Cloning and expression of the lectin gene from the mushroom Agrocybe aegerita and the activities of recombinant lectin in the resistance of shrimp white spot syndrome virus infection.

Lectin is a protein with multiple functions. In this study, the full-length cDNA of the Agrocybe aegerita lectin (AAL) gene was cloned, recombinant AAL (AAL-His) was expressed, and the activities of AAL-His were analyzed. Northern blot analysis showed that the major AAL transcript is approximately 900 bp. Sequence analysis showed that the coding region of AAL is 489 bp with a transcription start site located 39 nucleotides upstream of the translation initiation codon. In an agglutination test, AAL-His agglutinated rabbit erythrocytes at 12.5 µg/ml. AAL-His also showed antiviral activity in protecting shrimp from white spot syndrome virus (WSSV) infection. This anti-WSSV effect might be due to the binding of AAL-His on WSSV virions via the direct interactions with four WSSV structural proteins, VP39B, VP41B, VP53A and VP216. AAL demonstrates the potential for development as an anti-WSSV agent for shrimp culture. It also implies that these four AAL interaction WSSV proteins may play important roles in virus infection.

Investigation of synergistic effects of inductive and conductive factors in gelatin-based cryogels for bone tissue engineering.

Macroporous and biocompatible scaffolds for bone tissue engineering were prepared from 4% gelatin (G) and 4% gelatin/2% nanohydroxyapatite (nHAP), (GN), by cryogelation. The cryogels have interconnected pores with pore size around 100 µm and a high degree of cross-linking. The incorporation of nHAP slightly reduced the porosity, degree of crosslinking, swelling kinetics and equilibrium water uptake, but enhanced the toughness of the cryogel scaffolds. The osteo-regeneration potential of GN cryogels was further enhanced by binding with bone morphogenetic protein (BMP-2) to produce the gelatin/nHAP/BMP-2 (GNB) scaffold. The efficacy of BMP-2 incorporation was tested through in vitro release studies and a sustained release profile could be observed from the cumulative BMP-2 release curve. To elucidate the effect of cryogel composition on cell proliferation and differentiation, rabbit adipose-derived stem cells (ADSCs) were seeded in cryogel scaffolds. In vitro studies demonstrated a reduced proliferation rate and enhanced osteogenic differentiation of ADSCs in GNB cryogel scaffolds from the combined effect of nHAP and BMP-2, judging from the elevated alkaline phosphatase activity and the degree of mineralization. Confocal microscopy confirmed high viability and good cytoskeletal spreading of ADSCs on cryogels while osteocalcin (OCN) protein quantification affirmed the dominance of GNB in the osteogenic differentiation of ADSCs compared to G and GN cryogels. The maximum osteogenesis capability of GNB was also confirmed through the up-regulation of specific bone maker genes of early marker protein collagen I (COL I) and late marker protein osteopontin (OPN). From an in vivo animal model, computed tomography analysis confirmed the superior bone regeneration capability of ADSCs in GNB cryogels by implanting ADSCs/GNB cryogel constructs in rabbit calvarial critical size defects. Histological and immunohistochemical analysis demonstrated new bone formation and continued expression of COL I and OCN bone-specific proteins at the defect site. Taken together, the results demonstrate that G cryogels modified with osteo-conductive nHAP and osteo-inductive BMP-2 could provide cues to synergistically promote the osteogenesis of ADSCs in vitro and in vivo.

Preparation and characterization of gelatin/hyaluronic acid cryogels for adipose tissue engineering: in vitro and in vivo studies.

Macroporous elastic scaffolds containing gelatin (4% or 10%) and 0.25% hyaluronic acid (HA) were fabricated by cryogelation for application in adipose tissue engineering. These cryogels have interconnected pores (∼200 µm), high porosity (>90%) and a high degree of cross-linking (>99%). The higher gelatin concentration reduced the pore size, porosity and swelling ratio of the cryogel but improved its swelling kinetics. Compressive mechanical testing of cryogel samples demonstrated non-linear stress-strain behavior and hysteresis loops during loading-unloading cycles, but total recovery from large strains. The presence of more gelatin increased the elastic modulus, toughness and storage modulus and yielded a cryogel that was highly elastic, with a loss tangent equal to 0.03. Porcine adipose-derived stem cells (ADSCs) were seeded in the cryogel scaffolds to assess their proliferation and differentiation. In vitro studies demonstrated a good proliferation rate and the adipogenic differentiation of the ADSCs in the cryogel scaffolds, as shown by their morphological change from a fibroblast-like shape to a spherical shape, decreased actin cytoskeleton content, growth arrest, secretion of the adipogenesis marker protein leptin, Oil Red O staining for triglycerides and expression of early (LPL and PPARγ) and late (aP2 and leptin) adipogenic marker genes. In vivo studies of ADSCs/cryogel constructs implanted in nude mice and pigs demonstrated adipose tissue and new capillary formation, the expression of PPARγ, leptin and CD31 in immunostained explants, and the continued expression of adipocyte-specific genes. Both the in vitro and in vivo studies indicated that the gelatin/HA cryogel provided a structural and chemical environment that enabled cell attachment and proliferation and supported the biological functions and adipogenesis of the ADSCs.