Fascin Rigidity and L-plastin Flexibility Cooperate in Cancer Cell Invadopodia and Filopodia.

Invadopodia and filopodia are dynamic, actin-based protrusions contributing to cancer cell migration, invasion, and metastasis. The force of actin bundles is essential for their protrusive activity. The bundling protein fascin is known to play a role in both invadopodia and filopodia. As it is more and more acknowledged that functionally related proteins cooperate, it is unlikely that only fascin bundles actin in these protrusions. Another interesting candidate is L-plastin, normally expressed in hematopoietic cells, but considered a common marker of many cancer types. We identified L-plastin as a new component of invadopodia, where it contributes to degradation and invasiveness. By means of specific, high-affinity nanobodies inhibiting bundling of fascin or L-plastin, we further unraveled their cooperative mode of action. We show that the bundlers cannot compensate for each other due to strikingly different bundling characteristics: L-plastin bundles are much thinner and less tightly packed. Composite bundles adopt an intermediate phenotype, with fascin delivering the rigidity and strength for protrusive force and structural stability, whereas L-plastin accounts for the flexibility needed for elongation. Consistent with this, elevated L-plastin expression promotes elongation and reduces protrusion density in cells with relatively lower L-plastin than fascin levels.

Polydopamine-Gelatin as Universal Cell-Interactive Coating for Methacrylate-Based Medical Device Packaging Materials: When Surface Chemistry Overrules Substrate Bulk Properties.

Despite its widespread application in the fields of ophthalmology, orthopedics, and dentistry and the stringent need for polymer packagings that induce in vivo tissue integration, the full potential of poly(methyl methacrylate) (PMMA) and its derivatives as medical device packaging material has not been explored yet. We therefore elaborated on the development of a universal coating for methacrylate-based materials that ideally should reveal cell-interactivity irrespective of the polymer substrate bulk properties. Within this perspective, the present work reports on the UV-induced synthesis of PMMA and its more flexible poly(ethylene glycol) (PEG)-based derivative (PMMAPEG) and its subsequent surface decoration using polydopamine (PDA) as well as PDA combined with gelatin B (Gel B). Successful application of both layers was confirmed by multiple surface characterization techniques. The cell interactivity of the materials was studied by performing live-dead assays and immunostainings of the cytoskeletal components of fibroblasts. It can be concluded that only the combination of PDA and Gel B yields materials possessing similar cell interactivities, irrespective of the physicochemical properties of the underlying substrate. The proposed coating outperforms both the PDA functionalized and the pristine polymer surfaces. A universal cell-interactive coating for methacrylate-based medical device packaging materials has thus been realized.

Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization.

Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin-SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (K(d)~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP-9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin-bundling-independent role in MMP-9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.

Follicles of various maturation stages react differently to enzymatic isolation: a comparison of different isolation protocols.

Isolation of human follicles is based on digestion of the tissue by combinations of enzymes. Follicle vitality and morphology are often based on the analysis of pooled follicles of different maturation stages. Information is therefore lacking on the effect of the isolation protocol to individual follicles of different maturation stages. A study was conducted using five protocols combining different enzymes and varying concentrations. Isolated follicles were classified according to their maturation stages, counted and characterized for vitality, morphology, early apoptosis and organization of transzonal projections. No statistical differences were found between the protocols when outcome parameters were analysed on a pool of follicles regardless of their maturation status. Differences were observed in quality when the follicles were analysed separately according to their maturation status. Combining morphologic characteristics and vitality, both Liberase DH and Liberase TM combined with collagenase IV were better at isolating high-quality primordial follicles, compared with collagenase IV. No statistical difference between the isolation protocols was found for primary follicles. If only high-quality isolated secondary follicles are needed, collagenase IV is found to be most advantageous. Follicles of different maturation stages react differently when enzymatic isolation protocols are compared.

Interactions of highly and low virulent Flavobacterium columnare isolates with gill tissue in carp and rainbow trout.

