Cryopreservation based on freezing protocols for the long-term storage of microencapsulated myoblasts

One important challenge in biomedicine is the ability to cryogenically preserve not only cells, but also tissue-engineered constructs. In the present paper, alginate-poly-l-lysine-alginate (APA) microcapsules containing erythropoietin (Epo)-secreting C₂C₁₂ myoblasts were elaborated, characterized and tested both in vitro and in vivo. Dimethylsulfoxide (DMSO) was selected as cryoprotectant to evaluate the maintenance of physiological activity of cryopreserved microencapsulated myoblasts employing procedures based on freezing protocols up to a 45-day cryopreservation period. High chemical resistance of the cryopreserved microcapsules was observed using 10% DMSO as cryoprotectant following a standard slow-cooling procedure. Although a 42% reduction in Epo release from the microencapsulated cells was observed in comparison with the non-cryopreserved group, the in vivo biocompatibility and functionality of the encapsulated cells subcutaneously implanted in Balb/c mice was corroborated by high and sustained hematocrit levels over 194 days and lacking immunosuppressive protocols. No major host reaction was observed. Based on the results obtained in our study, a slow-cooling protocol using 10% DMSO as cryoprotectant (confirmed for cryopreservation periods up to 45 days) might be considered a suitable therapeutic strategy if the long-term storage of microencapsulated cells, such as C₂C₁₂ myoblasts is pretended.     

Liposome‐bound APO2L/TRAIL is an effective treatment in a rabbit model of rheumatoid arthritis

Objective

We previously observed that T lymphocytes present in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were sensitive to APO2L/TRAIL. In addition, there was a drastic decrease in the amount of bioactive APO2L/TRAIL associated with exosomes in SF from RA patients. This study was undertaken to evaluate the effectiveness of bioactive APO2L/TRAIL conjugated with artificial lipid vesicles resembling natural exosomes as a treatment in a rabbit model of antigen‐induced arthritis (AIA).

Methods

We used a novel Ni²⁺‐(N‐5‐amino‐1‐carboxypentyl)‐iminodiacetic acid)–containing liposomal system. APO2L/TRAIL bound to liposomes was intraarticularly injected into the knees of animals with AIA. One week after treatment, rabbits were killed, and arthritic synovial tissue was analyzed.

Results

Tethering APO2L/TRAIL to the liposome membrane increased its bioactivity and resulted in more effective treatment of AIA compared with soluble, unconjugated APO2L/TRAIL, with substantially reduced synovial hyperplasia and inflammation in rabbit knee joints. The results of biophysical studies suggested that the increased bioactivity of APO2L/TRAIL associated with liposomes was due to the increase in the local concentration of the recombinant protein, augmenting its receptor crosslinking potential, and not to conformational changes in the protein. In spite of this increase in bioactivity, the treatment lacked systemic toxicity and was not hepatotoxic.

Conclusion

Our findings indicate that binding APO2L/TRAIL to the liposome membrane increases its bioactivity and results in effective treatment of AIA.

     

Platelet-Rich Plasma to Improve the Bio-Functionality of Biomaterials

Growth factors and cytokines are active players in controlling the different stages of wound healing and tissue regeneration. Recent trends in personalized regenerative medicine involve using patient’s own platelet-rich plasma for stimulating wound healing and tissue regeneration. This technology provides a complex cocktail of growth factors and even a fibrin scaffold with multiple biologic effects. In the last few years, an increasing number of studies provide evidence of the potential of combining platelet-rich plasma with different biomaterials in order to improve their properties, including handling, administration, bioactivity, and level of osseointegration, among others. In this review, we discuss the use of platelet-rich plasma as an alternative, easy, cost-effective, and controllable strategy for the release of high concentrations of many endogenous growth factors. Additionally, we provide an overview of the current progress and future directions of research combining different types of biomaterials with platelet-rich plasma in tissue engineering and regenerative medicine.     

The potential impact of the preparation rich in growth factors (PRGF) in different medical fields

Platelet-rich preparations constitute a relatively new biotechnology for the stimulation and acceleration of tissue healing and bone regeneration. The versatility and biocompatibility of this approach has stimulated its therapeutic use in numerous medical and scientific fields including dentistry, oral implantology, orthopaedics, ulcer treatment, tissue engineering among others. Here we discuss the important progress that has been accomplished in the field of platelet-rich preparations in the last few years. Some of the most interesting therapeutic applications of this technology are discussed as are some of the limitations, future challenges and directions in the field.     

Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: putative high-affinity Ca(2+)-binding site in the protease domain.

