3D inkjet printing of biomaterials with strength reliability and cytocompatibility: Quantitative process strategy for Ti-6Al-4V.

Among additive manufacturing (AM) techniques, laser or electron beam based processes have been widely investigated for metallic implants. Despite the potential in manufacturing of patient-specific biomedical implants, 3D inkjet powder printing (3DIJPP, a variant of AM) of biomaterials is still in its infancy, as little is known quantitatively about the transient process physics and dynamics. An equally important challenge has been the ink formulation to manufacture biomaterials with reliable mechanical properties and desired biocompatibility. We have developed, for the very first time, the theoretical foundation and experimental formulation of a unique process strategy involving the 'on-demand' delivery of a novel in situ polymerisable acrylic ink system to print a model biomaterial, Ti-6Al-4V. The post-ejection in-flight dynamics of ink droplets have been captured in situ by employing high speed stroboscopic shadowgraphy, to quantitatively estimate the dimensionless numbers of fluid physics for 'printability' assessment. Washburn model was adapted extensively to quantify the capillary ink infiltration time in porous powder bed of finite thickness. On the other hand, particle tracking mode in diffusing wave spectroscopy (DWS) was exploited to analyse the timescale for effective binding of powder particles during in situ polymerisation. The clinically relevant combination of 3D porous architecture with 98.4% interconnectivity among 10-40 µm pores together with modest combination of elastic modulus (4 GPa) and strength reliability (Weibull modulus ∼8.1) establish the potential of inkjet printed Ti-6Al-4V as cortical bone analogue. A better cell attachment, viability, cytoskeletal spreading with pronounced proliferation of murine fibroblasts and pre-osteoblasts on 3DIJPP Ti-6Al-4V, when benchmarked against the metallurgically processed (commercial) or selective laser melted (SLM) Ti-6Al-4V, has been demonstrated, in vitro. The enhanced cellular activities on the 3DIJPP Ti-6Al-4V was explained in terms of an interplay among the elastic stiffness, surface roughness and wettability against the same benchmarking. It is conceived that the quantitative understanding of the integrated process physics and dynamics to print Ti-6Al-4V with reliable mechanical properties together with better cytocompatibility can lead to a paradigm shift in adapting the scalable 3DIJPP for manufacturing of metallic biomaterials.

Effect of testicular tissue lysate on developmental competence of porcine oocytes matured and fertilized in vitro.

The objective of the present study was to investigate the effect of testicular tissue lysate (TTL) on developmental competence of germinal vesicle (GV) stage porcine oocytes. Two types of TTL were prepared through repeated freeze-thaw in liquid nitrogen, one from whole testicular tissue (wTTL) and other from either of four different sections of testes, namely just beneath the tunica albuginea (TA), from the transitional area between the seminiferous cord/tubules and the mediastinum testis (TR) and from the intermediate area (parenchymal tissue origin) and CE (cauda epididymis origin). The whole or section-wise TTL treatments were given for 44 hr during in vitro maturation (IVM). Oocyte maturation was done in either of the two media, namely defined (high-performance basic medium for porcine oocyte maturation, commercially available) and serum containing (TCM199). After maturation, oocytes were co-incubated with fresh spermatozoa for 6 hr and then transferred to embryo culture media. Treatment of GV stage oocytes with wTTL (1 mg/ml) increased the cleavage and morula percentage rate (69.23 ± 6.23 and 48.15 ± 6.77, respectively) than that of their control (58.33 ± 8.08 and 32.54 ± 5.53, respectively) in defined media, and in serum-containing media, cleavage and morula percentage rate were almost equal in both treatment (54.56 ± 7.79 and 34.70 ± 6.78, respectively) and control (59.52 ± 8.21 and 38.52 ± 6.54, respectively). However, effect of wTTL was not significant. In case of section-wise TTL supplements, TR section significantly (p < .01) improved cleavage and morula rate (58.43 ± 7.98 and 36.14 ± 6.89, respectively) followed by TA. In conclusion, present study indicates that IVM, in vitro fertilization and in vitro culture of embryo are improved in the presence of TTL, particularly its TR section. Further study is expected to reveal the principal components of TTL which may prove useful for IVM.

Isolation, culture optimization and physico-chemical characterization of laccase enzyme from Pleurotus fossulatus.

