected genes was assessed during in vitro chondrogenic differentiation of MSCs and during dedifferentiation of expanded chondrocytes using quantitative and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: Eighty-seven genes were differentially expressed between MSCs and cartilage with a more than three-fold difference. Sixty-seven of them were higher expressed in cartilage and among them 15 genes were previously not detected in cartilage. Differential expression was confirmed for 69% of 26 reanalysed genes by RT-PCR. The profiles of three unknown transcripts and six protease-related molecules were characterised during differentiation. SERPINA1 and SERPINA3 mRNA expression correlated with chondrogenic differentiation of MSCs and dedifferentiation of chondrocytes, and SERPINA1 protein levels in culture supernatants could be correlated alike. CONCLUSIONS: cDNA-array analysis identified SERPINA1 and A3 as new differentiation-relevant genes for cartilage. Since SERPINA1 secretion correlated with both chondrogenesis of MSCs and dedifferentiation during chondrocyte expansion, it represents an attractive marker for refinement of chondrocyte differentiation.
Subject(s)
Cartilage, Articular/cytology , Chondrocytes/cytology , Chondrogenesis , Gene Expression Profiling , Mesenchymal Stem Cells/cytology , Osteoarthritis/genetics , Adult , Aged , Aged, 80 and over , Antigens, Differentiation/genetics , Cartilage, Articular/metabolism , Cell Differentiation , Chondrocytes/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Mesenchymal Stem Cells/metabolism , Microarray Analysis , Middle Aged , Oligonucleotide Array Sequence Analysis , Osteoarthritis/metabolism , Osteoarthritis/pathology , RNA, Messenger/analysis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Serpins/genetics , Serpins/metabolism , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/metabolismABSTRACT
In the search for new marker genes suitable to distinguish chondrocytes from osteoblasts and mesenchymal stem cells in culture, we have identified and characterized a novel gene called chondrocyte expressed protein-68 (CEP-68), harbouring an N-terminal leader peptide and an epidermal growth factor-like calcium-binding domain. CEP-68 defines a new family of proteins and complements collagen type II as a new marker for stem-cell-based chondrogenic tissue engineering.
Subject(s)
Calcium-Binding Proteins/genetics , Chondrocytes/metabolism , Amino Acid Sequence , Base Sequence , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Cell Differentiation/genetics , Cells, Cultured , DNA, Complementary/analysis , Gene Expression , Genetic Markers , Humans , Molecular Sequence Data , Osteoblasts/metabolism , Phenotype , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The melanoma antigen gene (MAGE) family comprises 12 known genes, of which 6 are expressed in tumors. In the course of a systematic analysis of transcripts in Xq28, we have identified cDNAs related to different MAGE genes. Analysis of cell hybrids, ordered YACs, and cosmids showed that all MAGE genes are located in Xq28 and are clustered in three main intervals within 3.5 Mb. The six genes expressed in tumors are contained in the two intervals closest to the telomere and are highly homologous to each other. Analysis of different species suggests that human MAGE sequences are conserved in primates, but less well conserved in other vertebrate species.
Subject(s)
Antigens, Neoplasm/genetics , Melanoma/genetics , Multigene Family , Neoplasm Proteins/genetics , X Chromosome , Animals , Base Sequence , CHO Cells , Chromosome Mapping , Chromosomes, Artificial, Yeast , Conserved Sequence , Cosmids , Cricetinae , DNA Fingerprinting , DNA, Complementary , Exons , Humans , Melanoma/immunology , Melanoma-Specific Antigens , Polymerase Chain Reaction , Primates , Sequence Homology, Nucleic Acid , VertebratesSubject(s)
Amidines/therapeutic use , Benzamidines , Trypanocidal Agents/therapeutic use , Trypanosomiasis, African/drug therapy , Amidines/toxicity , Animals , Female , Macaca mulatta , Male , Pentamidine/therapeutic use , Trypanocidal Agents/toxicity , Trypanosoma brucei brucei , Trypanosomiasis, African/diagnosisSubject(s)
Amidines/therapeutic use , Antiprotozoal Agents/therapeutic use , Leishmaniasis, Visceral/drug therapy , Malaria/drug therapy , Trypanosomiasis, African/drug therapy , Animals , Cricetinae , Drug Evaluation, Preclinical , Folic Acid/metabolism , Leishmania/drug effects , Mice , Plasmodium berghei/drug effects , Structure-Activity Relationship , Trypanosoma/drug effectsSubject(s)
Antiprotozoal Agents/therapeutic use , Protozoan Infections/drug therapy , Animals , Apicomplexa , Babesiosis/drug therapy , Chagas Disease/drug therapy , Coccidiosis/drug therapy , Female , Humans , Intestinal Diseases, Parasitic/drug therapy , Leishmaniasis/drug therapy , Malaria/drug therapy , Male , Pneumonia, Pneumocystis/drug therapy , Sarcocystosis/drug therapy , Toxoplasmosis/drug therapy , Trichomonas Infections/drug therapy , Trypanosomiasis, African/drug therapySubject(s)
Antiprotozoal Agents/therapeutic use , Liposomes/administration & dosage , Organometallic Compounds , Protozoan Infections/drug therapy , Animals , Antimony/therapeutic use , Antimony Sodium Gluconate/therapeutic use , Disease Models, Animal , Glycolipids/therapeutic use , Humans , Leishmaniasis/drug therapy , Leishmaniasis, Visceral/drug therapy , Lysosomes/metabolism , Malaria/drug therapy , Meglumine/analogs & derivatives , Meglumine/therapeutic use , Meglumine Antimoniate , Primaquine/therapeutic use , Trypanosomiasis/drug therapyABSTRACT
For identifying drugs which might be effective in the treatment of human Babesia microti infections, 20 selected antiprotozoal agents or combinations of agents were tested for activity against B. microti in Mongolian jirds (meriones unguiculatus). 4-Methyl-primaquine and aromatic diamidines, including diminazene and pentamidine, were the most effective compounds tested.
