[Show abstract][Hide abstract] ABSTRACT: The electrospinning of chitosan remains challenging due to its rigid crystalline structure,
insufficient viscosity and limited solubility in common organic solvents. This work
presents a “smart” chitosan modification that allows electrospinning irrespective of
molecular weight or deacetylation value and without blending with synthetic polymers. A
novel derivative, namely 2-nitrobenzyl-chitosan (NB), at various molar compositions of
chitosan:2-nitrobenzaldehyde (1:1 (NB-1), 1:0.5 (NB-2), 1:0.25 (NB-3)) was synthesized
by the reaction between amino groups of chitosan and aldehyde groups of 2-
Polymers for Advanced Technologies 03/2014; · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Electrospun nanofiber matrices have been produced using natural and synthetic polymers for a variety of biomedical applications. However, electrospinning of water-soluble polymers still remains as a major challenge. Polysaccharides are difficult to spin and often mixed with other synthetic polymers to produce electrospun nanofiber matrices. Chitosan, the deacetylated form of chitin, is reported to be a biocompatible, biodegradable, antimicrobial and non-toxic polysaccharide and thus used for a variety of biomedical applications including tissue engineering, drug delivery device and wound healing. Chitosan electrospinning remains challenging due to limited solubility and a rigid crystalline structure which does not allow sufficient polymer concentrations required for successful fiber formation by electrospinning. Here we present a novel and “smart” chitosan modification methodology that allows direct electrospinning of high molecular weight chitosan at very high concentrations. In brief, to facilitate chitosan electrospinning a new 2-nitrobenzyl-chitosan derivative was synthesized and electrospun nanofibers were produced by dissolving the derivative in trifluoroacetic acid (TFA) solvent. In this derivative, 2-nitrobenzyl aldehyde is used as a photolytic removal group to block the amino groups of chitosan and subsequently reduce its structural rigidity. Subsequently, the electrospun nanofiber matrices produced by 2-nitrobenzyl-chitosan was exposed to UV light to produce pure chitosan nanofiber matrix. Electrospinning parameters were optimized to produce defect-free cylindrical nanofibers at 15% (wt/v) chitosan solution concentration and 1 kV/cm electrical potential. In this work we report on electrospinning of three different 2-Nitrobenzyl-chitosan compositions namely 1:1, 1:0.5, and 1:0.25 and evaluating chitosan matrices for scaffold properties and cell compatibility. The mechanism of photolysis to obtain neat chitosan from 2-Nitrobenzyl-chitosan followed by UV exposure was confirmed by FTIR analysis. The morphology of the electrospun fibers, pore structure, and fiber diameter was examined using scanning electron microscopy. Biocompatibility was evaluated by measuring cell proliferation and metabolic activity of MC3T3 cells seeded onto the nanofiber scaffolds.The samples were screened for antimicrobial activity by disc diffusion method using B. subtilis as gram positive, E. coli as gram negative, C. albicans as yeast and A. niger as fungi. The results showed that the samples have a strong inhibitory activity against all pathogenic microorganisms used.
[Show abstract][Hide abstract] ABSTRACT: Repair and regeneration of human tissues and organs using biomaterials, cells and/or growth factors is the ultimate goal of tissue engineers. One of the grand challenges in this field is to closely mimic the structures and properties of native tissues. Regenerative engineering-the convergence of tissue engineering with advanced materials science, stem cell science, and developmental biology-represents the next valuable tool to overcome the challenges. This article reviews the recent progress in developing advanced chitosan structures using various fabrication techniques. These chitosan structures, together with stem cells and functional biomolecules, may provide a robust platform to gain insight into cell-biomaterial interactions and may function as excellent artificial extracellular matrices to regenerate complex human tissues and biological systems.
