Nano-/microfiber scaffold for tissue engineering: Physical and biological properties

Alginate hydrogel (AH) has intrinsic physical and biological limitations that hinder its broader application in tissue engineering. We hypothesized that the inclusion of nanofibers in the hydrogel and the use of a biotemplate that mimics nature would enhance the translational potential of alginate hydrogels. In this study, we have shown a method to obtain nano-/microfibers of titanium (nfTD) and hydroxyapatite (nfHY) using cotton as a biotemplate. These fibers were incorporated in the alginate hydrogel and the mechanical characteristics and biological response to these reinforced materials were evaluated. We observed that these nanofibers resembled the structure of natural collagen and did not mediate cell toxicity. The incorporation of nfTD or nfHY to the AH has not increased the viscosity of the hydrogel. Therefore, this is a feasible method to produce a scaffold with improved physical characteristics, while at the same time generating an enhanced environment for cell adhesion and proliferation. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3051-3058, 2012.     

Viscoelastic adhesive mechanics of aldehyde-mediated soft tissue sealants

Soft tissue sealants generally sacrifice adhesive strength for biocompatibility, motivating the development of materials which interact with tissue to a predictable and controllable extent. Crosslinked hydrogels comprising aminated star polyethylene glycol and high molecular weight dextran aldehyde polymers (PEG:dextran) display aldehyde-mediated adhesion and readily tunable reactivity with soft tissue ex-vivo. Evaluation of PEG:dextran compositional variants revealed that the burst pressure of repaired intestinal wounds and the extent of material-induced tissue deformation both increase nonlinearly with formulation aldehyde content and are consistently within the desired range established by traditional sealants. Adhesive test elements featuring PEG:dextran and intestinal tissue exhibited considerable viscoelasticity, prompting use of a standard linear solid (SLS) model to describe adhesive mechanics. Model elements were accurately represented as continuous functions of PEG:dextran chemistry, facilitating prediction of adhesive mechanics across the examined range of compositional formulations. SLS models of traditional sealants were also constructed to allow general correlative analyses between viscoelastic adhesive mechanics and metrics of sealant performance. Linear correlation of equilibrium SLS stiffness to sealant-induced tissue deformation indicates that dense adhesive crosslinking restricts tissue expansion, while correlation of instantaneous SLS stiffness to burst pressure suggests that the adhesive stress relaxation capacity of PEG:dextran enhances their overall performance relative to traditional sealants.     

Elastomeric biodegradable polyurethane blends for soft tissue applications

Four biodegradable polyurethane blends were made from segmented polyurethanes that contain amino acid-based chain extender and diisocyanate groups. The soft segments of these parent polyurethanes were either polyethylene oxide (PEO) or polycaprolactone (PCL) diols. The blends were developed to investigate the effect of varying soft segment compositions on the overall morphological, mechanical, and degradative properties of the materials, with a view to producing a family of materials with a wide range of properties. The highly hydrophilic PEO material was incorporated to increase the blend's susceptibility to degradation, while the PCL polyurethane was selected to provide higher moduli and percent elongations (strains) than the PEO parent materials can achieve. All four blends were determined to be semi-crystalline, elastomeric materials that possess similarly shaped stress-strain curves to that of the PCL-based parent polyurethane. As the percent composition of PEO polyurethane within the blend increased, the material became weaker and less extensible. The blends demonstrated rapid initial degradation in buffer followed by significantly slower, prolonged degradation, likely corresponding to an initial loss of primarily PEO-containing polymer, followed by the slower degradation of the PCL polyurethane. All four blends were successfully formed into three-dimensional porous scaffolds utilizing solvent casting/particulate leaching methods. Since these new blends possess a range of mechanical and degradation properties and can be shaped into three-dimensional objects, these materials may hold potential for use in soft tissue engineering scaffold applications.     

