Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis

In this study we investigated the use of a fibrin hydrogel to improve the potential of a polyurethane (PU) scaffold-based system for articular cartilage tissue engineering. PU-only ("no-fibrin") and PU-fibrin ("fibrin") composites were cultured for up to 28 days and analyzed for DNA content, glycosaminoglycan (GAG) content, type II collagen content, GAG release, and gene expression of aggrecan, collagen I, and collagen II. The use of fibrin allowed for higher viable cell-seeding efficiency (10% higher DNA content on day 2 in fibrin versus no-fibrin composites) and more even cell distribution on seeding, a more than 3-fold increase in the percentage of newly synthesized GAG retained in the constructs, and 2- to 6-fold higher levels of type II collagen and aggrecan gene expression through day 14. Addition of aprotinin to the medium inhibited fibrin degradation, most noticeably in the center of the constructs, but had little effect on biochemical composition or gene expression. Short-term mechanical compression (0-10% sinusoidal strain at 0.1 Hz for 1 h, applied twice daily for 3 days) doubled the rate of GAG release from the constructs, but had little effect on gene expression, regardless of the presence of fibrin. Although further work is needed to optimize this system, the addition of fibrin hydrogel to encapsulate cells in the stiff, macroporous PU scaffold is a step forward in our approach to articular cartilage tissue engineering.     

Functional domains of human interferon gamma probed with antipeptide antibodies

Functional domains of biologically active polypeptide molecules can be sought by raising antibodies to synthetic peptides. Human interferon gamma (HuIFN gamma) was thus studied, using two peptides based on candidate regions representing amino acids 7-16 and 121-130 of the HuIFN gamma molecule. These were conjugated to bovine serum albumin prior to immunization of rabbits. High titres of antipeptide antibodies which recognized the synthetic peptides were elicited in all of the four rabbits injected. The antipeptide antibodies from one of the rabbits immunized with the C-terminal (121-130) peptide detected native HuIFN gamma at a concn as low as 300 IU/ml, but the antipeptide antibodies from the rabbit immunized with the N-terminal (7-16) peptide did not detect HuIFN gamma. The IFN gamma-reactive antipeptide antibodies (anti-121-130) did not neutralize the antiviral activity of HuIFN gamma in a cytoprotection assay. These data and other studies establish that the C-terminus of HuIFN gamma is not essential for full antiviral activity and indicate an application of antipeptide antibodies in the analysis of structure-function relationships of cytokine molecules.     

Mutations in the cytochrome b gene of Plasmodium berghei conferring resistance to atovaquone

The molecular lesions which underlie the resistance of the malaria parasites to atovaquone, a coenzyme Q analogue, were investigated. Resistant clones of Plasmodium berghei ANKA strain were isolated following prolonged propagation in mice in the presence of increasing doses of the drug, and their cytochrome b gene sequenced. Three mutations were detected, T-C substitution at nt 431, G-A at nt 399 and G-T at nt 850, resulting in amino acid changes in the putative cytochrome b product at residues 133, 144 and 284. The V284F amino acid change is in the sixth transmembrane helix of the protein and was observed in all resistant clones. An additional M133I or L144S amino acid change within the Qo site at an extramembranous amphipathic helix significantly increases the resistance to atovaquone. Our results (a) provide evidence that the antimalarial activity of atovaquone indeed involves an interaction with the cytochrome b; (b) define atovaquone as an inhibitor of the ubiquinol oxidase activity of the cytochrome bc1 complex; and (c) define amino acid residues in the mammalian cytochrome b which might be critical in determining its relative resistance to atovaquone.     

Nanohydroxyapatite/poly(ester urethane) scaffold for bone tissue engineering

Biodegradable viscoelastic poly(ester urethane)-based scaffolds show great promise for tissue engineering. In this study, the preparation of hydroxyapatite nanoparticles (nHA)/poly(ester urethane) composite scaffolds using a salt-leaching-phase inverse process is reported. The dispersion of nHA microaggregates in the polymer matrix were imaged by microcomputed X-ray tomography, allowing a study of the effect of the nHA mass fraction and process parameters on the inorganic phase dispersion, and ultimately the optimization of the preparation method. How the composite scaffold’s geometry and mechanical properties change with the nHA mass fraction and the process parameters were assessed. Increasing the amount of nHA particles in the composite scaffold decreased the porosity, increased the wall thickness and consequently decreased the pore size. The Young’s modulus of the poly(ester urethane) scaffold was improved by 50% by addition of 10 wt.% nHA (from 0.95 ± 0.5 to 1.26 ± 0.4 MPa), while conserving poly(ester urethane) viscoelastic properties and without significant changes in the scaffold macrostructure. Moreover, the process permitted the inclusion of nHA particles not only in the poly(ester urethane) matrix, but also at the surface of the scaffold pores, as shown by scanning electron microscopy. nHA/poly(ester urethane) composite scaffolds have great potential as osteoconductive constructs for bone tissue engineering.     