The interactions of Flavobacterium columnare isolates of different virulence with the gills of carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum) were investigated. Both fish species were exposed to different high (HV) or low virulence (LV) isolates and sacrificed at seven predetermined times post-challenge. Histopathological and ultrastructural examination of carp and rainbow trout inoculated with the HV-isolate disclosed bacterial invasion and concomitant destruction of the gill tissue, gradually spreading from the filament tips towards the base, with outer membrane vesicles surrounding most bacterial cells. In carp, 5-10% of the fish inoculated with the LV-isolate became moribund and their gill tissue displayed the same features as described for the HV-isolate, albeit to a lesser degree. The bacterial numbers retrieved from the gill tissue were significantly higher for HV- compared to LV-isolate challenged carp and rainbow trout. TUNEL-stained and caspase-3-immunostained gill sections demonstrated significantly higher apoptotic cell counts in carp and rainbow trout challenged with the HV-isolate compared to control animals. Periodic acid-Schiff/alcian blue staining demonstrated a significantly higher total gill goblet cell count for HV- and LV-isolate challenged compared to control carp. Moreover, bacterial clusters were embedded in a neutral matrix while being encased by acid mucins, resembling biofilm formation. Eosinophilic granular cell counts were significantly higher in the HV-isolate compared to LV-isolate inoculated and control carp. The present data indicate a high colonization capacity, and the destructive and apoptotic-promoting features of the HV-isolate, and point towards important dynamic host mucin-F. columnare interactions warranting further research.

Non-Cytotoxic Crosslinkers for Heart Valve Tissue Engineering.

BACKGROUND AND AIM OF THE STUDY: Currently, no effective crosslinking reagents are available to treat xenogenic decellularized heart valve matrices. The study aim was to evaluate the crosslinking effect of quercetin, catechin, caffeic acid and tannic acid on porcine aortic valve matrices. METHODS: Cytotoxicity of the different crosslinkers was evaluated. The mechanical properties of crosslinked porcine matrices and control matrices (non-fixed) were examined by tensile strength testing, as was the cytocompatibility of the fixed matrices. Crosslinked and control matrices were implanted subcutaneously in Wistar rats (n = 9) and, after two weeks, their calcium contents were determined using inductively coupled plasma-mass spectrometry. The antibody reaction against porcine tissue in rat serum was also determined. RESULTS: Cytotoxicity studies showed that crosslinkers, even at high concentrations, did not inhibit cell viability. All crosslinkers except tannic acid improved the mechanical strength of acellular porcine matrices. Moreover, the tensile strength of quercetin-fixed matrices was comparable with that of glutaraldehyde (GTA)-fixed leaflets. Light microscopic evaluation showed that crosslinked matrices caused only a mild lymphocytic inflammatory reaction. Furthermore, quercetin-fixed leaflets exhibited a well-preserved matrix without infiltration of CD3+ cells. After two weeks, calcium levels were 206.33 µg/mg for controls (non-fixed), and 151.33 µg/mg, 181 µg/mg and 163.66 µg/mg for quercetin-, catechin-, and caffeic acid-fixed matrices, respectively. At two weeks after implantation the quercetin-crosslinked matrices also elicited the lowest levels of IgG antibodies. CONCLUSION: The study results identified quercetin as the most suitable crosslinker for heart valve tissue engineering, and a possible alternative to GTA. Further studies are essential to determine whether quercetin crosslinking will allow autologous cell repopulation in order to create a viable heart valve.

Mimicking herpes simplex virus 1 and herpes simplex virus 2 mucosal behavior in a well-characterized human genital organ culture.

We developed and morphologically characterized a human genital mucosa explant model (endocervix and ectocervix/vagina) to mimic genital herpes infections caused by herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Subsequent analysis of HSV entry receptor expression throughout the menstrual cycle in genital tissues was performed, and the evolution of HSV-1/-2 mucosal spread over time was assessed. Nectin-1 and -2 were expressed in all tissues during the entire menstrual cycle. Herpesvirus entry mediator expression was limited mainly to some connective tissue cells. Both HSV-1 and HSV-2 exhibited a plaque-wise mucosal spread across the basement membrane and induced prominent epithelial syncytia.