Subject(s)
Antiprotozoal Agents/therapeutic use , Babesiosis/drug therapy , Animals , Gerbillinae , Male , Structure-Activity RelationshipSubject(s)
Aminoquinolines/therapeutic use , Antimony/therapeutic use , Leishmaniasis, Visceral/drug therapy , Liposomes/administration & dosage , Tetracycline/therapeutic use , Aminoquinolines/administration & dosage , Animals , Antimony/administration & dosage , Cricetinae , Leishmaniasis, Visceral/prevention & control , Tetracycline/administration & dosageABSTRACT
Members of a class of compounds designated lepidines (8-amino-6-methoxy-4-methylquinoline derivatives) were tested in a hamster-Leishmania donovani model and found to have activity many-fold that of a reference drug meglumine antimoniate. One of them, 8-(7-isopropylaminoheptylamino)-6-methoxy-4-methylquinoline, was found to be 138 times as effective as the standard antimonial drug used.
Subject(s)
Aminoquinolines/therapeutic use , Antiprotozoal Agents/therapeutic use , Cricetinae , Leishmaniasis/veterinary , Mesocricetus , Rodent Diseases/drug therapy , Animals , Leishmaniasis/drug therapy , MaleABSTRACT
Of 303 compounds (66 active against cancer and 237 inactive against cancer) obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland, 25 were found to be active against Trypanosoma rhodesiense infections of ICR/Ha Swiss mice. Fifteen of these 25 compounds also had anticancer properties. The percentage of anticancer compounds found to have antitrypanosomiasis properties was 22.7. This percentage compares with 6% antitrypanosomiasis compounds among compounds selectecd by other methods.
Subject(s)
Antineoplastic Agents/pharmacology , Trypanocidal Agents , Trypanosomiasis/drug therapy , Animals , Drug Evaluation, Preclinical , Female , Male , Mice , Mice, Inbred ICRABSTRACT
Liposomes containing neutral glycolipids with a terminal glucose or galactose, when injected intravenously, prevented the appearance of erythrocytic forms of malaria (Plasmodium berghei) in mice previously injected with sporozoites. Inhibitory glycolipids included glucosyl, galactosyl, or lactosyl ceramide. Inhibition was not observed with liposomes containing ceramide, phosphocholine ceramide, sulfogalactosyl ceramide (sulfatide), or ganglioside GM1. Liposomes containing glycolipids did not inhibit infection transmitted by injecting blood containing erythrocytic stages of malaria. These results may have therapeutic implications in the treatment of malaria. Analysis of the mechanism of interference with the life cycle of malaria by liposomal glycolipids may yield information about the interactions of parasites with cellular membranes.
Subject(s)
Glycolipids/therapeutic use , Malaria/therapy , Animals , Ceramides/therapeutic use , Erythrocytes/parasitology , Liposomes/therapeutic use , Liver/parasitology , Malaria/parasitology , Mice , Plasmodium berghei , Structure-Activity RelationshipABSTRACT
Antitrypanosomal activity for benzyltriphenylphosphonium salts is reported for the first time. Testing was conducted using Trypanosoma rhodesiense infected mice. Of 70 phosphorus-containing compounds tested, 21 were active. Sixteen of these active chemical species were benzyltriphenylphosphonium salts. Four were nonbenzyl triphenyl compounds. The remaining active drug was a benzyldiphenylphosphonium salt.