Jayarama Reddy Venugopal

Electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue engineering

The development of bioinspired or biomimetic materials is essential and has formed one of the most important paradigms in todays tissue engineering research. This paper reports a novel biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan (HAp/CTS) prepared by combining an in situ co-precipitation synthesis approach with an electrospinning process. A model HAp/CTS nanocomposite with the HAp mass ratio of 30 wt% was synthesized through the co-precipitation method so as to attain homogenous dispersion of the spindle-shaped HAp nanoparticles (ca. 100 x 30 nm) within the chitosan matrix. By using a small amount (10 wt%) of ultrahigh molecular weight poly(ethylene oxide) (UHMWPEO) as a fiber-forming facilitating additive, continuous HAp/CTS nanofibers with a diameters of 214+/-25 nm had been produced successfully and the HAp nanoparticles with some aggregations were incorporated into the electrospun nanofibers. Further SAED and XRD analysis confirmed that the crystalline nature of HAp remains and had survived the acetic acid-dominant solvent system. Biological in vitro cell culture with human fetal osteoblast (hFOB) cells for up to 15 days demonstrated that the incorporation of HAp nanoparticles into chitosan nanofibrous scaffolds led to significant bone formation oriented outcomes compared to that of the pure electrospun CTS scaffolds. The electrospun nanocomposite nanofibers of HAp/CTS, with compositional and structural features close to the natural mineralized nanofibril counterparts, are of potential interest for bone tissue engineering applications.

Electrospun nanostructured scaffolds for bone tissue engineering

The current challenge in bone tissue engineering is to fabricate a bioartificial bone graft mimicking the extracellular matrix (ECM) with effective bone mineralization, resulting in the regeneration of fractured or diseased bones. Biocomposite polymeric nanofibers containing nanohydroxyapatite (HA) fabricated by electrospinning could be promising scaffolds for bone tissue engineering. Nanofibrous scaffolds of poly-l-lactide (PLLA, 860+/-110 nm), PLLA/HA (845+/-140 nm) and PLLA/collagen/HA (310+/-125 nm) were fabricated, and the morphology, chemical and mechanical characterization of the nanofibers were evaluated using scanning electron microscopy, Fourier transform infrared spectroscopy and tensile testing, respectively. The in vitro biocompatibility of different nanofibrous scaffolds was also assessed by growing human fetal osteoblasts (hFOB), and investigating the proliferation, alkaline phosphatase activity (ALP) and mineralization of cells on different nanofibrous scaffolds. Osteoblasts were found to adhere and grow actively on PLLA/collagen/HA nanofibers with enhanced mineral deposition of 57% higher than the PLLA/HA nanofibers. The synergistic effect of the presence of an ECM protein, collagen and HA in PLLA/collagen/HA nanofibers provided cell recognition sites together with apatite for cell proliferation and osteoconduction necessary for mineralization and bone formation. The results of our study showed that the biocomposite PLLA/collagen/HA nanofibrous scaffold could be a potential substrate for the proliferation and mineralization of osteoblasts, enhancing bone regeneration.

Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts.

Nanotechnology has enabled the engineering of nanostructured materials to meet current challenges in bone replacement therapies. Biocomposite nanofibrous scaffolds of poly(l-lactic acid)-co-poly(epsilon-caprolactone), gelatin and hydroxyapatite (HA) were fabricated by combining the electrospinning and electrospraying techniques in order to create a better osteophilic environment for the growth and mineralization of osteoblasts. Electrospraying of HA nanoparticles on electrospun nanofibers helped to attain rough surface morphology ideal for cell attachment and proliferation and also achieve improved mechanical properties than HA blended nanofibers. Nanofibrous scaffolds showed high pore size and porosity up to 90% with fiber diameter in the range of 200-700 nm. Nanofibrous scaffolds were characterized for their functional groups and chemical structure by FTIR and XRD analysis. Studies on cell-scaffold interaction were carried out by culturing human fetal osteoblast cells (hFOB) on both HA blended and sprayed PLACL/Gel scaffolds and assessing their growth, proliferation, mineralization and enzyme activity. The results of MTS, ALP, SEM and ARS studies confirmed, not only did HA sprayed biocomposite scaffolds showed better cell proliferation but also enhanced mineralization and alkaline phosphatase activity (ALP) proving that electrospraying in combination with electrospinning produced superior and more suitable biocomposite nanofibrous scaffolds for bone tissue regeneration.

