Perbandingan filogenetik protein antigen-I yang berpotensi sebagai calon diagnostik dan vaksin terhadap parasit cryptocaryon irritans

Protein antigen-i parasit ikan C. irritans berpotensi tinggi digunakan sebagai calon dalam pembangunan vaksin komersial terhadap C. irritans. Walau bagaimanapun, kewujudan variasi pada antigen-i serotip C. irritans yang berbeza mempengaruhi tahap perlindungan yang bakal diberikan terhadap varians C. irritans yang berbeza apabila antigen-i digunakan sebagai vaksin. Kajian ini dijalankan untuk membandingkan jujukan pelbagai antigen-i pencilan C. irritans di Malaysia berbanding antigen-i pencilan C. irritans yang pernah dilaporkan. Perbandingan filogenetik dijalankan untuk meramalkan potensi protein tersebut dalam usaha membangunkan calon serodiagnostik dan pemvaksinan terhadap pencilan C. irritans yang berlainan. Penjajaran jujukan berbilang bagi jujukan asid amino antigen-i dilakukan dengan perisian CLUSTALX dan analisis filogenetik antigen-i dilakukan menggunakan kaedah parsimoni maksimum (MP) dan kaedah Bayes. Sembilan transkrip unik (TU) C. irritans yang mempunyai padanan signifikan dengan antigen-i di pangkalan data protein NCBI didapati mempunyai peratus kesamaan antara 41% hingga 71%. Kedua-dua pohon MP dan Bayesian yang dijana menunjukkan varians antigen-i cn56 and cn57 terkelompok bersama dalam satu kumpulan manakala varians antigen-i yang lain terbahagi kepada dua kumpulan berasingan dan pengkelompokan ini disokong oleh kehadiran asid amino yang terpulihara dalam kumpulan masing-masing. Kajian lanjutan boleh dilakukan untuk mengenal pasti varians antigen-i yang sesuai sebagai calon serodiagnosis dan juga dapat memberi perlindungan silang terhadap pelbagai pencilan C. irritans di serata dunia

Development of nerve conduit using decellularized human umbilical cord artery seeded with Centella asiatica induced-neurodifferentiated human mesenchymal stem cell

Various natural biological conduits have been investigated to bridge peripheral nerve injury especially in critical gap (greater than 3 cm in human). Autograft, the current gold standard, has several drawbacks including limited availability of donor graft, donor-site morbidity and mismatch in size in clinical practices. The aim of this study was to analyze the development of nerve conduit using decellularized human umbilical cord (HUC) artery seeded with neurodifferentiated human MSCs (ndMSCs) in bridging peripheral nerve gap. Artery conduits obtained from HUC were decellularized to remove native cells (n=3), then characterized by Hematoxylin and Eosin (H&E) staining and nuclei counterstaining with DAPI. The decellularized artery conduit was measured for every 2 weeks until 12 weeks. Next, mesenchymal stem cells (MSCs) were differentiated into neural lineage using 400 μg/mL of Centella asiatica. Then, 1.5×106 of MSCs or ndMSCs were seeded into decellularized artery conduit to study cell attachment. H&E staining and nuclei counterstaining with DAPI showed that all cellular components were removed from the HUC arteries. The decellularized artery conduit did not collapse and the lumen remained rigid for 12 weeks. Immunocytochemistry analysis with neural markers namely S100β, P75 NGFR, MBP and GFAP showed that MSCs had differentiated into neural lineage cells. H&E staining showed that the seeded MSCs and ndMSCs attached to the lumen of the conduits as early as 2 days. In conclusion, this study showed that nerve conduit using decellularized HUC artery seeded with neurodifferentiated human MSCs was successfully developed and have the potential to bridge critical nerve gap

The effect of Centella asiatica (L.) Urban on the organotypic model of spinal cord injury