Injectable carboxymethylcellulose hydrogels for soft tissue filler applications

Disease, trauma and aging all lead to deficits in soft tissue. As a result, there is a need to develop materials that safely and effectively restore areas of deficiency. While autogenous fat is the current gold standard, hyaluronic acid (HA) fillers are commonly used. However, the animal and bacterial origin of HA-based materials can induce adverse reactions in patients. With the aim of developing a safer and more affordable alternative, this study characterized the properties of a plant-derived, injectable carboxymethylcellulose (CMC) soft tissue filler. Specifically, methacrylated CMC was synthesized and crosslinked to form stable hydrogels at varying macromer concentrations (2–4% w/v) using an ammonium persulfate and ascorbic acid redox initiation system. The equilibrium Young’s modulus was shown to vary with macromer concentration (ranging from ∼2 to 9.25 kPa), comparable to values of native soft tissue and current surgical fillers. The swelling properties were similarly affected by macromer concentration, with 4% gels exhibiting the lowest swelling ratio and mesh size, and highest crosslinking density. Rheological analysis was performed to determine gelation onset and completion, and was measured to be within the ISO standard for injectable materials. In addition, hydrolytic degradation of these gels was sensitive to macromer concentration, while selective removal using enzymatic treatment was also demonstrated. Moreover, favorable cytocompatibility of the CMC hydrogels was exhibited by co-culture with human dermal fibroblasts. Taken together, these findings demonstrate the tunability of redox-crosslinked CMC hydrogels by varying fabrication parameters, making them a versatile platform for soft tissue filler applications.     

Elastomeric biodegradable polyurethane blends for soft tissue applications

Four biodegradable polyurethane blends were made from segmented polyurethanes that contain amino acid-based chain extender and diisocyanate groups. The soft segments of these parent polyurethanes were either polyethylene oxide (PEO) or polycaprolactone (PCL) diols. The blends were developed to investigate the effect of varying soft segment compositions on the overall morphological, mechanical, and degradative properties of the materials, with a view to producing a family of materials with a wide range of properties. The highly hydrophilic PEO material was incorporated to increase the blend's susceptibility to degradation, while the PCL polyurethane was selected to provide higher moduli and percent elongations (strains) than the PEO parent materials can achieve. All four blends were determined to be semi-crystalline, elastomeric materials that possess similarly shaped stress-strain curves to that of the PCL-based parent polyurethane. As the percent composition of PEO polyurethane within the blend increased, the material became weaker and less extensible. The blends demonstrated rapid initial degradation in buffer followed by significantly slower, prolonged degradation, likely corresponding to an initial loss of primarily PEO-containing polymer, followed by the slower degradation of the PCL polyurethane. All four blends were successfully formed into three-dimensional porous scaffolds utilizing solvent casting/particulate leaching methods. Since these new blends possess a range of mechanical and degradation properties and can be shaped into three-dimensional objects, these materials may hold potential for use in soft tissue engineering scaffold applications.     

Hybrid carbon-based scaffolds for applications in soft tissue reconstruction

Current biomedical scaffolds utilized in surgery to repair soft tissues commonly fail to meet the optimal combination of biomechanical and tissue regenerative properties. Carbon is a scaffold alternative that potentially optimizes the balance between mechanical strength, durability, and function as a cell and biologics delivery vehicle that is necessary to restore tissue function while promoting tissue repair. The goals of this study were to investigate the feasibility of fabricating hybrid fibrous carbon scaffolds modified with biopolymer, polycaprolactone and to analyze their mechanical properties and ability to support cell growth and proliferation. Environmental scanning electron microscopy, micro-computed tomography, and cell adhesion and cell proliferation studies were utilized to test scaffold suitability as a cell delivery vehicle. Mechanical properties were tested to examine load failure and elastic modulus. Results were compared to an acellular dermal matrix scaffold control (GraftJacket(?) [GJ] Matrix), selected for its common use in surgery for the repair of soft tissues. Results indicated that carbon scaffolds exhibited similar mechanical maximums and capacity to support fibroblast adhesion and proliferation in comparison with GJ. Fibroblast adhesion and proliferation was collinear with carbon fiber orientation in regions of sparsely distributed fibers and occurred in clusters in regions of higher fiber density and low porosity. Overall, fibroblast adhesion and proliferation was greatest in lower porosity carbon scaffolds with highly aligned fibers. Stepwise multivariate regression showed that the variability in maximum load of carbon scaffolds and controls were dependent on unique and separate sets of parameters. These finding suggested that there were significant differences in the functional implications of scaffold design and material properties between carbon and dermis derived scaffolds that affect scaffold utility as a tissue replacement construct.     

A novel simulation algorithm for soft tissue compression

This paper presents a novel general approach to simulation of soft tissue compression. A theoretical framework of the compression algorithm has been developed and implemented, based on the concept of a simple spring. The volume subjected to compression is divided into a number of “model elements”, each one consisting of 27 nodes. The nodes are connected with springs. The mechanical properties of the tissues are assumed to be linear and isotropic. The compressed volume remains constant due to the introduced concept of spring variable equilibrium lengths. Initial settings for compression simulation are introduced in order that the algorithm converges faster. The developed compression algorithm was used to model breast compression during a standard mammography examination. Specifically, the method was applied to a high-resolution three-dimensional software breast phantom, composed to have a medium glandularity and calcification abnormalities. The compression was set to 50%. Results showed that the abnormalities maintain their shape and dimensions during the compression, while the surrounding breast tissues undergo considerable deformation and displacement. A “decompression” algorithm was also applied to test the reversibility of the model.     