Applications of polymer nanofibers in biomedicine and biotechnology

Recent advancements in the electrospinning method enable the production of ultrafine solid and continuous fibers with diameters ranging from a few nanometers to a few hundred nanometers with controlled surface and internal molecular structures. A wide range of biodegradable biopolymers can be electrospun into mats with specific fiber arrangement and structural integrity. Through secondary processing, the nanofiber surface can be functionalized to display specific biochemical characteristics. It is hypothesized that the large surface area of nanofibers with specific surface chemistry facilitates attachment of cells and control of their cellular functions. These features of nanofiber mats are morphologically and chemically similar to the extracellular matrix of natural tissue, which is characterized by a wide range of pore diameter distribution, high porosity, effective mechanical properties, and specific biochemical properties. The current emphasis of research is on exploiting such properties and focusing on determining appropriate conditions for electrospinning various polymers and biopolymers for eventual applications including multifunctional membranes, biomedical structural elements (scaffolds used in tissue engineering, wound dressing, drug delivery, artificial organs, vascular grafts), protective shields in specialty fabrics, and filter media for submicron particles in the separation industry. This has resulted in the recent applications for polymer nanofibers in the field of biomedicine and biotechnology.

Mesenchymal stem cell differentiation to neuronal cells on electrospun nanofibrous substrates for nerve tissue engineering

Bone marrow Mesenchymal stem cells capable of differentiating into neuronal cells on engineered nanofibrous scaffolds have great potential for bionanomaterial-cell transplantation therapy of neurodegenerative diseases and injuries of the nervous system. MSCs have been the highlight of many tissue engineering studies mainly because of their multipotential properties. We investigated the potential of human bone marrow derived Mesenchymal stem cells (MSCs) for neuronal differentiation in vitro on poly(L-lactic acid)-co-poly-(3-caprolactone)/Collagen (PLCL/Coll) nanofibrous scaffolds. PLCL and PLCL/Coll nanofibrous scaffolds were fabricated by electrospinning process and their chemical and mechanical characterizations were carried out using SEM, contact angle, FTIR, and tensile instrument. The differentiation of MSCs was carried out using neuronal inducing factors including beta-mercaptoethanol, epidermal growth factor, nerve growth factor and brain derived growth factor in DMEM/F12 media. The proliferations of MSCs evaluated by MTS assay showed that the cells grown on PLCL/Coll nanofibrous scaffolds were comparatively higher (80%) than those on PLCL. Scanning electron microscopy results showed that MSCs differentiated on PLCL/Coll nanofibrous scaffolds showed neuronal morphology, with multipolar elongations and expressed neurofilament and nestin protein by immuno-fluorescent microscopy. Our studies on the differentiation of MSCs to neuronal cells on nanofibrous scaffolds suggest their potential application towards nerve regeneration.

Applications of conducting polymers and their issues in biomedical engineering

Conducting polymers (CPs) have attracted much interest as suitable matrices of biomolecules and have been used to enhance the stability, speed and sensitivity of various biomedical devices. Moreover, CPs are inexpensive, easy to synthesize and versatile because their properties can be readily modulated by (i) surface functionalization techniques and (ii) the use of a wide range of molecules that can be entrapped or used as dopants. This paper discusses the various surface modifications of the CP that can be employed in order to impart physico-chemical and biological guidance cues that promote cell adhesion/proliferation at the polymer–tissue interface. This ability of the CP to induce various cellular mechanisms widens its applications in medical fields and bioengineering.

Aligned and random nanofibrous substrate for the in vitro culture of Schwann cells for neural tissue engineering

The current challenge in peripheral nerve tissue engineering is to produce an implantable scaffold capable of bridging long nerve gaps that will produce results similar to autograft without requiring the harvest of autologous donor tissue. Aligned and random polycaprolactone/gelatin (PCL/gelatin) nanofibrous scaffolds were fabricated for the in vitro culture of Schwann cells that assist in directing the growth of regenerating axons in nerve tissue engineering. The average fiber diameter attained by electrospinning of polymer blend (PCL/gelatin) ranged from 232+/-194 to 160+/-86nm with high porosity (90%). Blending PCL with gelatin resulted in increased hydrophilicity of nanofibrous scaffolds and yielded better mechanical properties, approaching those of PCL nanofibers. The biocompatibility of fabricated nanofibers was assessed for culturing and proliferation of Schwann cells by MTS assay. The results of the MTS assay and scanning electron microscopy confirmed that aligned and random PCL/gelatin nanofibrous scaffolds are suitable substrates for Schwann cell growth as compared to PCL nanofibrous scaffolds for neural tissue engineering.