[Show abstract][Hide abstract] ABSTRACT: Scaffold based bone tissue engineering (BTE) has made great progress in regenerating lost bone tissue. Materials of natural and synthetic origin have been used for scaffold fabrication. Scaffolds derived from natural polymers offer greater bioactivity and biocompatibility with mammalian tissues to favor tissue healing, due to their similarity to native extracellular matrix (ECM) components. Often it is a challenge to fabricate natural polymer based scaffolds for BTE applications without compromising their bioactivity, while maintaining adequate mechanical properties. In this work, we report the fabrication and characterization of cellulose and collagen based micro-nano structured scaffolds using human osteoblasts (HOB) for BTE applications. These porous micro-nano structured scaffolds (average pore diameter 190 +/- 10 microm) exhibited mechanical properties in the mid range of human trabecular bone (compressive modulus 266.75 +/- 33.22 MPa and strength 12.15 3 +/- 2.23 MPa). These scaffolds supported the greater adhesion and phenotype maintenance of cultured HOB as reflected by higher levels of osteogenic enzyme alkaline phosphatase and mineral deposition compared to control polyester micro-nano structured scaffolds of identical pore properties. These natural polymer based micro-nano structured scaffolds may serve as alternatives to polyester based scaffolds for BTE applications.
Journal of Biomedical Nanotechnology 04/2013; 9(4):719-31. · 7.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue engineering aims to repair, restore, and regenerate lost or damaged tissues by using biomaterials, cells, mechanical forces and factors (chemical and biological) alone or in combination. Growth factors are routinely used in the tissue engineering approach to expedite the process of regeneration. The growth factor approach has been hampered by several complications including high dose requirements, lower half-life, protein instability, higher costs and undesired side effects. Recently a variety of alternative small molecules of both natural and synthetic origin have been explored as alternatives to growth factors for tissue regeneration applications. Small molecules are simple biochemical components that elicit certain cellular responses through signaling cascades. Small molecules present a viable alternative to biological factors. Small molecule strategies can reduce various side effects, maintain bioactivity in a biological environment and minimize cost issues associated with complex biological growth factors. This manuscript focuses on three-osteoinductive small molecules, namely melatonin, resveratrol (from natural sources) and purmorphamine (synthetically designed) as inducers of bone formation and osteogenic differentiation of stem cells. Efforts have been made to summarize possible biological pathways involved in the action of each of these drugs. Melatonin is known to affect Mitogen Activated Protein (MAP) kinase, Bone morphogenic protein (BMP) and canonical wnt signaling. Resveratrol is known to activate cascades involving Int mammalian homologue of drosophila wingless protein (Wnt) and NAD-dependent deacetylase sirtuin-1 (Sirt1). Purmorphamine is a Hedgehog (Hh) pathway agonist as it acts on Smoothened (Smo) receptors. These mechanisms and the way they are affected by the respective small molecules will also be discussed in the manuscript.
Current pharmaceutical design 02/2013; · 4.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poly[(ethyl alanato)(1)(p-methyl phenoxy)(1)] phosphazene (PNEA-mPh) was used to modify the surface of electrospun poly(ε-caprolactone) (PCL) nanofiber matrices having an average fiber diameter of 3000 ± 1700 nm for the purpose of tendon tissue engineering and augmentation. This study reports the effect of polyphosphazene surface functionalization on human mesenchymal stem cell (hMSC) adhesion, cell-construct infiltration, proliferation and tendon differentiation, as well as long term cellular construct mechanical properties. PCL fiber matrices functionalized with PNEA-mPh acquired a rougher surface morphology and led to enhanced cell adhesion as well as superior cell-construct infiltration when compared to smooth PCL fiber matrices. Long-term in vitro hMSC cultures on both fiber matrices were able to produce clinically relevant moduli. Both fibrous constructs expressed scleraxis, an early tendon differentiation marker, and a bimodal peak in expression of the late tendon differentiation marker tenomodulin, a pattern that was not observed in PCL thin film controls. Functionalized matrices achieved a more prominent tenogenic differentiation, possessing greater tenomodulin expression and superior phenotypic maturity according to the ratio of collagen I to collagen III expression. These findings indicate that PNEA-mPh functionalization is an efficient method for improving cell interactions with electrospun PCL matrices for the purpose of tendon repair.
