isolation-expansion-differentiation-mesenchymal-stem-cells-from

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

Heterogeneity in tumors has led to the development of combination therapies that enable enhanced cell death. Previously explored combination therapies mostly involved the use of bioactive molecules. In this work, we explored a non-conventional strategy of using carbon nanostructures (CNs) [single walled carbon nanotube (SWNT) and graphene oxide (GO)] for potentiating the efficacy of a bioactive molecule [paclitaxel (Tx)] for the treatment of lung cancer. The results demonstrated enhanced cell death following combination treatment of SWNT/GO and Tx indicating a synergistic effect. In addition, synergism was abrogated in the presence of an anti-oxidant, N-acetyl cysteine (NAC), and was therefore shown to be reactive oxygen species (ROS) dependent. It was further demonstrated using bromodeoxyuridine (BrdU) incorporation assay that treatment with CNs was associated with enhanced mitogen associated protein kinase (MAPK) activation that was ROS mediated. Hence, these results for the first time demonstrated the potential of SWNT/GO as co-therapeutic agents with Tx for the treatment of lung cancer.

Combination of single walled carbon nanotubes/graphene oxide with paclitaxel: a reactive oxygen species mediated synergism for treatment of lung cancer.

Porous hydrogels have been explored for bone tissue engineering; however their poor mechanical properties make them less suitable as bone graft substitutes. Since incorporation of fillers is a well-accepted method for improving mechanical properties of hydrogels, in this work pullulan hydrogels were reinforced with nano-crystalline hydroxyapatite (nHAp) (5 wt% nHAp in hydrogel) and poly(3-hydroxybutyrate) (PHB) fibers (3 wt% fibers in hydrogel) containing nHAp (3 wt% nHAp in fibers). Addition of these fillers to pullulan hydrogel improved compressive modulus of the scaffold by 10 fold. However, the hydrophilicity of pullulan did not support adhesion and spreading of cells. To overcome this limitation, porous composite scaffolds were modified using a double diffusion method that enabled deposition of hydroxyapatite on pore walls. This method resulted in rapid and uniform coating of HAp throughout the three-dimensional scaffolds which not only rendered them osteoconductive in vitro but also led to an improvement in their compressive modulus. These results demonstrate the potential of mineralized pullulan-based composite scaffolds in non-load bearing bone tissue engineering.

Pullulan-based composite scaffolds for bone tissue engineering: Improved osteoconductivity by pore wall mineralization.

Scaffolds with aligned pores are being explored in musculoskeletal tissue engineering due to their inherent structural anisotropy. However, influence of their structure on mechanical behavior remains poorly understood. In this work, we elucidate this dependence using chitosan-gelatin based random and aligned scaffolds. For this, scaffolds with horizontally or vertically aligned pores were fabricated using unidirectional freezing technique. Random, horizontal and vertical scaffolds were characterized for their mechanical behavior under compressive, tensile and shear loading regimes. The results revealed conserved trends in compressive, tensile and shear moduli, with horizontal scaffolds showing the least moduli, vertical showing the highest and random showing intermediate. Further, these scaffolds demonstrated a highly viscoelastic behavior under cyclic compressive loading, with a pore orientation dependent relative energy dissipation. These results established that mechanical behavior of porous scaffolds can be modulated by varying pore orientation alone. This finding paved the way to recreate the structural and consequent mechanical anisotropy of articular cartilage tissue using zonally varied pore orientation in scaffolds. To this end, monolithic multizonal scaffolds were fabricated using a novel sequential unidirectional freezing technique. The superficial zone of this scaffold had horizontally aligned pores while the deep zone consisted of vertically aligned pores, with a transition zone between the two having randomly oriented pores. This depth-dependent pore architecture closely mimicked the collagen alignment of native articular cartilage which translated into similar depth-dependent mechanical anisotropy as well. A facile fabrication technique, biomimetic pore architecture and associated mechanical anisotropy make this multizonal scaffold a promising candidate for cartilage tissue engineering. 

