Biodegradable microfluidic scaffolds for tissue engineering from amino alcohol-based poly(ester amide) elastomers

Biodegradable polymers with high mechanical strength, flexibility and optical transparency, optimal degradation properties and biocompatibility are critical to the success of tissue engineered devices and drug delivery systems. Most biodegradable polymers suffer from a short half life due to rapid degradation upon implantation, exceedingly high stiffness, and limited ability to functionalize the surface with chemical moieties. This work describes the fabrication of microfluidic networks from poly(ester amide), poly(1,3-diamino-2-hydroxypropane-co-polyol sebacate) (APS), a recently developed biodegradable elastomeric poly(ester amide). Microfluidic scaffolds constructed from APS exhibit a much lower Young’s Modulus and a significantly longer degradation half-life than those of previously reported systems. The device is fabricated using a modified replica-molding technique, which is rapid, inexpensive, reproducible, and scalable, making the approach ideal for both rapid prototyping and manufacturing of tissue engineering scaffolds.     

Polyhydroxyalkanoate (PHA)/inorganic phase composites for tissue engineering applications

Polyhydroxyalkanoates are emerging as a class of biodegradable polymers for applications in tissue engineering. Members of the polyhydroxyalkanoates family encompass a wide variety of materials, from hard and brittle materials to soft and elastomeric. Over the years, efforts have been made to extend the group of polyhydroxyalkanoates and to investigate their use in numerous biomedical applications, such as sutures, cardiovascular patches, wound dressings, guided tissue repair/regeneration devices, and tissue engineering scaffolds. Along with the development of polyhydroxyalkanoates, researchers have looked into the possibility of designing composites in combination with inorganic phases to further improve the mechanical properties, rate of degradation, and also impart bioactivity. Poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) are some of the polymers which have been studied extensively to fabricate composites in combination with hydroxyapatite, bioactive glass, and glass-ceramic fillers or coatings. This paper reviews international research carried out toward development of polyhydroxyalkanoates/inorganic phase composites in terms of systems investigated, microstructures, properties achieved, and applications, with special focus on tissue engineering scaffolds. A comparison between different composite systems developed in the past few years is presented. The paper also addresses the prospect of potential further development of polyhydroxyalkanoates/inorganic phase composites with optimized microstructure and properties for improved tissue engineering scaffolds.     

Synthesis, characterization, and in vitro degradation of a biodegradable photo-cross-linked film from liquid poly(epsilon-caprolactone-co-lactide-co-glycolide) diacrylate

There has been little study on the effect of composition or molecular weight on the biodegradation rate of photo-cross-linked biodegradable aliphatic polyesters though such information is important for tissue engineering scaffolds. We have synthesized a new series of photopolymerizable linear poly(epsilon-caprolactone-co-lactide-co-glycolide) diacrylates with different molecular weights (Mn = 1800, 4800, and 9300 Da) and compositions (20%, 40%, and 60% epsilon-CL) and studied their biodegradation rates. The resultant oligomers were amorphous and appeared as viscous liquids at room temperature. Liquid-to-solid polymerization was carried out by UV irradiation in the presence of a photoinitiator. The photocuring yield was high (greater than 95%), and the photo-cross-linked polymers were amorphous and rubbery. Mechanical measurements showed that the polymers can be stretchable or rigid; the high molecular weight/low epsilon-CL network has a strain of 176% and a modulus of 1.66 MPa while the low molecular weight/high epsilon-CL network has a strain of 21% and a modulus of 12.3 MPa. In a 10 week in vitro biodegradation study, the polymers exhibited a two-stage degradation behavior. In the first stage, the polymer weight and strain remained almost constant, but a linear decrease in the Young's modulus (E) and ultimate stress (sigma) were observed. Lower oligomer molecular weight or epsilon-CL content correlated with a faster decrease in Young's modulus. In the second stage, which began when the Young's modulus dropped below 1 MPa, there was rapid weight loss and strain increase. The lower the epsilon-CL content, the earlier the second stage happened. Low molecular weight and high epsilon-CL content correlated with a longer modulus half-life (time for the modulus to degrade to 50% of its initial value). The degradation results suggest principles that may be helpful in predicting the biodegradation behavior of similar polymeric cross-linked networks. Films formed from these new polymers have excellent biocompatibility with smooth muscle cells.     

