Fabrication and characterization of porous 3D TCP-CMC- alginate fibrous constructs for implant applications

This study reports the fabrication of 3D constructs using Tri-calcium phosphate/carboxymethyl cellulose composite with alginate. Microporous scaffold fibers were developed by incorporating gas bubbles within fibers, stabilizing it with surfactants, and subsequently removing the gas by vacuum treatment. The prepared paste was dispensed through a specially designed sieve plate by applying pressure and extruded in a calcium chloride/acetic acid bath. Gas is evolved as a result of reaction between sodium bicarbonate in paste and acetic acid in solution. The porosity of the fiber is tuned using 0.9, 1.8 and 3.6 weight% sodium bicarbonate (NaHCO3). Fibers were characterized using scanning electron microscopy (SEM) and mercury intrusion porosimetry (MIP). The morphology of the scaffold was characterized using scanning electron microscopy (SEM). Pore morphology was found to be better in scaffolds with 0.9 wt% NaHCO3 as it revealed an interconnected structure. MIP results showed an increase in pore volume with increasing concentration of NaHCO3. Study on the mechanical properties of the constructs was carried out to evaluate the compressive strength. In-vitro bioactivity studies were carried out in simulated body fluid (SBF) for 2/4 weeks. The study showed that the scaffold provides favorable substrate conditions to form bone like mineral HA phase, which plays a significant role in osteointegration

Hierarchically Engineered Biomimetic 3-D Scaffolds for Guided Regeneration of Bone Tissue

Considering the complex hierarchical structure of bone, biomimicking the micro and nano level features should be an integral part of scaffold fabrication for successful bone regeneration. In this thesis, we aim to biomimic the micro and nanostructure of bone and study the effect of physical and mechanical cues on cell alignment, proliferation and differentiation. To achieve this, in the first part of our study we have combined effect of anisotropy (micropattern, using photolithography technique) and the isotropy (nanofibers using electrospinning technique) on mesenchymal stem cell (MSC) alignment, proliferation and osteodifferentiation on SU-8 polymer scaffolds. We hypothesize that biomimicking the hierarchical features of bone at nano and microlevel will provide a better niche for MSC alignment and induce early osteodifferentiation of MSCs even in absence of external chemical factors. We divided the scaffolds into groups: electrospun SU-8 nanofibers, electrospun SU-8 nanofibers with UV treatment and micropatterned (20 µm sized ridges and grooves) SU-8 nanofibers by photolithography with UV treatment. Two types of culture conditions were applied: with and without osteoinduction medium. In-vitro cell proliferation assays, protein estimation, ALP osteodifferentiation assay, live dead assay and cell alignment studies were performed on these micro-patterned nanofiber domains. Our findings show that patterned surface induced an early osteodifferentiation of MSCs even in absence of osteoinduction medium. An interesting similarity with the helicoidal plywood model of the bone was observed. The cells showed layering and rotation along the patterns with time. This resembles the in-vivo anisotropic multilamellar bone tissue architecture thus, closely mimicking the sub-cellular features of bone. This might serve as a smart biomaterial surface for MSC differentiation in therapeutics where the addition of external chemical factors is a challenge. With this background, the next set of experiments deals with 3-D cell culture of spheroids on patterned scaffolds. 2-D cell culture has been widely developed with various micropatterning and microfabrication techniques over the past few decades for creating and controlling cellular microenvironments including cell-matrix interactions, cell-cell interactions, and bio-mimicking the in-vivo tissue hierarchy and functions. However, the drawbacks of 2-D culture has currently paved the way to 3-D cell culture which is considered clinically and biologically more relevant. Here we report a 3-D double strategy for osteodifferentiation of MSC spheroids on nano- and micropatterned PLGA/Collagen/nHAp electrospun fiber mats. A comparison of cell alignment, proliferation and differentiation of 2-D and 3-D MSCs on patterned and non-patterned substrate was done. The study demonstrates the synergistic effect of geometric cues and 3-D culture on differentiation of MSC spheroids into osteogenic lineage even in absence of osteoinduction medium. Hence, in this work, we made an effort to successfully combine the strength of 3-D spheroid culture with the hierarchically patterned micro and nanostructures to create a biologically and clinically relevant functional material for regeneration of bone tissue. In the last set of experiment, we have investigated the effect of mechanical strain along with patterned substrates on differentiation of MSCs to osteogenic lineage. We fabricated a thin, stretchable patterned PDMS membrane which warrants high-throughput to study different geometric pattern dimensions in a single sheet of the fabricated membrane. The membrane was integrated at the bottom of a customized 96-well plate device. Our previously developed multiwell stretch device was used for applying uniform strain across individual wells of the developed 96-well patterned stretch plates. We tested the effect of MSC differentiation to osteogenic lineage on 12 different micropattern dimensions at two strain frequencies (0.1Hz and 1 Hz) and a strain magnitude of 7.5%. Hence, we tried to biomimic the physiological conditions found invivo to investigate the effect on MSC differentiation to osteogenic lineage. Hence the findings from this work can be used as a model system to study the effect of topographical parameters and mechanical stimuli not only for bone, but also for other tissues owing to the accuracy and ease of the fabrication technique used. Together, the finding from this study, bio-mimicking the micro and nano features of bone tissue, will improve the efficiency of the currently available scaffolds for bone tissue regeneration and repair