Centella asiatica (L.) Urban (CA) is a well- known plant used to improve brain and memory functions in traditional medicine. Scientifically it was proven to show neurogenic effect on neural cell lines and in rat’s hippocampus. Its effect on spinal cord (SC) neurons, however, have not been studied. Aim of this study was to investigate the effects of raw extract of CA (RECA) on neurite outgrowths in an organotypic model of SC injury (OMSCI). OMSCI was prepared using SC slices obtained from postnatal-day 8 rat pups. Spinal cord tissues were embedded in gelatine gel and sliced to produce 300 μm thick slices. These slices were 100% viable for 8 days in culture. RECA, in concentrations of 0-800 μg/mL was added to the OMSCI media for 7 days, followed by immunostaining for TUJ-1 and GFAP. The investigated parameters were mean neurite count, mean neurite length, mean longest neurite and growth ratio. The tested RECA concentrations showed no cytotoxicity. ANOVA and Kruskal-Wallis tests showed no significant difference between groups in all the tested parameters. This may be due to low content of neurotrophic bioactive compounds content in the extract, which probably due to differences in geographical location, extraction method and absence of neurotrophic factors in the media. In conclusion, the tested RECA concentration were found to be safe; but without notable neurotrophic effects on the spinal cord organotypic model as demonstrated in this study

Nasal fibroblast conditioned medium promotes cell attachment and migration of human respiratory epithelium

Endoscopic sinus surgery (ESS) is a well-known surgical treatment for chronic rhinosinusitis disease after failed medical and antibiotics treatment. However, improper wound healing might induce synechiae or adhesion. Conditioned medium from cultured cells is known to promote wound healing and potentially able to accelerate wound healing in ESS and other airway epithelial injuries. This study was to investigate the effect of human nasal fibroblast conditioned medium on the attachment, proliferation and migration of respiratory epithelial cells (RECs) in an in vitro model. RECs and fibroblasts were co-cultured in Defined Keratinocytes Medium and F-12 and Dulbecco’s Modified Eagle’s Medium. Once confluent, the fibroblasts were removed, leaving the colonies of RECs to reach confluency. RECs and fibroblasts were cultured separately and the conditioned medium was acquired by culturing fibroblast either in DKSFM or F12: DMEM, denoted as NFCM_DKSFM and NFCM_FD, respectively. RECs were supplemented with 20% conditioned medium for attachment, proliferation and migration assay. The results showed significantly higher cell attachment in NFCM_DKSFM (3452.77±588.1 cell/cm2) compared to NFCM_FD (2336.1±440.4 cell/cm2) and DKSFM alone (2819.8±509.5 cell/cm2). After 7 days, the specific growth rate was higher in DKSFM (0.019±5.16×10-4 h-1) compared to NFCM_DKSFM (0.015±8.94×10-4 h-1) and NFCM_FD (0.013±1.03×10-3 h-1). The mean of migration rate was significantly higher in NFCM_DKSFM (4341.81±385.7 μm2/hr) compared to NFCM_FD (1803.38±408.1 μm2/hr) and DKSFM (1933.48±271.9 μm2/hr). Hence, NFCM_DKSFM supplementation provides suitable culture conditions for RECs through increased cell attachment and migration, which suggest that the factors secreted in conditioned medium may play a major role in enhancing airway epithelial wound healing

Physicochemical and structural characterization of surface modified electrospun PMMA nanofibre