Cell distribution and regenerative activity following meniscus replacement.

PURPOSE: Meniscus replacement is of clinical benefit, but universal efficacy remains elusive. A greater understanding of the biological activity within implanted allografts or synthetic scaffolds may assist the development of improved surgical strategies. MATERIALS: Biopsies of fresh-frozen allograft (n=20), viable allograft (n=18) and polyurethane scaffolds (n=20) were obtained at second-look arthroscopy. Histological evaluation of tissue morphology and cell density/distribution was performed using haematoxylin-eosin (H&E) staining. Immunohistochemistry was used to detect the presence of CD34 (on progenitor cells and blood vessels) and smooth muscle actin (SMA)-positive structures and aggrecan. Collagen presence was investigated using picrosirius red staining. RESULTS: Cell density in the deep zone of the meniscus replacement was significantly higher in polyurethane scaffolds versus allograft transplants (p<0.01) and also significantly higher in viable allograft compared with deep-frozen allograft (p<0.01). CD34 staining was significantly higher in polyurethane and viable allografts versus deep-frozen allograft (progenitor cells p<0.05; blood vessels p<0.01). There were no significant differences in SMA or aggrecan staining across groups. All three specimen types demonstrated strong presence of collagen type I. CONCLUSIONS: Both viable allograft and a polyurethane meniscal scaffold show enhanced morphological, cell-distribution and regenerative patterns over deep-frozen allograft following surgical implantation. Given the limitations in viable allograft availability, these findings support the continued development of synthetic scaffolds for meniscus replacement surgery.

Bioactive porcine matrices in heart valve tissue engineering.

BACKGROUND AND AIM OF THE STUDY: Platelet gel (PG), a storage vehicle of growth factors, can be considered for the application of growth factors in combination with mesenchymal stem cells (MSCs) to accelerate tissue regeneration. Moreover, the addition of bioactive factors to porcine aortic valves could result in a more rapid repopulation. The study aim was to load acellular porcine aortic valve matrices with the PG-rich growth factors and to evaluate the effect on MSC repopulation. METHODS: Ovine mesenchymal stem cells (oMSCs) were isolated from sheep bone marrow. Acellular porcine heart valve matrices (n = 3) were preloaded with heparin and incubated with the PG for 2 h. A quantitative sandwich enzyme immunoassay was used to examine the release of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) from the matrices, oMSC repopulation was stimulated by static and dynamic culture. RESULTS: The immunoassays revealed that heparin-preloaded PG-incubated matrices showed a sustained release of 56.28 pg/ml bFGF and 30.66 ng/ml TGF-beta1 after 24 h. Dynamic culture induced oMSC invasion in growth factor-loaded matrices. Cell density results showed that dynamic culture significantly enhanced the repopulation of growth factor-loaded matrices (75 +/- 21 cells/mm2) when compared to static culture (26 +/- 10 cells/mm2). CONCLUSION: The incubation of a porcine aortic valve matrix with a PG concentrate creates a bioactive matrix. However, further fine-tuning of the PG concentration is necessary to take full advantage of platelet growth factor interaction between cells and the extracellular matrix in order to optimize cellular repopulation.

Decellularization of heart valve matrices: search for the ideal balance.