Pore orientation mediated control of mechanical behavior of scaffolds and its application in cartilage-mimetic scaffold design.

Electrospun poly(hydroxybutyrate) (PHB) fiber meshes have shown some success in wound dressing applications, however, their use is limited by their high hydrophobicity and brittle nature. In this study we investigated the effect of hydrophilization of electrospun PHB fibers by blending with Pluronic F-108 (PF) for use as a wound dressing material. Blending of PHB with different concentrations of PF (0.5%PF-PHB and 1.0% PF-PHB) before electrospinning led to a significant increase in the water wettability and swelling properties of fibers as compared to pristine PHB fibers. Further, it was observed that though the tensile moduli of PF blended PHB fibers were relatively lower as compared to PHB fibers, they show higher resistance to failure measured in terms of strain to failure and energy to failure. Moreover, PF blending significantly improved the in vitro blood clotting rate on PHB fibers when compared to control PHB fibers. Furthermore, the fabricated fiber systems were found to be cytocompatible and supported adhesion of fibroblasts in vitro. Finally, it was demonstrated that the PF blended fiber systems were suitable for the encapsulation of an antibiotic (doxycycline) to render them with antibacterial properties. Taken together, this study demonstrates that PF blending can be used to significantly improve properties of PHB fibers for wound dressing applications. 

Fabrication and characterization of Pluronic modified poly(hydroxybutyrate) fibers for potential wound dressing applications.

Post-translational modifications such as phosphorylation and sialylation impart crucial functions such as mineral deposition and osteogenic differentiation to non-collagenous bone matrix proteins. In this work, the influence of phosphorylation and polysialylation of gelatin on mineralization in simulated body fluid (SBF) and on osteogenic differentiation of mesenchymal stem cells (MSC) was studied. It was observed that increase in phosphorylation could be directly correlated with the mineralization ability of phosphorylated gelatin in SBF. The total calcium and phosphate deposited increased with increase in degree of phosphorylation and was >3 fold higher on the highest degree of phosphorylation. Whereas, polysialylation did not have any significant influence on mineral deposition in SBF. On the other hand, when MSCs were cultured on polysialylated surfaces they showed relatively higher cell elongation with 1.5 fold higher cell aspect ratio, higher alkaline phosphatase activity and 3 fold higher mineral deposition when compared to control and phosphorylated gelatin surfaces. In conclusion, phosphorylation and polysialylation of gelatin show a significant influence on mineralization and osteogenic differentiation respectively which can be advantageously used for bone tissue engineering. 

Understanding the influence of phosphorylation and polysialylation of gelatin on mineralization and osteogenic differentiation.

Matrix stiffness is known to play a pivotal role in cellular differentiation. Studies have shown that soft scaffolds (<2-3kPa) promote cellular aggregation and chondrogenesis, whereas, stiffer ones (>10kPa) show poor chondrogenesis in vitro. In this work we investigated if fibrin matrix from clotted blood can act as a soft surrogate which nullifies the influence of the underlying stiff scaffold, thus promoting chondrogenesis irrespective of bulk scale scaffold stiffness. For this we performed in vitro chondrogenesis on soft (∼1.5kPa) and stiff (∼40kPa) gelatin scaffolds in the presence and absence of pericellular plasma clot. Our results demonstrated that in absence of pericellular plasma clot, chondrocytes showed efficient condensation and cartilaginous matrix secretion only on soft scaffolds, whereas, in presence of pericellular plasma clot, cell rounding and cartilaginous matrix secretion was observed in both soft and stiff scaffolds. More specifically, significantly higher collagen II, chondroitin sulfate and aggrecan deposition was observed in soft scaffolds, and soft and stiff scaffolds with pericellular plasma clot as compared to stiff scaffolds without pericellular plasma clot. Moreover, collagen type I, a fibrocartilage/bone marker was significantly higher only in stiff scaffolds without plasma clot. Therefore, it can be concluded that chondrocytes surrounded by a soft fibrin network were unable to sense the stiffness of the underlying scaffold/substrate and hence facilitate chondrogenesis even on stiff scaffolds. This understanding can have significant implications in the design of scaffolds for cartilage tissue engineering.