Electrospun Biocomposite Nanofibrous Scaffolds for Neural Tissue Engineering

Bridging of nerve gaps after injury is a major problem in peripheral nerve regeneration. Considering the potential application of a bio-artificial nerve guide material, polycaprolactone (PCL)/chitosan nanofibrous scaffolds was designed and evaluated in vitro using rat Schwann cells (RT4-D6P2T) for nerve tissue engineering. PCL, chitosan, and PCL/chitosan nanofibers with average fiber diameters of 630, 450, and 190 nm, respectively, were fabricated using an electrospinning process. The surface chemistry of the fabricated nanofibers was determined using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Simple blending of PCL with chitosan proved an easy and efficient method for fabricating PCL/chitosan nanofibrous scaffolds, whose surface characteristics proved more hydrophilic than PCL nanofibers. Evaluation of mechanical properties showed that the Youngs modulus and strain at break of the electrospun PCL/chitosan nanofibers were better than those of the chitosan nanofibers. Results of cell proliferation studies on nanofibrous scaffolds using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay showed 48% more cell proliferation on PCL/chitosan scaffolds than on PCL scaffolds after 8 days of culture. PCL/chitosan scaffolds showed better cell proliferation than PCL scaffolds and maintained their characteristic cell morphology, with spreading bipolar elongations to the nanofibrous substrates. This electrospun nanofibrous matrix thus proved of specific interest in tissue engineering for peripheral nerve regeneration.

Nanobioengineered electrospun composite nanofibers and osteoblasts for bone regeneration.

Bone defects represent a medical and socioeconomic challenge. Engineering bioartificial bone tissues may help to solve problems related to donor site morbidity and size limitations. Nanofibrous scaffolds were electrospun into a blend of synthetic biodegradable polycaprolactone (PCL) with hydroxyapatite (HA) and natural polymer gelatin (Gel) at a ratio of 1:1:2 (PCL/HA/Gel) compared to PCL (9%), PCL/HA (1:1), and PCL/Gel (1:2) nanofibers. These fiber diameters were around 411 +/- 158 to 856 +/- 157 nm, and the pore size and porosity around 5-35 microm and 76-93%, respectively. The interconnecting porous structure of the nanofibrous scaffolds provides large surface area for cell attachment and sufficient space for nutrient transportation. The tensile property of composite nanofibrous scaffold (PCL/HA/Gel) was highly flexible and allows penetrating osteoblasts inside the scaffolds for bone tissue regeneration. Fourier transform infrared analysis showed that the composite nanofiber contains an amino group, a phosphate group, and carboxyl groups for inducing proliferation and mineralization of osteoblasts for in vitro bone formation. The cell proliferation (88%), alkaline phosphatase activity (77%), and mineralization (66%) of osteoblasts were significantly (P < 0.001) increased in composite nanofibrous scaffold compared to PCL nanofibrous scaffolds. Field emission scanning electron microscopic images showed that the composite nanofibers supported the proliferation and mineralization of osteoblast cells. These results show that the fabrication of electrospun PCL/HA/Gel composite nanofibrous scaffolds has potential for the proliferation and mineralization of osteoblasts for bone regeneration.

Precipitation of nanohydroxyapatite on PLLA/PBLG/Collagen nanofibrous structures for the differentiation of adipose derived stem cells to osteogenic lineage.

Tissue engineering and nanotechnology have enabled engineering of nanostructured materials to meet the current challenges in bone treatment owing to rising occurrence of bone diseases, accidental damages and defects. Poly(L-lactic acid)/Poly-benzyl-L-glutamate/Collagen (PLLA/PBLG/Col) scaffolds were fabricated by electrospinning and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). The abundance and accessibility of adipose derived stem cells (ADSC) may prove to be novel cell therapeutics for bone repair and regeneration. ADSCs were cultured on these scaffolds and were induced to undergo osteogenic differentiation in the presence of PBLG/n-HA for BTE. The cell-biomaterial interactions were analyzed using cell proliferation, SEM and CMFDA dye extraction techniques. Osteogenic differentiation of ADSC was confirmed using alkaline phosphatase activity (ALP), mineralization (ARS) and dual immunofluorescent staining using both ADSC marker protein and Osteocalcin, which is a bone specific protein. The utmost significance of this study is the bioactive PBLG/n-HA biomolecule introduced on the polymeric nanofibers to regulate and improve specific biological functions like adhesion, proliferation and differentiation of ADSC into osteogenic lineage. This was evident from the immunostaining and CMFDA images of ADSCs showing cuboidal morphology, characteristic of osteogenic lineage. The observed results proved that the PLLA/PBLG/Col/n-HA scaffolds promoted greater osteogenic differentiation of ADSC as evident from the enzyme activity and mineralization profiles for bone tissue engineering.