OBJECTIVE: Currently used decellularization procedures have negative effects on extracellular matrix (ECM) integrity. The objective of this study is to evaluate four decellularization methods and their effect on the collagen ultrastructure, mechanical behavior and antigenicity of porcine aortic valves. METHODS: Aortic valves were placed in a trypsin, osmotic, trypsin-osmotic or detergent-osmotic solution. Leaflets were processed for histology and mechanical testing. Matrices were implanted subdermally in rats to evaluate immune reaction and calcification. RESULTS: Trypsin-osmotic methodology effected near-complete decellularization. Trypsin treatment resulted in cell removal only in the spongiosa layer. Osmotic and detergent-osmotic treatments did not remove any cells from the cusps. Mechanical strength was significantly inferior in the trypsin (p50,03) and trypsin-osmotic treated group (p50,04). Trypsin and trypsin-osmotic decellularized matrices evoked a strong CD31 inflammatory cell infiltration. CONCLUSION: Enzymatic-osmotic decellularization appears to be the only effective method to remove cellular components. However, the near cell free scaffolds still evokes a strong CD31 T-cell inflammatory reaction.

Slow cooling cryopreservation of cell-microcarrier constructs.

Ideally, tissue engineered constructs should be readily available to meet the need for fast intervention in complex bone defects. To circumvent the long culture period of these constructs before implantation, we investigated the possibility of cryopreserving cell-loaded constructs. Goat bone marrow-derived mesenchymal stem cells (BMSC) and mouse osteoblast-like cells from the MC3T3-E1 cell line were cultured on gelatin CultiSpher-S(R) microcarriers. These constructs were cryopreserved using the slow cooling technique, i.e. cooling to -80 degrees C at a rate of -1.5 degrees C/min, and were then stored in liquid nitrogen for 1 week. Four different cryomedia were tested, i.e. 90 vol% serum with 10 vol% dimethylsulphoxide (Me(2)SO) with or without ascorbic acid (AA) and 90 vol% serum supplemented with 5 vol% Me(2)SO and 5 vol% hydroxyethyl starch or 5 vol% sucrose (60 mM). Cell viability on the constructs was assessed with fluorescent live/dead staining and the colorimetric MTS assay. Cell viability was compared before freezing and at fixed time points after thawing. Immediately after thawing, the viability percentages in all groups were significantly lower than before cryopreservation (p = 0.0369). No significant differences were observed between the viability percentages on the cell constructs cryopreserved in the different media; however, there was a general tendency for higher cell survival and faster recolonization of constructs cryopreserved in Me(2)SO with or without AA than of the constructs cryopreserved in the other media. For constructs cryopreserved in 10 vol% Me(2)SO with or without AA, the recolonization period was 3 days for the BMSC constructs and 3.6 and 3.8 days, respectively, for the MC3T3-E1 constructs.

Genipin blues: an alternative non-toxic crosslinker for heart valves?

BACKGROUND AND AIM OF THE STUDY: One approach in tissue-engineering involves the implantation of decellularized, xenogenic scaffolds, with the expectation of repopulation in vivo. However, a major limitation of this method is the propensity to induce a strong immune host response. The study aim was to mitigate this immunogenicity by employing a crosslinking treatment with genipin. METHODS: Porcine matrices were prepared using a detergent-enzymatic treatment and fixed in 0.01% or 0.001% aqueous genipin. The mechanical properties of the matrices were monitored by tensile strength testing. The survival of chicken fibroblasts was used to determine cell-friendliness of the matrices. Non-fixed, decellularized biological scaffolds (n = 3) were implanted in a sheep model and compared to an equal number of genipin-fixed scaffolds (n = 6). Matrices implanted in the pulmonary position were explanted after six weeks and examined using light and transmission electron microscopy. The antibody reaction against porcine tissue in sheep serum was also determined. RESULTS: Statistically significant differences were found between non-fixed leaflets, 0.001% genipin-and 0.6% glutaraldehyde (GA)-fixed leaflets for work to maximum load (non-fixed 0.00646 J; genipin-fixed 0.00509 J; GA-fixed 0.00543 J) and stiffness (non-fixed 9281 N/m; genipin-fixed 16214 N/m; GA-fixed 14401 N/m). Genipin-treated matrices were not cytotoxic. For all concentrations of genipin a high proportion of viable cells was present (79-100%). Low-dose GA (10 microg/ml) showed a distinct cytotoxicity (24.8% viability). At explant, an intense chronic inflammatory response was observed in non-fixed matrices, in contrast to genipin-fixed scaffolds. The sheep serum showed a marked decrease in IgG response in both 0.001% and 0.01% genipin-fixed matrices (IgG 30 and 20, respectively) when compared to non-fixed matrices (IgG 40). CONCLUSION: Genipin crosslinking of the matrices attenuated, but did not eliminate, the inflammatory host reaction. Whether genipin treatment might extend the durability of xenogenic scaffolds remains to be investigated.