Computer aided design of architecture of degradable tissue engineering scaffolds

One important factor affecting the process of tissue regeneration is scaffold stiffness loss, which should be properly balanced with the rate of tissue regeneration. The aim of the research reported here was to develop a computer tool for designing the architecture of biodegradable scaffolds fabricated by melt-dissolution deposition systems (e.g. Fused Deposition Modeling) to provide the required scaffold stiffness at each stage of degradation/regeneration. The original idea presented in the paper is that the stiffness of a tissue engineering scaffold can be controlled during degradation by means of a proper selection of the diameter of the constituent fibers and the distances between them. This idea is based on the size-effect on degradation of aliphatic polyesters. The presented computer tool combines a genetic algorithm and a diffusion-reaction model of polymer hydrolytic degradation. In particular, we show how to design the architecture of scaffolds made of poly(DL-lactide-co-glycolide) with the required Young’s modulus change during hydrolytic degradation.     

Functionalized synthetic biodegradable polymer scaffolds for tissue engineering

Scaffolds (artificial ECMs) play a pivotal role in the process of regenerating tissues in 3D. Biodegradable synthetic polymers are the most widely used scaffolding materials. However, synthetic polymers usually lack the biological cues found in the natural extracellular matrix. Significant efforts have been made to synthesize biodegradable polymers with functional groups that are used to couple bioactive agents. Presenting bioactive agents on scaffolding surfaces is the most efficient way to elicit desired cell/material interactions. This paper reviews recent advancements in the development of functionalized biodegradable polymer scaffolds for tissue engineering, emphasizing the syntheses of functional biodegradable polymers, and surface modification of polymeric scaffolds.     

Biodegradable fumarate-based polyHIPEs as tissue engineering scaffolds

PolyHIPEs show great promise as tissue engineering scaffolds due to the tremendous control of pore size and interconnectivity afforded by this technique. Highly porous, fully biodegradable scaffolds were prepared by polymerization of the continuous phase of high internal phase emulsions (HIPEs) containing the macromer poly(propylene fumarate) (PPF) and the cross-linker propylene fumarate diacrylate (PFDA). Toluene was used as a diluent to reduce the viscosity of the organic phase to enable HIPE formation. A range of polyHIPE scaffolds of different pore sizes and morphologies were generated by varying the diluent concentration (40-60 wt %), cross-linker concentration (25-75 wt %), and macromer molecular weight ( M n = 800-1000 g/mol). Although some formulations resulted in macroporous monoliths (pore diameter >500 microm), the majority of the polyHIPEs studied were rigid, microporous monoliths with average pore diameters in the range 10-300 microm. Gravimetric analysis confirmed the porosity of the microporous monoliths as 80-89% with most scaffolds above 84%. These studies demonstrate that emulsion templating can be used to generate rigid, biodegradable scaffolds with highly interconnected pores suitable for tissue engineering scaffolds.     

Development of biodegradable scaffolds based on patient-specific arterial configuration

Biodegradable scaffolds are of great value in tissue engineering. We have developed a method for fabricating patient-specific vascular scaffolds from a biocompatible and biodegradable polymer, poly(L-lactide-co-epsilon-caprolactone). This method's usefulness is due to flexibility in the choice of materials and vascular configurations. Here, we present a way to fabricate scaffolds of human carotid artery by combining processes of rapid prototyping, lost wax, dip coating, selective dissolution, and salt leaching. The result was the successful development of porous biodegradable scaffolds, with mechanical strength covering the range of human blood vessels (1-3 MPa). Human umbilical vein endothelial cells were also cultured on the scaffolds and their biocompatibility was confirmed by cell growth. The Young's modulus of scaffolds could be controlled by changing polymer concentration and porosity. The wall thickness of the tubular scaffold was also controllable by adjusting polymer concentration and pull-up velocity during dip coating. We believe that this fabrication technique can be applied to patient-specific regeneration of blood vessels.     