3D printers for surgical practice

Surgical operations are challenging for newly recruited residents and experienced surgeons alike, as they involve unexpected findings in a surgical field, timely decisions, and unpredictable outcomes. In addition, they pose problems for experienced surgeons, who are dealing with congenital anomalies or complicated cancer cases, where the inter-anatomical relationships might not be as per traditional knowledge. Although a number of imaging modalities exist to accurately predict the anatomy in 3D, surgeons find it difficult to grasp and plan the details because of the limitations of 3D anatomy visualization and inter-tissue relationships on a 2D screen. 3D printing technology comes to aid in these circumstances. Surgeons could use the technology for a number of applications, including creation of patient specific pathological models for surgical planning, designing of customized prostheses, planning for surgical guide templates, and fabrication of accurate low-cost anatomical models as teaching aids. In addition, the patients as customers of medical therapy can clearly understand the complexity of surgery and the goals of surgical planning in complicated surgeries using these 3D models. Surgeons can clearly communicate with them about their condition through these palpable models. In summary, 3D printing technology could play an important role for both patient satisfaction and surgeons’ ability to provide better surgical car

Enhanced osteodifferentiation of MSC spheroids on patterned electrospun fiber mats - An advanced 3D double strategy for bone tissue regeneration

2D cell culture has been widely developed with various micropatteming and microfabrication techniques over the past few decades for creating and controlling cellular microenvironments including cell-matrix interactions, cell-cell interactions, and bio-mimicking the in-vivo tissue hierarchy and functions. However, the drawbacks of 2D culture have currently paved the way to 3D cell culture which is considered clinically and biologically more relevant. Here we report a 3D double strategy for osteodifferentiation of MSC spheroids on nano- and micro-patterned PLGA/Collagen/nHAp electrospun fiber mats. A comparison of cell alignment, proliferation and dif ferentiation of 2D and 3D MSCs on patterned and non-patterned substrate was done. The study demonstrates the synergistic effect of geometric cues and 3D culture on differentiation of MSC spheroids into osteogenic lineage even in absence of osteoinduction medium

Electrospun fibers for recruitment and differentiation of stem cells in regenerative medicine

Electrospinning is a popular technique used to mimic the natural sub-micron features of the native tissue. The ultra-fine fibers provide a favorable extracellular matrix-like environment for regulation of cellular functions. This article summarizes and reviews the current advances in electrospun fiber application and focuses on the novel strategies applied for tissue regeneration and repair. It explores the different factors affecting the attachment and proliferation of mesenchymal stem cells (MSCs) on the electrospun substrates. The influence of different features of electrospun fibers in the differentiation of MSCs into specific lineages (bone, cartilage, tendon/ligament and nerves) has been elaborated. In addition, the different techniques to mimic the hierarchical features of tissues and its effect on cellular functions are reviewed. Additionally, the new developments like three- dimensional (3D) electrospinning, 3D spheroid double strategy and the comparative analysis of dynamic and static culture on electrospun scaffolds are discussed. With the intricate understanding of the interaction between the cells and the electrospun fiber matrix we can aim to combine the newer strategies to overcome the existing challenges and improve the potential application of electrospun fibers in the field of tissue regeneration and repair

Effect of Patterned Electrospun Hierarchical Structures on Alignment and Differentiation of Mesenchymal Stem Cells: Biomimicking Bone

Considering the complex hierarchical structure of bone, biomimicking the micro and nano level features should be an integral part of scaffold fabrication for successful bone regeneration. We aim to biomimic the micro- and nano-structure of bone and study the effect of physical cues on cell alignment, proliferation and differentiation. To achieve this, we have divided the scaffolds into groups: electrospun SU-8 nanofibers, electrospun SU-8 nanofibers with UV treatment and micropatterned (20 μm sized ridges and grooves) SU-8 nanofibers by photolithography with UV treatment. Two types of culture conditions were applied: with and without osteoinduction medium. In-vitro cell proliferation assays, protein estimation, ALP osteodifferentiation assay, live dead assay and cell alignment studies were performed on these micro-patterned nanofiber domains. Our findings show that patterned surface induced an early osteodifferentiation of MSCs even in absence of osteoinduction medium. An interesting similarity with the helicoidal plywood model of the bone was observed. The cells showed layering and rotation along the patterns with time. This resembles the in-vivo anisotropic multi-lamellar bone tissue architecture thus, closely mimicking the sub-cellular features of bone. This might serve as a smart biomaterial surface for MSC differentiation in therapeutics where the addition of external chemical factors is a challenge