Although electrospun poly(methyl methacrylate) (PMMA) may mimic structural features of extracellular matrix, its highly hydrophobic nature causes reduced cell attachment. This study analysed the physicochemical and structural changes of the surface modified PMMA nanofiber. The electrospun PMMA nanofibers (PM) were surface-treated as follows: PM alone, collagen coated-PM (PM-C), UV-irradiated PM (PM-UV), collagen coated UV-irradiated PM (PM-C-UV) and collagen coated-PM crosslinked with genipin (PM-C-GEN). They were subjected to scanning electron microscopy, Fourier transform infrared (FTIR), cell attachment analysis, X-ray photoelectron spectroscopy (XPS), atomic force microscopy and X-ray powder diffraction (XRD). The surface roughness was lower in PM-C-UV group compared to others. Based on FTIR results, all expected functional group were present in all groups. XPS result showed that there are changes in the mass concentration of UV-treated surfaces and in the collagen coated surfaces. All PM groups showed amorphous nature through XRD. UV irradiation and collagen coating were shown to increase PM’s functional groups and modify its surface, which contributed to the increased attachment of cells onto the inert PM scaffold. As conclusion, collagen coated UV irradiated PMMA provided a better surface for cell to attach hence are suitable to be used further as scaffold for in vitro model

Recent advances in natural polymer-based hydroxyapatite scaffolds:Properties and applications

New materials that mimic natural bone properties, matching functional, mechanical, and biological properties have been continuously developed to rehabilitate bone defects. Desirably, 'tissue engineering' has been a multidisciplinary ground that uses the principles of life sciences and engineering for the biological replacements that restore or replace the tissue function or a whole organ. Nevertheless, the bone grafting treatment has numerous restrictions, counting the major hazards of morbidity from the sites where donor bone grafts are removed, the likelihood for an immune rejection or bacterial transport from the donor site (in case of allogeneic grafting), and the inadequate availability of donor bone grafts that can meet the current demands. Since the proper growth of synthetic materials for implantable bones encourages the reconstruction of bone tissues by providing strong structural support without any damages to the interferences of biological tissue. To serve for such behavior, the biodegradable matrices provide temporary scaffolds within which the bone tissues can be regenerated. Typically, the thermoplastic aliphatic polyesters are found to serve this purpose. The great significance of this field lies in the in vitro growth of precise cells on porous matrices (scaffolds) to generate three-dimensional (3D) tissues that can be entrenched into the location of tissue/bone damage. Numerous gifts have been gifted by our nature to advance and preserve the well-being of all living things either directly or indirectly. This review focuses on the recent advances in polymer-based hydroxyapatite scaffolds including their properties and applications

Effects of secretome from dynamic 3D cell culture system onto growth and cytoprotection of nasal fibroblast

Three dimensional (3D) models mimic the features of native tissue environment. Thus, morphology and signalling of cells from 3D culture are often more physiological than routine two dimensional (2D) cell culture. It is also known that the cell-secreted products have paracrine effect on other cells growth. In this experimental study, we optimised the nasal fibroblast culture on a 3D cell culture system and studied the effects of secretome from the 3D culture (3DCM) onto fibroblast growth and cytoprotection. Nasal fibroblast was isolated from human nasal turbinates. The suitable microcarrier was selected by culturing the fibroblasts in passage 3 on various types PolyGEM™ polystyrene microcarriers. Then, the cells were cultured on selected microcarrier using a 3D culture system and the conditioned medium (CM) was collected. 3DCM were supplemented to fibroblasts to study for attachment, proliferation, and cytoprotective effects against cytotoxicity of Centella asiatica. Bicinchonic Acid Assay (BCA) was performed to quantify protein amount in CMs. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for preliminary profiling and comparison of 2DCM and 3DCM protein profile. Our study showed that the 3DCM did not significantly enhance cell attachment and proliferation. The secretome of both 2DCM and 3DCM found to have significant cytoprotective effect onto nasal fibroblast against cytotoxicity of C.asiatica extract. 3DCM had higher protein concentration than 2DCM. SDS-PAGE showed three exclusive proteins in 3DCM and four exclusive proteins in 2DCM. Future study should be conducted on utility of nasal fibroblast secretome on cytoprotection against harmful agents in environment and cytotoxicity of natural products