Osteostimulation scaffolds of stem cells: BMP‐7‐derived peptide‐decorated alginate porous scaffolds promote the aggregation and osteo‐differentiation of human mesenchymal stem cells

The scaffolds for stem cell‐based bone tissue engineering should hold the ability to guide stem cells osteo‐differentiating. Otherwise, stem cells will differentiate into unwanted cell types or will form tumors in vivo. Alginate, a natural polysaccharide with great biocompatibility, was widely used in biomedical applications. However, the limited bioactivity and poor osteogenesis capability of pristine alginate hampered its further application in tissue engineering. In this work, a bone forming peptide‐1 (BFP‐1), derived from bone morphogenetic protein‐7, was grafted to alginate polymer chains to prepare peptide‐decorated alginate porous scaffolds (pep‐APS) for promoting osteo‐differentiation of human mesenchymal stem cells (hMSCs). SEM images of pep‐APS exhibited porous structure with about 90% porosity (pore size 100–300 μm), which was appropriate for hMSCs ingrowth. The adhesion, proliferation and aggregation of hMSCs grown on pep‐APS were enhanced in vitro. Moreover, pep‐APS promoted the alkaline phosphatase (ALP) activity of hMSCs, and the osteo‐related genes expression was obviously up‐regulated. The immunochemical staining and western blot analysis results showed high expression level of OCN and Col1a1 in the hMSCs grown on pep‐APS. This work provided a facile and valid strategy to endow the alginate polymers themselves with specific bioactivity and prepare osteopromoting scaffold with enhanced osteogenesis ability, possessing potential applications in stem cell therapy and regenerative medicine.     

PHA applications: addressing the price performance issue: I. Tissue engineering.

This paper describes the development of medical applications for polyhydroxyalkanoates (PHAs), a class of natural polymers with a wide range of thermoplastic properties. Methods are described for preparing PHAs with high purity, modifying these materials to change their surface and degradation properties, and methods for fabricating them into different forms, including tissue engineering scaffolds. Preliminary reports characterizing their in vivo behavior are given, as well as methods for using the natural polymers in tissue engineering applications.     

In vitro cytotoxicity of unsaturated oligo[poly(ethylene glycol) fumarate] macromers and their cross-linked hydrogels

Currently, oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels are being investigated as an injectable and biodegradable system for tissue engineering applications. In this study, cytotoxicity of each component of the OPF hydrogel formulation and the resulting cross-linked network was examined. Specifically, OPF synthesized with poly(ethylene glycol) (PEG) of different molecular weights (MW), the cross-linking agent [PEG-diacrylate (PEG-DA)], and the redox initiator pair [ammonium persulfate (APS) and ascorbic acid (AA)] were evaluated for cytotoxicity at 2 and 24 h using marrow stromal cells (MSCs) as model cells. The effect of leachable byproducts of OPF hydrogels on cytotoxicity was also investigated. Upon exposure to various concentrations of OPF for 2 h, greater than 50% of the MSCs were viable, regardless of OPF molecular weight or concentration in the media. After 24 h, the MSCs maintained more than 75% viability except for OPF concentrations higher than 25% (w/v). When examining the cross-linking agent, PEG-DA of higher MW (3400) demonstrated significantly higher viability compared to PEG-DA with MW 575 at all concentrations tested. Considering initiators, when MSCs were exposed to AA and APS, as well as the combination of AA and APS, higher viability was observed at lower concentrations. Once cross-linked, the leachable products from the OPF hydrogels had minimal adverse effects on the viability of MSCs (percentage of live cells was higher than 90% regardless of hydrogel types). The results suggest that, after optimization of cross-linking parameters, OPF-based hydrogels hold promise as novel injectable scaffolds or cell carriers in tissue engineering.     