[Show abstract][Hide abstract] ABSTRACT: Electrospun polycaprolactone nanofiber matrices surface functionalized with poly[(ethyl alanato), (p-methyl phenoxy),] phosphazene were fabricated for the purpose of soft skeletal tissue regeneration. This preliminary study reports the effect of fiber diameter and polyphosphazene surface functionalization on significant scaffold properties such as morphology, surface hydrophilicity, porosity, tensile properties, human mesenchymal stem cell adhesion and proliferation. Six fiber matrices comprised of average fiber diameters in the range of 400-500, 900-1000, 1400-1500, 1900-2000, 2900-3000 and 3900-4000 nm were considered for primary evaluation. After achieving the greatest proliferation while maintaining moderate tensile modulus, matrices in the diameter range of 2900-3000 nm were selected to examine the effect of coating with 1%, 2% and 3% (weight/volume) polyphosphazene solutions. Polyphosphazene functionalization resulted in rougher surfaces that correlated with coating solution concentration. Analytical techniques such as energy dispersive X-ray analysis, Fourier transform infrared spectroscopy, elemental analysis, differential scanning calorimetry, water contact angle goniometry and confocal microscopy confirmed the presence of polyphosphazene and its distribution on the functionalized fiber matrices. Functionalization achieved through 2% polymer solutions did not affect average pore diameter, tensile modulus, suture retention strength or cell proliferation compared to PCL controls. Surface polyphosphazene functionalization significantly improved the matrix hydrophilicity evidenced through decreased water contact angle of PCL matrices from 130 degrees to 97 degrees. Further, enhanced total protein synthesis by cells during in vitro culture was seen on 2% PPHOS functionalized matrices over controls. Improving PCL matrix hydrophilicity via proposed surface functionalization may be an efficient method to improve cell-PCL matrix interactions.
Journal of Biomedical Nanotechnology 02/2012; 8(1):107-24. · 7.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Successful regeneration necessitates the development of three-dimensional (3-D) tissue-inducing scaffolds that mimic the hierarchical architecture of native tissue extracellular matrix (ECM). Cells in nature recognize and interact with the surface topography they are exposed to via ECM proteins. The interaction of cells with nanotopographical features such as pores, ridges, groves, fibers, nodes, and their combinations has proven to be an important signaling modality in controlling cellular processes. Integrating nanotopographical cues is especially important in engineering complex tissues that have multiple cell types and require precisely defined cell-cell and cell-matrix interactions on the nanoscale. Thus, in a regenerative engineering approach, nanoscale materials/scaffolds play a paramount role in controlling cell fate and the consequent regenerative capacity. Advances in nanotechnology have generated a new toolbox for the fabrication of tissue-specific nanostructured scaffolds. For example, biodegradable polymers such as polyesters, polyphosphazenes, polymer blends and composites can be electrospun into ECM-mimicking matrices composed of nanofibers, which provide high surface area for cell attachment, growth, and differentiation. This review provides the fundamental guidelines for the design and development of nanostructured scaffolds for the regeneration of various tissue types in human upper and lower extremities such as skin, ligament, tendon, and bone. Examples focusing on the collective work of our laboratory in those areas are discussed to demonstrate the regenerative efficacy of this approach. Furthermore, preliminary strategies and significant challenges to integrate these individual tissues into one complex organ through regenerative engineering-based integrated graft systems are also discussed.
IEEE transactions on nanobioscience 01/2012; 11(1):3-14. · 1.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tendon injuries range from acute traumatic ruptures and lacerations to chronic overuse injuries, such as tendinosis. Even with improved nonsurgical, surgical, and rehabilitation techniques, outcomes following tendon repair are inconsistent. Primary repair remains the standard of care. However, repaired tendon tissue rarely achieves functionality equal to that of the preinjured state. Poor results have been linked to alterations in cellular organization within the tendon that occur at the time of injury and throughout the early stages of healing. Enhanced understanding of the biology of tendon healing is needed to improve management and outcomes. The use of growth factors and mesenchymal stem cells and the development of biocompatible scaffolds could result in enhanced tendon healing and regeneration. Recent advances in tendon bioengineering may lead to improved management following tendon injury.
