The use of fibrin and poly(lactic-co-glycolic acid) hybrid scaffold for articular cartilage tissue engineering: an in vivo analysis

Our preliminary results indicated that fibrin and poly(lactic co-glycolic acid) (PLGA) hybrid scaffold promoted early chondrogenesis of articular cartilage constructs in vitro. The aim of this study was to evaluate in vivo cartilaginous tissue formation by chondrocyte-seeded fibrin/PLGA hybrid scaffolds. PLGA scaffolds were soaked carefully, in chondrocyte-fibrin suspension, and polymerized by dropping thrombin-calcium chloride (CaCl2) solution.PLGA-seeded chondrocytes were used as a control. Resulting constructs were implanted subcutaneously, at the dorsum of nude mice, for 4 weeks. Macroscopic observation, histological evaluation, gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 4 weeks postimplantation.Cartilaginous tissue formation in fibrin/PLGA hybrid construct was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs was confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene encoded cartilage-specific markers, collagen type II and aggrecan core protein. The sGAG production in fibrin/PLGA hybrid constructs was higher than in the PLGA group. In conclusion, fibrin/PLGA hybrid scaffold promotes cartilaginous tissue formation in vivo and may serve as a potential cell delivery vehicle and a structural basis for articular cartilage tissue-engineering

Fibrin and poly(lactic-co-glycolic acid) hybrid scaffold promotes early chondrogenesis of articular chondrocytes: an in vitro study

<p>Abstract</p> <p>Background</p> <p>Synthetic- and naturally derived- biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. Poly(lactic-co-glycolic acid) (PLGA) are bioresorbable and biocompatible, rendering them as a promising tool for clinical application. To minimize cells lost during the seeding procedure, we used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds. We evaluated <it>in vitro </it>chondrogenesis of rabbit articular chondrocytes in PLGA scaffolds using fibrin as cell transplantation matrix.</p> <p>Methods</p> <p>PLGA scaffolds were soaked in chondrocytes-fibrin suspension (1 × 10⁶cells/scaffold) and polymerized by dropping thrombin-calcium chloride (CaCl₂) solution. PLGA-seeded chondrocytes was used as control. All constructs were cultured for a maximum of 21 days. Cell proliferation activity was measured at 1, 3, 7, 14 and 21 days <it>in vitro </it>using 3-(4,5-dimethylthiazole-2-yl)-2-, 5-diphenyltetrazolium-bromide (MTT) assay. Morphological observation, histology, immunohistochemistry (IHC), gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 3 weeks to elucidate <it>in vitro </it>cartilage development and deposition of cartilage-specific extracellular matrix (ECM).</p> <p>Results</p> <p>Cell proliferation activity was gradually increased from day-1 until day-14 and declined by day-21. A significant cartilaginous tissue formation was detected as early as 2-week in fibrin/PLGA hybrid construct as confirmed by the presence of cartilage-isolated cells and lacunae embedded within basophilic ECM. Cartilage formation was remarkably evidenced after 3 weeks. Presence of cartilage-specific proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs were confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrix. Chondrogenic properties were further demonstrated by the expression of genes encoded for cartilage-specific markers, collagen type II and aggrecan core protein. Interestingly, suppression of cartilage dedifferentiation marker; collagen type I was observed after 2 and 3 weeks of <it>in vitro </it>culture. The sulphated-glycosaminoglycan (sGAG) production in fibrin/PLGA was significantly higher than in PLGA.</p> <p>Conclusion</p> <p>Fibrin/PLGA promotes early <it>in vitro </it>chondrogenesis of rabbit articular chondrocytes. This study suggests that fibrin/PLGA may serve as a potential cell delivery vehicle and a structural basis for <it>in vitro </it>tissue-engineered articular cartilage.</p

The effects of various growth factors on the proliferation of human nasal septum chondrocytes and reconstruction of human cartilage via tissue engineering technology

Tissue loss and organ failure are one of the costly health care management in all over the world. One of the recent strategies for the treatment of organ failure and damaged tissues is via tissue engineering technology. The primary goal of all approaches in tissue engineering is to repair and restore tissue functions through the delivery of living elements which become integrated into the patient (Langer and Vacanti, 1993)

Supplementation of TGF-Beta3 in low serum media promotes chondrogenesis of BMSCs

ABSTRACT Tissue engineering has emerged as a new promising field that allow in vitro construction of whole transplantable tissue. Recently, bone marrow stem cells (BMSCs) fulfil the requirements as appropriate cell source that is renewable for cartilage tissue engineering since they were able to form hyaline-like cartilage in vitro and in vivo when cultured in media supplemented with specific growth factors. This study aimed to examine the potential of BMSCs chondrogenesis in vitro and in vivo. BMSCs were isolated from sheep and divided into a test group and control group. Control group was cultured in medium supplemented with 10% fetal bovine serum (FBS). Test group was cultured in medium supplemented with 1% FBS, 1% ITS, 5ng/mL TGF-ß3, 50ng/mL IGF-1, 40 ng/mL L-proline, 100 nM dexamethasone and 50 μg/mL ascorbic acid-2 phosphate. The experiment was carried out on athymic nude mice. Cell aggregates were formed in test group and indicated the early chondrogenesis in cell culture. Later, cells-fibrin constructs were made and implanted subcutaneously into nude mice for 5 wks, then explanted for histological examination and glycosaminoglycans (GAG) quantification. Test group constructs showed higher cartilage matrix synthesis as confirmed by Safranin O staining and GAG production. These results demonstrated the effectiveness of low serum media supplemented with TGF-ß3 in promoting chondrogenesis in BMSCs. Keywords: bone marrow, cartilage, chondrogenesis, growth factors, low serum media, stem cell

