Roshan James

Electrospun nanofiber scaffolds: engineering soft tissues

Electrospinning has emerged to be a simple, elegant and scalable technique to fabricate polymeric nanofibers. Pure polymers as well as blends and composites of both natural and synthetics have been successfully electrospun into nanofiber matrices. Physiochemical properties of nanofiber matrices can be controlled by manipulating electrospinning parameters to meet the requirements of a specific application. Such efforts include the fabrication of fiber matrices containing nanofibers, microfibers, combination of nano-microfibers and also different fiber orientation/alignments. Polymeric nanofiber matrices have been extensively investigated for diversified uses such as filtration, barrier fabrics, wipes, personal care, biomedical and pharmaceutical applications. Recently electrospun nanofiber matrices have gained a lot of attention, and are being explored as scaffolds in tissue engineering due to their properties that can modulate cellular behavior. Electrospun nanofiber matrices show morphological similarities to the natural extra-cellular matrix (ECM), characterized by ultrafine continuous fibers, high surface-to-volume ratio, high porosity and variable pore-size distribution. Efforts have been made to modify nanofiber surfaces with several bioactive molecules to provide cells with the necessary chemical cues and a more in vivo like environment. The current paper provides an overlook on such efforts in designing nanofiber matrices as scaffolds in the regeneration of various soft tissues including skin, blood vessel, tendon/ligament, cardiac patch, nerve and skeletal muscle.

Electrospun poly(lactic acid-co-glycolic acid) scaffolds for skin tissue engineering.

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.

Tendon: Biology, Biomechanics, Repair, Growth Factors, and Evolving Treatment Options

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.

Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems.

Tendon tissue engineering with a biomaterial scaffold that mimics the tendon extracellular matrix (ECM) and is biomechanically suitable, and when combined with readily available autologous cells, may provide successful regeneration of defects in tendon. Current repair strategies using suitable autografts and freeze-dried allografts lead to a slow repair process that is sub-optimal and fails to restore function, particularly in difficult clinical situations such as zone II flexor tendon injuries of the hand. We have investigated the effect of GDF-5 on cell proliferation and gene expression by primary rat adipose-derived stem cells (ADSCs) that were cultured on a poly(DL-lactide-co-glycolide) PLAGA fiber scaffold and compared to a PLAGA 2D film scaffold. The electrospun scaffold mimics the collagen fiber bundles present in native tendon tissue, and supports the adhesion and proliferation of multipotent ADSCs. Gene expression of scleraxis, the neotendon marker, was upregulated seven- to eightfold at 1 week with GDF-5 treatment when cultured on a 3D electrospun scaffold, and was significantly higher at 2 weeks compared to 2D films with or without GDF-5 treatment. Expression of the genes that encode the major tendon ECM protein, collagen type I, was increased by fourfold starting at 1 week on treatment with 100 ng mL(-1) GDF-5, and at all time points the expression was significantly higher compared to 2D films irrespective of GDF-5 treatment. Thus stimulation with GDF-5 can modulate primary ADSCs on a PLAGA fiber scaffold to produce a soft, collagenous musculoskeletal tissue that fulfills the need for tendon regeneration.

Adipose-Derived Mesenchymal Stem Cells Treated with Growth Differentiation Factor-5 Express Tendon-Specific Markers

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSION 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.

Recent Patents on Electrospun Biomedical Nanostructures: An Overview

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.

Nanostructured Polymeric Scaffolds for Orthopaedic Regenerative Engineering

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.

Electrospun Nanofibrous Scaffolds for Engineering Soft Connective Tissues

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.

Micro- and nanofabrication of chitosan structures for regenerative engineering.

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.

Tissue engineering solutions for tendon repair.

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.