The Journal of the American Academy of Orthopaedic Surgeons 03/2011; 19(3):134-42. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The synthesis and organization of extracellular matrix (ECM) of tendon, in resting and states of repair, are governed by fibroblasts. Growth differentiation factor-5 (GDF-5) may enhance the cellular response to tendon injury, thus improving the structural outcome of the regenerative tissue. This study was an attempt to identify potential mechanisms controlling the response of fibroblasts to injury and GDF-5, in the pursuit of improved tissue regeneration. There were two sets of experiments. Isolated mice Achilles tendon fibroblasts were treated with different concentrations of rGDF-5 (0-100 ng/ml) for 0-12 days in cell culture. The temporal effect of rGDF-5 on ECM gene expression was analysed for type I collagen and aggrecan expression. Microarray and gene expression analysis were performed on cells treated with 100 ng/ml for 4 days. Forty-five mice underwent bilateral mid-substance Achilles tendon tenotomy and suture repair. Repair sites were injected with 10 µg rGDF-5 or saline. Tendons were assessed histologically at 2, 4 and 6 weeks. Expression of ECM genes procollagen IX, aggrecan, matrix metalloproteinase 9 and fibromodulin were upregulated. Proinflammatory reaction genes were downregulated. rGDF-5 led to an increase in total DNA, glycosaminoglycan (GAG) and hydroxyproline (OHP). The OHP:DNA ratio of fibroblast cultures was increased over all time points, with increased GAG:DNA at day 12. rGDF-5 treatment showed improved collagen organization over controls. The results delineate the mode of action of rGDF-5 at the cellular and gene level. rGDF-5 could play a role in tendon repair and be used for future therapies that promote tendon healing.
Journal of Tissue Engineering and Regenerative Medicine 03/2011; 5(3):191-200. · 4.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue-engineered medical implants, such as polymeric nanofiber scaffolds, are potential alternatives to autografts and allografts, which are short in supply and carry risks of disease transmission. These scaffolds have been used to engineer various soft connective tissues such as skin, ligament, muscle, and tendon, as well as vascular and neural tissue. Bioactive versions of these materials have been produced by encapsulating molecules such as drugs and growth factors during fabrication. The fibers comprising these scaffolds can be designed to match the structure of the native extracellular matrix (ECM) closely by mimicking the dimensions of the collagen fiber bundles evident in soft connective tissues. These nanostructured implants show improved biological performance over the bulk materials in aspects of cellular infiltration and in vivo integration, and the topography of such scaffolds has been shown to dictate cellular attachment, migration, proliferation, and differentiation, which are critical steps in engineering complex functional tissues and crucial to improved biocompatibility and functional performance. Nanofiber matrices can be fabricated using a variety of techniques, including drawing, molecular self-assembly, freeze-drying, phase separation, and electrospinning. Among these processes, electrospinning has emerged as a simple, elegant, scalable, continuous, and reproducible technique to produce polymeric nanofiber matrices from solutions and their melts. We have shown the ability of this technique to be used to fabricate matrices composed of fibers from a few hundred nanometers to several microns in diameter by simply altering the polymer solution concentration. This chapter will discuss the use of the electrospinning technique in the fabrication of ECM-mimicking scaffolds. Furthermore, selected scaffolds will be seeded with primary adipose-derived stromal cells, imaged using scanning electron microscopy and confocal microscopy, and evaluated in terms of their capacity toward supporting cellular proliferation over time.