In vivo soft tissue damage assessment for applications in surgery

In robotic and conventional minimally invasive surgery the risk of complications caused by collateral tissue damage remains high. This paper studies the concept of imposing damage thresholds on surgical instruments to avoid tissue overload. More specifically, the correlation between mechanical loading and damage in case of vascular clamping is investigated.With a computer controlled device, a high and a low clamping load were applied in vivo on the abdominal aorta of 43 rats. Samples of both loading levels were compared with zero load control samples and with samples clamped by a mosquito clamp w.r.t. functionality and histological integrity.Analysis of the samples shows that high clamping forces result in endothelial and smooth muscle cell destruction. Clamping with a mosquito clamp will cause even more damage to the elastic lamellae. Samples loaded at the lower load showed significantly less smooth muscle cell damage and a lower degree of endothelial damage.This paper is the first to statistically quantify the correlation between the degree of mechanical loading and the degree of tissue damage, thus setting the first steps towards tissue overload prevention during surgery. Future experiments will also include the effects of loading duration, recovery and patient-specificity.     

Interfacial optimization of fiber-reinforced hydrogel composites for soft fibrous tissue applications

Meniscal tears are the most common orthopedic injuries to the human body, yet the current treatment of choice is a partial meniscectomy, which is known to lead to joint degeneration and osteoarthritis. As a result, there is a significant clinical need to develop materials capable of restoring function to the meniscus following an injury. Fiber-reinforced hydrogel composites are particularly suited for replicating the mechanical function of native fibrous tissues due to their ability to mimic the native anisotropic property distribution present. A critical issue with these materials, however, is the potential for the fiber–matrix interfacial properties to severely limit composite performance. In this work, the interfacial properties of an ultra-high-molecular-weight polyethylene (UHMWPE) fiber-reinforced poly(vinyl alcohol) (PVA) hydrogel are studied. A novel chemical grafting technique, confirmed using X-ray photoelectron spectroscopy, is used to improve UHMWPE–PVA interfacial adhesion. Interfacial shear strength is quantified using fiber pull-out tests. Results indicate significantly improved fiber–hydrogel interfacial adhesion after chemical grafting, where chemically grafted samples have an interfacial shear strength of 256.4 ± 64.3 kPa compared to 11.5 ± 2.9 kPa for untreated samples. Additionally, scanning electron microscopy of fiber surfaces after fiber pull-out reveal cohesive failure within the hydrogel matrix for treated fiber samples, indicating that the UHMWPE–PVA interface has been successfully optimized. Lastly, inter-fiber spacing is observed to have a significant effect on interfacial adhesion. Fibers spaced further apart have significantly higher interfacial shear strengths, which is critical to consider when optimizing composite design. The results in this study are applicable in developing similar chemical grafting techniques and optimizing fiber–matrix interfacial properties for other hydrogel-based composite systems.     

Elastic properties of synthetic materials for soft tissue modeling

Mechanical models of soft tissue are useful for studying vibro-acoustic phenomena. They may be used for validating mathematical models and for testing new equipment and techniques. The objective of this study was to measure density and visco-elastic properties of synthetic materials that can be used to build such models. Samples of nine different materials were tested under dynamic (0.5 Hz) compressive loading conditions. The modulus of elasticity of the materials was varied, whenever possible, by adding a softener during manufacturing. The modulus was measured over a nine month period to quantify the effect of ageing and softener loss on material properties. Results showed that a wide range of the compression elasticity modulus (10 to 1400 kPa) and phase (3.5 degrees -16.7 degrees ) between stress and strain were possible. Some materials tended to exude softener over time, resulting in a weight loss and elastic properties change. While the weight loss under normal conditions was minimal in all materials (<3% over nine months), loss under accelerated weight-loss conditions can reach 59%. In the latter case an elasticity modulus increase of up to 500% was measured. Key advantages and limitations of candidate materials were identified and discussed.     

A tensile machine with a novel optical load cell for soft biological tissues application

The uniaxial tensile testing machine is the most common device used to measure the mechanical properties of industrial and biological materials. The need for a low-cost uniaxial tension testing device for small research centers has always been the subject of research. To address this need, a novel uniaxial tensile testing machine was designed and fabricated to measure the mechanical properties of soft biological tissues. The device is equipped with a new low-cost load cell which works based on the linear displacement/force relationship of beams. The deflection of the beam load cell is measured optically by a digital microscope with an accuracy of 1?µm. The stiffness of the designed load cell was experimentally and theoretically determined at 100?N mm^?1. The stiffness of the load cell can be easily adjusted according to the tissue’s strength. The force-time behaviour of soft tissue specimens was obtained by an in-house image processing program. To demonstrate the efficiency of the fabricated device, the mechanical properties of amnion tissue was measured and compared with available data. The obtained results indicate a strong agreement with that of previous studies.     