New insight into functional limb regeneration: A to Z approaches

Limb/digit amputation is a common event in humans caused by trauma, medical illness, or surgery. Although the loss of a digit is not lethal, it affects quality of life and imposes high costs on amputees. In recent years, the increasing interest in limb regeneration has led to enhanced scientific knowledge. However, the limited ability to develop functional limb regeneration in the clinical setting suggests that a challenging issue remains in limb regeneration. Recently, the emergence of regenerative engineering is a promising field to address this challenge and close the gap between science and clinical applications. Cell signalling and molecular mechanisms involved in the limb regeneration process have been extensively studied; however, there is still insufficient data on cell therapy and tissue engineering for limb regeneration. In this review, we intend to focus on therapeutic approaches for limb regeneration that are closely related to gene, immune, and stem cell therapies, as well as tissue engineering approaches that take into consideration the peculiar developmental properties of the limbs. In addition, we attempt to identify the challenges of these strategies for limb regeneration studies in terms of clinical settings and as a road map to accomplish the goal of functional human limb regeneration.     

The extracellular matrix of cartilage in the growth plate before and during calcification: Changes in composition and degradation of type II collagen

Summary Calcification occurs in the extracellular matrix of the hypertrophic zone of the growth plate when the extra-cellular matrix volume is reduced to a minimum and alkaline phosphatase content is maximal. The present study shows that significant quantitative and qualitative changes occur in the composition and structure of macromolecules in the extracellular matrix before and during calcification in the proximal tibial growth plate of the bovine fetus. These were detected in part by using microchemical and microimmuno-chemical analyses of sequential transverse frozen sections at chemical analyses of sequential transverse frozen sections at defined sites throughout the growth plate. Concentrations of matrix molecules in the extracellular matrix have not previously been determined biochemically. They were measured per unit matrix volume by using combined immunochemical/chemical-histomorphometric analyses. The concentrations within the extracellular matrix of the C-propeptide of type II collagen, aggregating proteoglycan (aggrecan), and hyaluronic acid all progressively increased in the maturing and hypertrophic zones, being maximal (or near maximal) at the time of initiation of mineralization. These results for proteoglycan are contrary to some earlier reports of a loss of proteoglycan prior to mineralization which measured the tissue content of proteoglycan rather than that present in the extracellular matrix, the volume of which is progressively reduced as the growth plate matures. The C-propeptide data provides a quantitative confirmation of previous immunohistochemical studies. Total collagen concentration (measured as hydroxyproline) in the extracellular matrix initially increased through the proliferating and maturing zones but then rapidly decreased in the hypertrophic zone. Immunohistochemical studies revealed that this is associated with the unwinding of the triple helix of type II collagen (previously shown to result from cleavage) which starts in pericellular sites in the zone of maturation (when type X collagen is first synthesized) and then extends throughout the hypertrophic zone. The significance of these matrix changes in the development and mineralization of the growth plate is discussed.     

Ethnogeographical structure of hepatitis B virus genotype distribution in Indonesia and discovery of a new subgenotype, B9

The distribution of hepatitis B virus (HBV) in the populations of island Southeast Asia is of medical and anthropological interest and is associated with an unusually high genetic diversity. This study examined the association of this HBV genetic diversity with the ethnogeography of the populations of the Indonesian archipelago. Whole genome analysis of 21 HBV isolates from East Nusa Tenggara and Papua revealed two recently reported HBV/B subgenotypes unique to the former, B7 (7 isolates) and B8 (5 isolates), and uncovered a further novel subgenotype designated B9 (4 isolates). Further isolates were collected from 419 individuals with defined ethnic backgrounds representing 40 populations. HBV/B was predominant in Austronesian-language-speaking populations, whereas HBV/C was the major genotype in Papua and Papua-influenced populations of Moluccas; HBV/B3 was the predominant subgenotype in the western half of the archipelago (speakers of the Western Malayo-Polynesian [WMP] branch of Austronesian languages), whereas B7, B8 and B9 were specific to Nusa Tenggara (Central Malayo-Polynesian (CMP)). The result provides the first direct evidence that the distribution of HBV genotypes/subgenotypes in the Indonesian archipelago is related to the ethnic origin of its populations and suggests that the HBV distribution is associated with the ancient migratory events in the peopling of the archipelago.     

Mitochondrial studies in Kearns-Sayre syndrome: normal respiratory chain function with absence of a mitochondrial translation product

Intact mitochondria were isolated from skeletal muscle of two patients with Kearns-Sayre syndrome (retinitis pigmentosa, heart block, chronic external ophthalmoplegia), and mitochondrial protein translation was measured. Mitochondrial protein synthesis was up to 10 times greater than in control subjects and SDS-polyacrylamide gel electrophoresis revealed absence of a translation product with the mobility of a 5 KDa protein. State 3 respiration rates were normal with site 1 and site 2 substrates, suggesting that the absent protein was not a functional subunit of a respiratory chain complex.