Evaluation methods as quality control in the generation of decellularized peripheral nerve allografts.

Nowadays, the high incidence of peripheral nerve injuries and the low success ratio of surgical treatments are driving research to the generation of novel alternatives to repair critical nerve defects. In this sense, tissue engineering has emerged as a possible alternative with special attention to decellularization techniques. Tissue decellularization offers the possibility to obtain a cell-free, natural extracellular matrix (ECM), characterized by an adequate 3D organization and proper molecular composition to repair different tissues or organs, including peripheral nerves. One major problem, however, is that there are no standard quality control methods to evaluate decellularized tissues. Therefore, in this review, a brief description of current strategies for peripheral nerve repair is given, followed by an overview of different decellularization methods used for peripheral nerves. Furthermore, we extensively discuss the available and currently used methods to demonstrate the success of tissue decellularization in terms of the cell removal, preservation of essential ECM molecules and maintenance or modification of biomechanical properties. Finally, orientative guidelines for the evaluation of decellularized peripheral nerve allografts are proposed.

Differential expression of GAP-43 and neurofilament during peripheral nerve regeneration through bio-artificial conduits.

Nerve conduits are promising alternatives for repairing nerve gaps; they provide a close microenvironment that supports nerve regeneration. In this sense, histological analysis of axonal growth is a determinant to achieve successful nerve regeneration. To evaluate this process, the most-used immunohistochemical markers are neurofilament (NF), ß-III tubulin and, infrequently, GAP-43. However, GAP-43 expression in long-term nerve regeneration models is still poorly understood. In this study we analysed GAP-43 expression and its correlation with NF and S-100, using three tissue-engineering approaches with different regeneration profiles. A 10 mm gap was created in the sciatic nerve of 12 rats and repaired using collagen conduits or collagen conduits filled with fibrin-agarose hydrogels or with hydrogels containing autologous adipose-derived mesenchymal stem cells (ADMSCs). After 12 weeks the conduits were harvested for histological analysis. Our results confirm the long-term expression of GAP-43 in all groups. The expression of GAP-43 and NF was significantly higher in the group with ADMSCs. Interestingly, GAP-43 was observed in immature, newly formed axons and NF in thicker and mature axons. These proteins were not co-expressed, demonstrating their differential expression in newly formed nerve fascicles. Our descriptive and quantitative histological analysis of GAP-43 and NFL allowed us to determine, with high accuracy, the heterogenic population of axons at different stages of maturation in three tissue-engineering approaches. Finally, to perform a complete assessment of axonal regeneration, the quantitative immunohistochemical evaluation of both GAP-43 and NF could be a useful quality control in tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

In vitro characterization of a nanostructured fibrin agarose bio-artificial nerve substitute.