Honey ameliorate negative effects in neurodegenerative diseases: an evidence-based review

Neurodegenerative diseases are cluster of disorders arising from neuronal cell death in the central nervous system. Its prevalence increases with increasing age. Therapeutic options for neurodegenerative disease include protection against oxidative damage, attenuation of neuroinflammation, maintenance of essential neurotransmitters, and protection against environmental factors that induce neurotoxicity. Honey with its antioxidative, anti-inflammatory, and cytoprotective effects is a potential candidate for therapy in neurodegenerative diseases. The present evidence-based review summarizes the effects of honey on neurodegenerative diseases in non-human subjects. Three electronic databases, namely PubMed, Ovid Medline and Scopus were searched for records published from inception of database to May 2020 to identify reports on the association of honey and neurodegenerative diseases. Based on the preset eligibility criteria, 8 qualified articles were selected and discussed in this review. Honey from different geological origin around the globe was used by different researcher among the studies included. Honey confers protection against oxidative stress induced by hypoxia and metabolic syndrome, aluminium toxicity, and neuroinflammation. Honey also demonstrated potential ability to inhibit neurotransmitters degrading enzymes and restore memory impairment. This review showed a sparse body of evidence on the potential of honey as neurodegenerative disease therapy

Posterolateral spinal fusion with ostegenesis induced BMSC seeded TCP/HA in a sheep model

Autogenous bone graft is the gold standard for fusion procedure. However, pain at donor site and inconsistent outcome have left a surgeon to venture into some other technique for spinal fusion. The objective of this study was to determine whether osteogenesis induced bone marrow stem cells with the combination of ceramics granules (HA or TCP/HA), and fibrin could serve as an alternative to generate spinal fusion. The sheep's bone marrow mesenchymal stem cells (BMSCs) were aspirated form iliac crest and cultured for several passages until confluence. BMSCs were trypsinized and seeded on hydroxyapatite scaffold (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) for further osteogenic differentiation in the osteogenic medium one week before implantation. Six adult sheep underwent three-level, bilateral, posterolateral intertransverse process fusions at L1–L6. Three fusion sites in each animal were assigned to three treatments: (a) HA constructs group/L1–L2, (b) TCP/HA constructs group/L2–L3, and (c) autogenous bone graft group/L5–L6. The spinal fusion segments were evaluated using radiography, manual palpation, histological analysis and scanning electron microscopy (SEM) 12 weeks post implantation. The TCP/HA constructs achieved superior lumbar intertransverse fusion compared to HA construct but autogenous bone graft still produced the best fusion among all

Tricalcium phosphate/hydroxyapatite (TCP-HA) bone scaffold as potential candidate for the formation of tissue engineered bone

Background & objectives: Various materials have been used as scaffolds to suit different demands in tissue engineering. One of the most important criteria is that the scaffold must be biocompatible. This study was carried out to investigate the potential of HA or TCP/HA scaffold seeded with osteogenic induced sheep marrow cells (SMCs) for bone tissue engineering. Methods: HA-SMC and TCP/HA-SMC constructs were induced in the osteogenic medium for three weeks prior to implantation in nude mice. The HA-SMC and TCP/HA-SMC constructs were implanted subcutaneously on the dorsum of nude mice on each side of the midline. These constructs were harvested after 8 wk of implantation. Constructs before and after implantation were analyzed through histological staining, scanning electron microscope (SEM) and gene expression analysis. Results: The HA-SMC constructs demonstrated minimal bone formation. TCP/HA-SMC construct showed bone formation eight weeks after implantation. The bone formation started on the surface of the ceramic and proceeded to the centre of the pores. H&E and Alizarin Red staining demonstrated new bone tissue. Gene expression of collagen type 1 increased significantly for both constructs, but more superior for TCP/HA-SMC. SEM results showed the formation of thick collagen fibers encapsulating TCP/HA-SMC more than HA-SMC. Cells attached to both constructs surface proliferated and secreted collagen fibers. Interpretation & conclusions: The findings suggest that TCP/HA-SMC constructs with better osteogenic potential compared to HA-SMC constructs can be a potential candidate for the formation of tissue engineered bone

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

Supplementation of TGF-Beta3 in low serum media promotes chondrogenesis of BMSCs

Tissue engineering has emerged as a new promising field that allow in vitro construction of whole transplantable tissue. Recently, bone marrow stem cells (BMSCs) fulfil the requirements as appropriate cell source that is renewable for cartilage tissue engineering since they were able to form hyaline-like cartilage in vitro and in vivo when cultured in media supplemented with specific growth factors. This study aimed to examine the potential of BMSCs chondrogenesis in vitro and in vivo. BMSCs were isolated from sheep and divided into a test group and control group. Control group was cultured in medium supplemented with 10% fetal bovine serum (FBS). Test group was cultured in medium supplemented with 1% FBS, 1% ITS, 5ng/mL TGF-ß3, 50ng/mL IGF-1, 40 ng/mL L-proline, 100 nM dexamethasone and 50 μg/mL ascorbic acid-2 phosphate. The experiment was carried out on athymic nude mice. Cell aggregates were formed in test group and indicated the early chondrogenesis in cell culture. Later, cells-fibrin constructs were made and implanted subcutaneously into nude mice for 5 wks, then explanted for histological examination and glycosaminoglycans (GAG) quantification. Test group constructs showed higher cartilage matrix synthesis as confirmed by Safranin O staining and GAG production. These results demonstrated the effectiveness of low serum media supplemented with TGF-ß3 in promoting chondrogenesis in BMSCs

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