Pericellular plasma clot negates the influence of scaffold stiffness on chondrogenic differentiation.

Cell source plays a deterministic role in defining the outcome of a cell-based cartilage regenerative therapy and its clinical translational ability. Recent efforts in the direction of co-culture of two or more cell types attempt to combine the advantages of constituent cell types and negate their demerits.

Co-culture of infrapatellar fat pad-derived mesenchymal stromal cells and articular chondrocytes in plasma clot for cartilage tissue engineering.

In this work, a plant-derived polysaccharide carboxymethylcellulose (CMC) was chemically modified to incorporate sulfate groups to facilitate binding of cationic growth factors. The sulfated CMC (heparin mimic) was then used with CMC (glycosaminoglycan mimic) and gelatin (collagen mimic) to fabricate injectable pre-formed, macroporous scaffolds for cartilage tissue engineering. These scaffolds demonstrated high resilience and shape memory, thereby making them injectable through a 14G needle for up to 4-6 aspiration and injection cycles. Further, the scaffolds could sequester cationic proteins and growth factors (TGF-β1) through affinity-based interactions. When seeded with infrapatellar fat pad derived MSCs, the scaffolds demonstrated enhanced chondrogenesis after 28 days of in vitro culture when compared to controls. Taken together; these results demonstrate a polysaccharide-based minimally-invasive and translatable pre-formed injectable scaffold-based cell and growth factor delivery system for cartilage regeneration.

Sulfated polysaccharide mediated TGF-β1 presentation in pre-formed injectable scaffolds for cartilage tissue engineering.

Extracellular matrix (ECM) and its architecture have a vital role in articular cartilage (AC) structure and function. We hypothesized that a multi-layered chitosan-gelatin (CG) scaffold that resembles ECM, as well as native collagen architecture of AC, will achieve superior chondrogenesis and AC regeneration. We also compared its in vitro and in vivo outcomes with randomly aligned CG scaffold.

Article	Year
Combination of single walled carbon nanotubes/graphene oxide with paclitaxel: a reactive oxygen species mediated synergism for treatment of lung cancer. Nanoscale\| PubMed ID: 23443459	2013
Pullulan-based composite scaffolds for bone tissue engineering: Improved osteoconductivity by pore wall mineralization. Carbohydrate polymers\| PubMed ID: 25843850	2015
Pore orientation mediated control of mechanical behavior of scaffolds and its application in cartilage-mimetic scaffold design. Journal of the mechanical behavior of biomedical materials\| PubMed ID: 26256472	2015
Fabrication and characterization of Pluronic modified poly(hydroxybutyrate) fibers for potential wound dressing applications. Materials science & engineering. C, Materials for biological applications\| PubMed ID: 27040219	2016
Understanding the influence of phosphorylation and polysialylation of gelatin on mineralization and osteogenic differentiation. Materials science & engineering. C, Materials for biological applications\| PubMed ID: 27157722	2016
Pericellular plasma clot negates the influence of scaffold stiffness on chondrogenic differentiation. Acta biomaterialia\| PubMed ID: 27693666	2016
Co-culture of infrapatellar fat pad-derived mesenchymal stromal cells and articular chondrocytes in plasma clot for cartilage tissue engineering. Cytotherapy\| PubMed ID: 28479049	2017
Sulfated polysaccharide mediated TGF-β1 presentation in pre-formed injectable scaffolds for cartilage tissue engineering. Carbohydrate polymers\| PubMed ID: 29773398	2018
In vivo cartilage regeneration in a multi-layered articular cartilage architecture mimicking scaffold. Bone & joint research\| PubMed ID: 33014353	2020

Aditya Arora

Biography

JoVE Journal Publications

Other Publications