Pleurotus fossulatus (Cooke) Sace is member of oyster mushroom can produced extracellular laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) in submerged fermentation. To analyze the optimum production for laccase P. fossulatus was cultured both in stationary and shaking condition in different media. Partial purification of laccase was done after 0-80% ammonium sulphate precipitation, followed by DEAE (Diethylaminoethyl) Sephadex (A-50) anion exchange chromatography. Potato-sucrose peptone (PSP) medium and Potato-dextrose (PD) medium showed highest laccase production in shaking and stationary conditions, respectively. Though the time required for optimum laccase production in stationary condition was much more than the shaking condition but the amount of laccase was about 2.75t greater in former condition. The laccase produced in stationary condition was more stable than the enzyme produced in shaking condition. The partially purified enzyme showed highest affinity towards o-dianisidine than guaiacol and ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) as evidenced by their K(m). The physico-chemical properties of the laccase suggested the significance of this enzyme in industrial applications.

Role of α-crystallin B as a regulatory switch in modulating cardiomyocyte apoptosis by mitochondria or endoplasmic reticulum during cardiac hypertrophy and myocardial infarction.

Cardiac hypertrophy and myocardial infarction (MI) are two major causes of heart failure with different etiologies. However, the molecular mechanisms associated with these two diseases are not yet fully understood. So, this study was designed to decipher the process of cardiomyocyte apoptosis during cardiac hypertrophy and MI in vivo. Our study revealed that mitochondrial outer membrane channel protein voltage-dependent anion channel-1 (VDAC1) was upregulated exclusively during cardiac hypertrophy, whereas 78 kDa glucose-regulated protein (GRP78) was exclusively upregulated during MI, which is an important upstream regulator of the endoplasmic reticulum (ER) stress pathway. Further downstream analysis revealed that mitochondrial pathway of apoptosis is instrumental in case of hypertrophy, whereas ER stress-induced apoptosis is predominant during MI, which was confirmed by treatment with either siRNA against VDAC1 or ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Very interestingly, our data also showed that the expression and interaction of small heat-shock protein α-crystallin B (CRYAB) with VDAC1 was much more pronounced during MI compared with either hypertrophy or control. The study demonstrated for the first time that two different organelles--mitochondria and ER have predominant roles in mediating cardiomyocyte death signaling during hypertrophy and MI, respectively, and activation of CRYAB acts as a molecular switch in bypassing mitochondrial pathway of apoptosis during MI.

Sarcoptic mange infestation in pigs in a hilly region of Meghalaya.

Skin lesions were collected from 15 adult crossbred pigs maintained at the pig farm of ICAR Research Complex for the North-Eastern Hill Region, Umiam, a hilly region of the state of Meghalaya, India. Skin scrapings of these 15 animals were examined by KOH digestion method. Presence of mites were detected in five animals which were indistinguishable from Sarcoptes scabiei var. suis. Infected pigs were treated with a single dose of Ivermectin at 200 ug/kg body weight, subcutaneously. No mites were recovered from treated pigs after a period of 10 days of post-treatment. The presence of S. scabiei var. suis in pigs from this part of the country has never been reported. This infestation has great significance because it affects the growth rate and feed conversion significantly, specially to the grower and weaned pigs. It can be concluded that S. scabiei var. suis infestation is prevalent in hilly region of Meghalaya. The pig producers are generally unaware about the severity of the problem of mite infestation, so precaution should be taken to prevent their valuable pigs from S. scabiei var. suis infestation.

Preservation of boar semen at 18 degrees C induces lipid peroxidation and apoptosis like changes in spermatozoa.

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (DeltaPsi(m)) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 degrees C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, DeltaPsi(m), and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of DeltaPsi(m) was done using 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly (p<0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10(9) sperm) were low in sperm (99.83+/-2.69) and seminal plasma (191.98+/-11.58), which gradually increased up to the 96 h of storage. Highest negative correlation (r value) was observed between MDA levels and sperm motility (-0.97), live percent (-0.97), acrosomal integrity (-0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (-0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage (r=0.98). There was a significant (p<0.05) increase in the sperm cells with low DeltaPsi(m) from 0 to 96 h of preservation. Before preservation, 14.85+/-4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00+/-6.25%. The apoptotic sperm population was 8.33+/-2.31% in fresh semen, while this population was 25.19+/-4.25% at 96 h of preservation and the difference was significant (p<0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 degrees C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.