Neural tissue engineering is focused on the design of novel biocompatible substitutes to repair peripheral nerve injuries. In this paper we describe a nanostructured fibrin-agarose bioartificial nerve substitute (NFABNS), based on nanostructured fibrin-agarose hydrogels (FAHs) with human adipose-derived mesenchymal stem cells (HADMSCs). These NFABNSs were mechanically characterized and HADMSCs behaviour was evaluated using histological and ultrastructural techniques. Mechanical characterization showed that the NFABNSs were resistant, flexible and elastic, with a high deformation capability. Histological analyses carried out in vitro during 16 days revealed that the number of HADMSCs decreased over time, with a significant increase after 16 days. HADMSCs formed cell clusters and degraded the surrounding scaffold during this time; additionally, HADMSCs showed active cell proliferation and cytoskeletal remodelling, with a progressive synthesis of extracellular matrix molecules. Finally, this study demonstrated that it is possible to generate biologically active and mechanically stable tissue-like substitutes with specific dimensions, based on the use of HADMSCs, FAHs and a nanostructure technique. However, in vivo analyses are needed to demonstrate their potential usefulness in peripheral nerve repair. Copyright © 2015 John Wiley & Sons, Ltd.

Remote histology learning from static versus dynamic microscopic images.

Histology is the study of microscopic structures in normal tissue sections. Curriculum redesign in medicine has led to a decrease in the use of optical microscopes during practical classes. Other imaging solutions have been implemented to facilitate remote learning. With advancements in imaging technologies, learning material can now be digitized. Digitized microscopy images can be presented in either a static or dynamic format. This study of remote histology education identifies whether dynamic pictures are superior to static images for the acquisition of histological knowledge. Test results of two cohorts of second-year Bachelor in Medicine students at Ghent University were analyzed in two consecutive academic years: Cohort 1 (n = 190) and Cohort 2 (n = 174). Students in Cohort 1 worked with static images whereas students in Cohort 2 were presented with dynamic images. ANCOVA was applied to study differences in microscopy performance scores between the two cohorts, taking into account any possible initial differences in prior knowledge. The results show that practical histology scores are significantly higher with dynamic images as compared to static images (F (1,361) = 15.14, P < 0.01), regardless of student's gender and performance level. Several reasons for this finding can be explained in accordance with cognitivist learning theory. Since the findings suggest that knowledge construction with dynamic pictures is stronger as compared to static images, dynamic images should be introduced in a remote setting for microscopy education. Further implementation within a larger electronic learning management system needs to be explored in future research. Anat Sci Educ 9: 222-230. © 2015 American Association of Anatomists.

Near-infrared bulk optical properties of goat wound tissue and human serum: consequences for an implantable optical glucose sensor.

Near-infrared (NIR) spectroscopy offers a promising technological platform for continuous glucose monitoring in the human body. Moreover, these measurements could be performed in vivo with an implantable single-chip based optical sensor. However, a thin tissue layer may grow in the optical path of the sensor. As most biological tissues are highly scattering, they only allow a small fraction of the collimated light to pass, significantly reducing the light throughput. To quantify the effect of a thin tissue layer in the optical path, the bulk optical properties of serum and tissue samples grown on implanted dummy sensors were characterized using double integrating sphere and unscattered transmittance measurements. The estimated bulk optical properties were then used to calculate the light attenuation through a thin tissue layer. The combination band of glucose was found to be the better option, relative to the first overtone band, as the absorptivity of glucose molecules is higher, while the reduction in unscattered transmittance due to tissue growth is less. Additionally, as the wound tissue was found to be highly scattering, the unscattered transmittance of the tissue layer is expected to be very low. Therefore, a sensor configuration which measures the diffuse transmittance and/or reflectance instead was recommended. (a) Dummy sensor; (b) explanted dummy sensor in tissue lump; (c) removal of dummy sensor from tissue lump; and (d) 900 µm slices of tissue lump.

Generation of composites for bone tissue-engineering applications consisting of gellan gum hydrogels mineralized with calcium and magnesium phosphate phases by enzymatic means.