Photoacoustic tomography of foreign bodies in soft biological tissue

In detecting small foreign bodies in soft biological tissue, ultrasound imaging suffers from poor sensitivity (52.6%) and specificity (47.2%). Hence, alternative imaging methods are needed. Photoacoustic (PA) imaging takes advantage of strong optical absorption contrast and high ultrasonic resolution. A PA imaging system is employed to detect foreign bodies in biological tissues. To achieve deep penetration, we use near-infrared light ranging from 750 to 800 nm and a 5-MHz spherically focused ultrasonic transducer. PA images were obtained from various targets including glass, wood, cloth, plastic, and metal embedded more than 1 cm deep in chicken tissue. The locations and sizes of the targets from the PA images agreed well with those of the actual samples. Spectroscopic PA imaging was also performed on the objects. These results suggest that PA imaging can potentially be a useful intraoperative imaging tool to identify foreign bodies.     

Tensile properties of bioactive fibers for tissue engineering applications

Cell transplantation using biocompatible, biodegradable scaffolds offers the possibility of creating or regenerating tissue to replace organ function when deficiency arises. The role of these temporary substrates is to support and guide the expanding cell culture until it becomes structurally integrated with the host tissue. 45S5 Bioglass(R) is a 4-component, melt-derived bioactive glass, which has been approved for human clinical use by the Food and Drug Administration. The biocompatibility and biodegradability of 45S5 Bioglass(R) are long established, whereas research into its performance as an extracellular scaffold is currently underway. In this study the tensile strengths (93 +/- 8 and 82 +/- 14 MPa), elongation to fracture (0.7 +/- 0.05%) and Weibull's moduli (3.0 and 3.5) of 45S5 Bioglass(R) fibers (mean diameters 193 and 280 microm) for tissue engineering applications are reported. The tensile strengths of the fibers are compared with those of bulk 45S5 Bioglass(R) and a range of biodegradable polymer materials currently used in the field of tissue engineering. Aspects of glass and fiber technology relevant to the design and manufacture of extracellular ceramic scaffolds are also discussed.     

Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications

In the engineering of soft tissues, scaffolds with high elastance and strength coupled with controllable biodegradable properties are necessary. To fulfill such design criteria we have previously synthesized two kinds of biodegradable polyurethaneureas, namely poly(ester urethane)urea (PEUU) and poly(ether ester urethane)urea (PEEUU) from polycaprolactone, polycaprolactone-b-polyethylene glycol-b-polycaprolactone, 1,4-diisocyanatobutane and putrescine. PEUU and PEEUU were further fabricated into scaffolds by thermally induced phase separation using dimethyl sulfoxide (DMSO) as a solvent. The effect of polymer solution concentration, quenching temperature and polymer type on pore morphology and porosity was investigated. Scaffolds were obtained with open and interconnected pores having sizes ranging from several mum to more than 150 microm and porosities of 80-97%. By changing the polymer solution concentration or quenching temperature, scaffolds with random or oriented tubular pores could be obtained. The PEUU scaffolds were flexible with breaking strains of 214% and higher, and tensile strengths of approximately 1.0 MPa, whereas the PEEUU scaffolds generally had lower strengths and breaking strains. Scaffold degradation in aqueous buffer was related to the porosity and polymer hydrophilicity. Smooth muscle cells were filtration seeded in the scaffolds and it was shown that both scaffolds supported cell adhesion and growth, with smooth muscle cells growing more extensively in the PEEUU scaffold. These biodegradable and flexible scaffolds demonstrate potential for future application as cell scaffolds in cardiovascular tissue engineering or other soft tissue applications.     

Dielectric properties of a liquid crystalline siloxane copolymer

An investigation on the molecular dynamics of a liquid crystalline side chain polymer using the dielectric relaxation method on oriented samples in the frequency range of 0,1 to 10 000 kHz is presented. The compound under investigation is a polysiloxane copolymer with two different mesogenic side chains and a phase sequence glassy-smectic A-nematic-isotropic. Three main relaxation processes are found and assigned to the rotation of the side chains around the main chain, the glass transition process coupled with side chain motions, and a local motion in the glassy state, respectively. Evidence is found for a layered arrangement of the polymer main chain in the smectic phase and for a parallel correlation between the transverse components of the dipole moments.     