[Show abstract][Hide abstract] ABSTRACT: This study was designed to examine the cellular and molecular response of tendon fibroblasts to growth/differentiation factor-5 (GDF-5). Rat Achilles tendon fibroblasts (ATFs) were treated in culture with varying concentrations of GDF-5 (0-1000 ng/ml) over varying periods of time (0-12 days). Cell proliferation, evaluated through use of a standard MTT colorimetric assay, confirmed that GDF-5 stimulates ATF proliferation in a concentration- and time-dependent fashion. Temporal and concentration analysis revealed that GDF-5 increases total DNA, glycosaminoglycan (GAG), and hydroxyproline (HYP) content. Ratios of HYP/DNA and GAG/DNA increased with increasing concentrations of GDF-5 (0-1000 ng/ml). Expression of the following 12 extracellular matrix (ECM) and cell-adhesion-related genes was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR): collagen I (col I), collagen III (col III), matrix metalloproteinases (MMP)-3 and -13, aggrecan, tissue inhibitor of matrix metalloproteinase (TIMP)-2, syndecan-4, N-cadherin, tenascin-C, biglycan, versican, and decorin. RT-PCR data revealed an increase in the expression of col I, col III, MMP-3, MMP-13, TIMP-2, syndecan-4, N-cadherin, tenascin-C, and aggrecan genes by day 6. A statistically significant decrease in TIMP-2 and MMP-13 was observed on day 12. Decorin expression was depressed at all time points in cells treated with GDF-5. There was no significant change in biglycan expression in ATFs supplemented with GDF-5. These findings suggest that GDF-5 induces cellular proliferation and ECM synthesis as well as expression of ECM and cell-adhesion-related genes in ATFs. This study further defines the influence of GDF-5 on rat ATFs through its action on the expression of genes that are associated with tendon ECM.
Connective tissue research 01/2011; 52(4):353-64. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adipose-derived mesenchymal stem cells (ADMSCs) are a unique population of stem cells with therapeutic potential in the treatment of connective tissue injuries. Growth differentiation factor-5 (GDF)-5 is known to play a role in tendon repair and maintenance. The aim of this study was to investigate the effects of GDF-5 on proliferation and tendonogenic gene expression of rat ADMSCs.
ADMSCs were treated in culture with different concentrations of GDF-5 (0-1000 ng/mL) for 12 days. Biochemical, temporal, and concentration kinetic studies were done. Extracellular matrix (ECM) synthesis, tendonogenic differentiation, and matrix remodeling gene and protein expression were analyzed.
GDF-5 led to increased ADMSC proliferation in a dose- and time-dependent manner. ADMSCs demonstrated enhanced ECM (collagen type I, decorin, and aggrecan) and tendonogenic marker (scleraxis, tenomodulin, and tenascin-C) gene expression with 100 ng/mL of GDF-5 (p < 0.05). ECM and tendon-specific markers showed time-dependent increases at various time points (p < 0.05), although decorin decreased at day 9 (p < 0.05). GDF-5 did alter expression of matrix remodeling genes, with no specific trends observed. Western blot analysis confirmed dose- and time-dependent increases in protein expression of tenomodulin, tenascin-C, Smad-8, and matrix metalloproteinase-13.
In vitro GDF-5 treatment can induce cellular events leading to the tendonogenic differentiation of ADMSCs. The use of combined GDF-5 and ADMSCs tissue-engineered therapies may have a role in the future of tendon repair.
Tissue Engineering Part A 09/2010; 16(9):2941-51. · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nanostructures in the form of tubes, wires, crystals, rods, spheres, and fibers have been fabricated and assembled into various macrostructures for a variety of high technology applications. Nanofeatures impart several amazing properties to these macrostructures including high surface area, surface functionality, and superior mechanical, optical, electrical, and magnetic properties over the parent bulk material. Polymeric nanofibers in the form of nonwoven cloth, membrane, braids and tubes are extensively used for daily needs, and in addition used as filters, protective clothing, and for a variety of industrial and biomedical applications. Electrospinning or electrostatic spinning has emerged as a very popular technique to fabricate polymeric nanofiber matrices. More than 100 different polymers of natural, synthetic origin, their blends and composites have been electrospun into different three dimensional (3-D) macrostructures. Electrospinning provides opportunities to manipulate and control surface area, fiber diameter, porosity and pore size of nanofiber matrices. These nanofiber matrices closely mimic the structure of extracellular matrix (ECM) and influence cellular activities both in vitro and in vivo. Nanofiber macrostructures have been used as a vehicle to deliver therapeutic agents, as scaffolds for engineering various tissues and also serve as an integrated part of biomedical implants. Present review will cover some of the recent important patents that use electrospun nanofiber matrices for various biomedical applications.