Mineralization of hydrogels, desirable for bone regeneration applications, may be achieved enzymatically by incorporation of alkaline phosphatase (ALP). ALP-loaded gellan gum (GG) hydrogels were mineralized by incubation in mineralization media containing calcium and/or magnesium glycerophosphate (CaGP, MgGP). Mineralization media with CaGP:MgGP concentrations 0.1:0, 0.075:0.025, 0.05:0.05, 0.025:0.075 and 0:0.1 (all values mol/dm3 , denoted A, B, C, D and E, respectively) were compared. Mineral formation was confirmed by IR and Raman, SEM, ICP-OES, XRD, TEM, SAED, TGA and increases in the the mass fraction of the hydrogel not consisting of water. Ca was incorporated into mineral to a greater extent than Mg in samples mineralized in media A-D. Mg content and amorphicity of mineral formed increased in the order A < B < C < D. Mineral formed in media A and B was calcium-deficient hydroxyapatite (CDHA). Mineral formed in medium C was a combination of CDHA and an amorphous phase. Mineral formed in medium D was an amorphous phase. Mineral formed in medium E was a combination of crystalline and amorphous MgP. Young's moduli and storage moduli decreased in dependence of mineralization medium in the order A > B > C > D, but were significantly higher for samples mineralized in medium E. The attachment and vitality of osteoblastic MC3T3-E1 cells were higher on samples mineralized in media B-E (containing Mg) than in those mineralized in medium A (not containing Mg). All samples underwent degradation and supported the adhesion of RAW 264.7 monocytic cells, and samples mineralized in media A and B supported osteoclast-like cell formation. Copyright © 2014 John Wiley & Sons, Ltd.

The biocompatibility of dibutyryl chitin in the context of wound dressings.

Dibutyryl chitin (DBC) is a modified chitin carrying butyryl groups at 3 and 6 positions; its peculiarity is that it dissolves promptly in common solvents, while being insoluble in aqueous systems. The high biocompatibility of dibutyryl chitin in the form of films and non-wovens has been demonstrated for human, chick and mouse fibroblasts by the Viability/Cytotoxicity assay, In situ Cell Proliferation assay, Neutral Red Retention assay, Lactate Dehydrogenase Release assay, MTS cytotoxicity assay, and scanning electron microscopy. DBC was hardly degradable by lysozyme, amylase, collagenase, pectinase and cellulase over the observation period of 48 days at room temperature, during which no more than 1.33% by weight of the DBC filaments (0.3 mm diameter) was released to the aqueous medium. DBC non-wovens were incorporated into 5-methylpyrrolidinone chitosan solution and submitted to freeze-drying to produce a reinforced wound dressing material. The latter was tested in vivo in full thickness wounds in rats. The insertion of 4x4 mm pieces did not promote any adverse effect on the healing process, as shown histologically. DBC is therefore suitable for contacting intact and wounded human tissues.

Calcification as an indicator of osteoinductive capacity of biomaterials in osteoblastic cell cultures.

Mineralized extracellular matrix formation is representative for the osteoinductive capacity of biomaterials and is often tested in vitro. Characteristics of in vitro mineralization of primary rat osteoblastic cells (bone marrow, calvaria, periosteum, fetal and adult long bone) and UMR-106 cells were compared by von Kossa staining, FTIR, X-ray diffractometry, TEM and related to parameters of early (ALP and collagen I formation) and late (osteocalcin secretion) osteoblast expression. All cultures expressed high alkaline phosphatase activity and were able to form bone apatite. However, a nodular versus diffuse mineralization pattern was observed. Bone marrow, calvaria and periosteum (early passage) derived cells mineralized restrictively on the three-dimensional area of a nodule. The extracellular matrix consisted of collagen I fibers, among matrix vesicles loaded with needle-like crystals. Long bone, late passage periosteum derived and UMR-106 cells exhibited a diffuse mineralization pattern. Needle-like crystals were observed between the cells but collagen fibers and matrix vesicles could not be detected. Secretion of osteocalcin was detected in cultures derived from bone marrow and absent in UMR-106 and long bone derived cell cultures. The present study demonstrates that dystrophic calcification can not be distinguished from cell-mediated calcification with von Kossa, FTIR and X-ray diffractometry. Primary osteoblastic cells capable of forming nodules are recommended to evaluate the osteoinductive properties of biomaterials.