Photocrosslinkable chitosan as a biological adhesive

A photocrosslinkable chitosan to which both azide and lactose moieties were introduced (Az-CH-LA) was prepared as a biological adhesive for soft tissues and its effectiveness was compared with that of fibrin glue. Introduction of the lactose moieties resulted in a much more water-soluble chitosan at neutral pH. Application of ultraviolet light (UV) irradiation to photocrosslinkable Az-CH-LA produced an insoluble hydrogel within 60 s. This hydrogel firmly adhered two pieces of sliced ham with each other, depending upon the Az-CH-LA concentration. The binding strength of the chitosan hydrogel prepared from 30-50 mg/mL of Az-CH-LA was similar to that of fibrin glue. Compared to the fibrin glue, the chitosan hydrogel more effectively sealed air leakage from pinholes on isolated small intestine and aorta and from incisions on isolated trachea. Neither Az-CH-LA nor its hydrogel showed any cytotoxicity in cell culture tests of human skin fibroblasts, coronary endothelial cells, and smooth muscle cells. Furthermore, all mice studied survived for at least 1 month after implantation of 200 microL of photocrosslinked chitosan gel and intraperitoneal administration of up to 1 mL of 30 mg/mL of Az-CH-LA solution. These results suggest that the photocrosslinkable chitosan developed here has the potential of serving as a new tissue adhesive in medical use.     

Adhesion of synthetic organic polymer on soft tissue. I. a fast setting polyurethane adhesive

Conventional polyurethane prepolymers have been shown to adhere to living biological tissues. However, their setting is not sufficiently expedient to permit convenient applications in vivo. A prepolymer prepared from the highly reactive 6-chloro-2,4,5-trifluoro-1,3-phenylene diisocyanate, castor oil, and a trace of pyridine has afforded an adhesive which sets in about 2 min in vivo. The fast setting has resulted in poor adhesion on biological tissue. The bonding has been improved by the inclusion of tolylene diisocyanate in the composition without affecting the fast curing rate of the prepolymer. The dispersion of the adhesive and its cohesion after solidification have been adjusted by other minor additives. Preliminary evaluation on animals indicates that this adhesive is most useful as a hemostatic coating in hepatic lacerations.     

Physical and biological properties of barium cross-linked alginate membranes

We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability. Clinical grade, ultra-high viscosity alginate is gelled by diffusion of Ba2+ followed by use of the "crystal gun" [Zimmermann H. et al., Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging. Biomaterials 2003;24:2083-96]. Burst pressure of well-hydrated membranes is between 34 and 325kPa depending on manufacture and storage details. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG 6000, which may be related to a Hagen-Poiseuille flow. Hydraulic conductivity, L(p), from PEG-induced flows ranges between 2.4x10(-12) and 6.5x10(-12) m Pa(-1)s(-1). Hydraulic conductivity measured with hydrostatic pressure up to 6 kPa is 2-3 orders of magnitude higher and decreases with increasing pressure to about 3x10(-10) m Pa(-1)s(-1) at 4kPa. Mechanical introduction of 200 microm-diameter pores increases hydraulic conductivity dramatically without loss of mechanical stability or flexibility. NMR imaging with Cu2+ as contrast agent shows a layered structure in membranes cross-linked for 2h. Phase contrast and atomic force microscopy in liquid environment reveals surface protrusions and cavities correlating with steps of the production process. Murine L929 cells adhere strongly to the rough surface of crystal-bombarded membranes. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol allowing cell culture on the membranes.     

Physical and biological properties of collagen-phospholipid polymer hybrid gels

We successfully developed a novel method for immobilizing poly(2-methacryloyloxyethyl phosphorylcholine) [Poly(MPC)] polymer onto collagen using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linkers. In order to obtain the highest possible molar ratio of immobilized MPC moieties on the collagen gel, a collagen-phospholipid polymer hybrid gel was prepared by repeating the cross-linking process up to three times to create a dense network of collagen and PMA. Network formation by repeating the immobilization process was successful, resulting in decreased free amine group content and a low swelling ratio. The hybrid gel displayed very high stability against degradation by collagenase and possessed high hydrophilicity. Fibrinogen adsorption and cell adhesion were reduced and demonstrated less cell proliferation as compared to that by uncross-linked collagen gel. The collagen-phospholipid polymer hybrid gel did not exhibit toxicity, and the cell morphology remained intact (round); this implies that the interaction between the cell and the collagen-phospholipid polymer hybrid gel is safe and mild.