Recent Patents on Biomedical Engineering. 01/2010; 1(1).
[Show abstract][Hide abstract] ABSTRACT: Electrospun fiber matrices composed of scaffolds of varying fiber diameters were investigated for potential application of severe skin loss. Few systematic studies have been performed to examine the effect of varying fiber diameter electrospun fiber matrices for skin regeneration. The present study reports the fabrication of poly[lactic acid-co-glycolic acid] (PLAGA) matrices with fiber diameters of 150-225, 200-300, 250-467, 500-900, 600-1,200, 2,500-3,000 and 3,250-6,000 nm via electrospinning. All fiber matrices found to have a tensile modulus from 39.23+/-8.15 to 79.21+/-13.71 MPa which falls in the range for normal human skin. Further, the porous fiber matrices have porosity between 38 to 60% and average pore diameters between 10 to 14 microm. We evaluated the efficacy of these biodegradable fiber matrices as skin substitutes by seeding them with human skin fibroblasts (hSF). Human skin fibroblasts acquired a well spread morphology and showed significant progressive growth on fiber matrices in the 350-1,100 nm diameter range. Collagen type III gene expression was significantly up-regulated in hSF seeded on matrices with fiber diameters in the range of 350-1,100 nm. Based on the need, the proposed fiber skin substitutes can be successfully fabricated and optimized for skin fibroblast attachment and growth.
[Show abstract][Hide abstract] ABSTRACT: Surgical treatment of tendon ruptures and lacerations is currently the most common therapeutic modality. Tendon repair in the hand involves a slow repair process, which results in inferior repair tissue and often a failure to obtain full active range of motion. The initial stages of repair include the formation of functionally weak tissue that is not capable of supporting tensile forces that allow early active range of motion. Immobilization of the digit or limb will promote faster healing but inevitably results in the formation of adhesions between the tendon and tendon sheath, which leads to friction and reduced gliding. Loading during the healing phase is critical to avoid these adhesions but involves increased risk of rupture of the repaired tendon. Understanding the biology and organization of the native tendon and the process of morphogenesis of tendon tissue is necessary to improve current treatment modalities. Screening the genes expressed during tendon morphogenesis and determining the growth factors most crucial for tendon development will likely lead to treatment options that result in superior repair tissue and ultimately improved functional outcomes.
The Journal Of Hand Surgery 02/2008; 33(1):102-12. · 1.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This manuscript focuses on bone repair/regeneration using tissue engineering strategies, and highlights nanobiotechnology developments leading to novel nanocomposite systems. About 6.5 million fractures occur annually in USA, and about 550,000 of these individual cases required the application of a bone graft. Autogenous and allogenous bone have been most widely used for bone graft based therapies; however, there are significant problems such as donor shortage and risk of infection. Alternatives using synthetic and natural biomaterials have been developed, and some are commercially available for clinical applications requiring bone grafts. However, it remains a great challenge to design an ideal synthetic graft that very closely mimics the bone tissue structurally, and can modulate the desired function in osteoblast and progenitor cell populations. Nanobiomaterials, specifically nanocomposites composed of hydroxyapatite (HA) and/or collagen are extremely promising graft substitutes. The biocomposites can be fabricated to mimic the material composition of native bone tissue, and additionally, when using nano-HA (reduced grain size), one mimics the structural arrangement of native bone. A good understanding of bone biology and structure is critical to development of bone mimicking graft substitutes. HA and collagen exhibit excellent osteoconductive properties which can further modulate the regenerative/healing process following fracture injury. Combining with other polymeric biomaterials will reinforce the mechanical properties thus making the novel nano-HA based composites comparable to human bone. We report on recent studies using nanocomposites that have been fabricated as particles and nanofibers for regeneration of segmental bone defects. The research in nanocomposites, highlight a pivotal role in the future development of an ideal orthopaedic implant device, however further significant advancements are necessary to achieve clinical use.