The Fab fragment of a monoclonal antibody (mAb) reactive to the N-terminal half (residues 180-310) of the protease domain of human factor IX has been previously shown to inhibit the binding of factor IXa to its cofactor, factor VIIIa. These data suggested that this segment of factor IXa may participate in binding to factor VIIIa. We now report that the binding rate (kon) of the mAb is 3-fold higher in the presence of Ca2+ than in its absence for both factors IX and IXa; the half-maximal effect was observed at approximately 300 microM Ca2+. Furthermore, the off rate (koff) of the mAb is 10-fold higher for factor IXa than for factor IX with or without Ca2+. Moreover, like the kon for mAb binding, the incorporation of dansyl-Glu-Gly-Arg chloromethyl ketone (dEGR-CK) into factor IXa was approximately 3 times faster in the presence of Ca2+ than in its absence. Since steric factors govern the kon and the strength of noncovalent interactions governs the koff, the data indicate that the region of factor IX at residues 180-310 undergoes two separate conformational changes before expression of its biologic activity: one upon Ca2+ binding and the other upon zymogen activation. Furthermore, the dEGF-CK incorporation data suggest that both conformational changes also affect the active site residues. Analyses of the known three-dimensional structures of serine proteases indicate that in human factor IX a high-affinity Ca(2+)-binding site may be formed by the carboxyl groups of glutamates 235 and 245 and by the main chain carbonyl oxygens of residues 237 and 240. In support of this conclusion, a synthetic peptide including residues 231-265 was shown to bind Ca2+ with a Kd of approximately 500 microM. This peptide also bound to the mAb, although with approximately 500-fold reduced affinity. Moreover, like factor IX, the peptide bound to the mAb more strongly (approximately 3-fold) in the presence of Ca2+ than in its absence. Thus, it appears that a part of the epitope for the mAb described above is contained in the proposed Ca(2+)-binding site in the protease domain of human factor IX. This proposed site is analogous to the Ca(2+)-binding site in trypsin and elastase, and it may be involved in binding factor IXa to factor VIIIa.     

Association between neural reactivity and startle reactivity to uncertain threat in two independent samples

Prior studies indicate that anxiety disorders are associated with heightened sensitivity to uncertain threat (U threat). Individual differences in reactivity to U threat have been measured in the laboratory with two methodologies—startle eyeblink potentiation and fMRI. While startle and fMRI are purported to relate to each other, very little research exists on whether individual differences in one measure are associated with individual differences in another and, thus, whether startle and fMRI capture shared mechanisms. Therefore, the current study was designed to investigate if and where in the brain measures of startle potentiation and fMRI BOLD signal correlate during response to U threat across two independent samples. Participants in both studies completed two threat anticipation tasks—once during collection of startle potentiation and once during fMRI. In Study 1 (n = 43), the startle and fMRI tasks both used electric shock as the threat. As an extension, in Study 2 (n = 38), the startle task used electric shock but the fMRI task used aversive images. Despite these methodological differences, greater startle potentiation to U threat was associated with greater dorsal anterior cingulate, caudate, and orbitofrontal cortex reactivity to U threat in both samples. The findings suggest that startle and fMRI measures of responding to U threat overlap, and points toward an integrated brain‐behavior profile of aberrant U threat responding.     

Quantification of left and right ventricular function and myocardial mass: comparison of low-radiation dose 2nd generation dual-source CT and cardiac MRI

Objective

To prospectively evaluate the accuracy of left and right ventricular function and myocardial mass measurements based on a dual-step, low radiation dose protocol with prospectively ECG-triggered 2nd generation dual-source CT (DSCT), using cardiac MRI (cMRI) as the reference standard.

Materials and methods

Twenty patients underwent 1.5 T cMRI and prospectively ECG-triggered dual-step pulsing cardiac DSCT. This image acquisition mode performs low-radiation (20% tube current) imaging over the majority of the cardiac cycle and applies full radiation only during a single adjustable phase. Full-radiation-phase images were used to assess cardiac morphology, while low-radiation-phase images were used to measure left and right ventricular function and mass. Quantitative CT measurements based on contiguous multiphase short-axis reconstructions from the axial CT data were compared with short-axis SSFP cardiac cine MRI. Contours were manually traced around the ventricular borders for calculation of left and right ventricular end-diastolic volume, end-systolic volume, stroke volume, ejection fraction and myocardial mass for both modalities. Statistical methods included independent t-tests, the Mann–Whitney U test, Pearson correlation statistics, and Bland–Altman analysis.

Results

All CT measurements of left and right ventricular function and mass correlated well with those from cMRI: for left/right end-diastolic volume r = 0.885/0.801, left/right end-systolic volume r = 0.947/0.879, left/right stroke volume r = 0.620/0.697, left/right ejection fraction r = 0.869/0.751, and left/right myocardial mass r = 0.959/0.702. Mean radiation dose was 6.2 ± 1.8 mSv.

Conclusions

Prospectively ECG-triggered, dual-step pulsing cardiac DSCT accurately quantifies left and right ventricular function and myocardial mass in comparison with cMRI with substantially lower radiation exposure than reported for traditional retrospective ECG-gating.