3D Printed Bioscaffolds for Developing Tissue-Engineered Constructs

Tissue engineering techniques enable the fabrication of tissue substitutes integrating cells, biomaterials, and bioactive compounds to replace or repair damaged or diseased tissues. Despite the early success, current technology is unable to fabricate reproducible tissue-engineered constructs with the structural and functional similarity of the native tissue. The recent development of 3D printing technology empowers the opportunities of developing biofunctional complex tissue substitutes via layer-by-layer fabrication of cell(s), biomaterial(s), and bioactive compound(s) in precision. In this chapter, the current development of fabricating tissue-engineered constructs using 3D bioprinting technology for potential biomedical applications such as tissue replacement therapy, personalized therapy, and in vitro 3D modeling for drug discovery will be discussed. The current challenges, limitations, and role of stakeholders to grasp the future success also will be highlighted

Sciatic nerve repair with tissue engineered nerve: olfactory ensheating cells seeded poly (lactic-co-glygolic acid) conduit in an animal model

Background and Aim: Synthetic nerve conduits have been sought for repair of nerve defects as the autologous nerve grafts causes donor site morbidity and possess other drawbacks. Many strategies have been investigated to improve nerve regeneration through synthetic nerve guided conduits. Olfactory ensheathing cells (OECs) that share both Schwann cell and astrocytic characteristics have been shown to promote axonal regeneration after transplantation. The present study was driven by the hypothesis that tissue‑engineered poly(lactic-co-glycolic acid) (PLGA) seeded with OECs would improve peripheral nerve regeneration in a long sciatic nerve defect. Materials and Methods: Sciatic nerve gap of 15 mm was created in six adult female Sprague‑Dawley rats and implanted with PLGA seeded with OECs. The nerve regeneration was assessed electrophysiologically at 2, 4 and 6 weeks following implantation. Histopathological examination, scanning electron microscopic (SEM) examination and immunohistochemical analysis were performed at the end of the study. Results: Nerve conduction studies revealed a significant improvement of nerve conduction velocities whereby the mean nerve conduction velocity increases from 4.2 ± 0.4 m/s at week 2 to 27.3 ± 5.7 m/s at week 6 post-implantation (P < 0.0001). Histological analysis revealed presence of spindle‑shaped cells. Immunohistochemical analysis further demonstrated the expression of S100 protein in both cell nucleus and the cytoplasm in these cells, hence confirming their Schwann‑cell‑like property. Under SEM, these cells were found to be actively secreting extracellular matrix. Conclusion: Tissue‑engineered PLGA conduit seeded with OECs provided a permissive environment to facilitate nerve regeneration in a small animal model

Decellularized and genipin crosslinked human umbilical cord artery and vein for potential use as peripheral nerve conduit

Critical gap peripheral nerve injury, commonly caused by motor vehicle accidents, results in dysfunctional nerve and impaired body function. Our study aims to develop a conduit from decellularized and genipin crosslinked human umbilical cord artery and vein for future use in critical nerve gap injury treatments. Human umbilical cord arteries (HUCA) and veins (HUCV) were divided into native (nHUCA and nHUCV), decellularized (dHUCA and dHUCV) and genipin-crosslinked (clHUCA and clHUCV) groups. Both the decellularized and crosslinked groups were decellularized, and subsequently, the clHUCA and clHUCV groups were crosslinked with 0.1%, 0.4% and 0.7% (w/v) genipin. The HUCA and HUCV were then studied for decellularization efficiency, crosslinking index, biodegradation, swelling ratio, ultrastructure analysis, flexibility and mechanical strength. In addition, mesenchymal stem cells isolated from Wharton’s jelly were seeded into HUCA and HUCV for biocompatibility studies. The degradation test showed that nHUCV and dHUCV degraded at day 7 compared to other groups that did not show any degradation even after 21 days. Biocompatibility studies showed that the conduits crosslinked with 0.4% (w/v) genipin were successfully seeded and was having the most amount of seeded cells. In conclusion, the decellularization and genipin crosslinking of human umbilical cord artery and vein enabled successful in fabrication of conduit with suitable properties such as reduced swelling, flexibility, porosity and mechanical strength, with potential in tissue engineering applications