Bone tissue engineering using marrow stromal cells

Bone tissue defects cause a significant socioeconomic problem, and bone is the most frequently transplanted tissue beside blood. Autografting is considered the gold standard treatment for bone defects, but its utility is limited due to donor site morbidity. Hence much research has focused on bone tissue engineering as a promising alternative method for repair of bone defects. Marrow stromal cells (MSCs) are considered to be potential cell sources for bone tissue engineering. In bone tissue engineering using MSCs, bone is formed through intramembranous and endochondral ossification in response to osteogenic inducers. Angiogenesis is a complex process mediated by multiple growth factors and is crucial for bone regeneration. Vascular endothelial growth factor plays important roles in bone tissue regeneration by promoting the migration and differentiation of osteoblasts, and by inducing angiogenesis. Scaffold materials used for bone tissue engineering include natural components of bone, such as calcium phosphate and collagen I, and biodegradable polymers such as poly(lactide-coglycolide) However, ideal scaffolds for bone tissue engineering have yet to be found. Bone tissue engineering has been successfully used to treat bone defects in several human clinical trials to regenerate bone defects. Through investigation of MSC biology and the development of novel scaffolds, we will be able to develop advanced bone tissue engineering techniques in the future.     

Synthesis and evaluation of novel biodegradable hydrogels based on poly(ethylene glycol) and sebacic acid as tissue engineering scaffolds

Novel biodegradable poly(ethylene glycol) (PEG) based hydrogels, namely, PEG sebacate diacrylate (PEGSDA) were synthesized, and their properties were evaluated. Chemical structures of these polymers were confirmed by Fourier transform infrared and proton nuclear magnetic resonance (1H NMR) spectroscopy. After photopolymerization, the dynamic shear modulus of the hydrogels was up to 0.2 MPa for 50% PEGSDA hydrogel, significantly higher than conventional hydrogels such as PEG diacrylate (PEGDA). The swelling ratios of these macromers were significantly lower than PEGDA. The in vitro degradation study demonstrated that these hydrogels were biodegradable with weight losses about 66% and 32% for 25% and 50% PEGSDA after 8 weeks of incubation in phosphate-buffered saline at 37 degrees C. In vitro biocompatibility was assessed using cultured rat bone marrow stromal cells (MSCs) in the presence of unreacted monomers or degradation products. Unlike conventional PEGDA hydrogels, PEGSDA hydrogel without RGD peptide modification induced MSC cell adhesion similar to tissue culture polystyrene. Finally, complex three-dimensional structures of PEGSDA hydrogels using solid free form technique were fabricated and their structure integrity was better maintained than PEGDA hydrogels. These hydrogels may find use as scaffolds for tissue engineering applications.     

Hybrid chitosan–ß‐glycerol phosphate–gelatin nano‐/micro fibrous scaffolds with suitable mechanical and biological properties for tissue engineering

Scaffold‐based tissue engineering is considered as a promising approach in the regenerative medicine. Graft instability of collagen, by causing poor mechanical properties and rapid degradation, and their hard handling remains major challenges to be addressed. In this research, a composite structured nano‐/microfibrous scaffold, made from a mixture of chitosan–ß‐glycerol phosphate–gelatin (chitosan–GP–gelatin) using a standard electrospinning set‐up was developed. Gelatin–acid acetic and chitosan ß‐glycerol phosphate–HCL solutions were prepared at ratios of 30/70, 50/50, 70/30 (w/w) and their mechanical and biological properties were engineered. Furthermore, the pore structure of the fabricated nanofibrous scaffolds was investigated and predicted using a theoretical model. Higher gelatin concentrations in the polymer blend resulted in significant increase in mean pore size and its distribution. Interaction between the scaffold and the contained cells was also monitored and compared in the test and control groups. Scaffolds with higher chitosan concentrations showed higher rate of cell attachment with better proliferation property, compared with gelatin‐only scaffolds. The fabricated scaffolds, unlike many other natural polymers, also exhibit non‐toxic and biodegradable properties in the grafted tissues. In conclusion, the data clearly showed that the fabricated biomaterial is a biologically compatible scaffold with potential to serve as a proper platform for retaining the cultured cells for further application in cell‐based tissue engineering, especially in wound healing practices. These results suggested the potential of using mesoporous composite chitosan–GP–gelatin fibrous scaffolds for engineering three‐dimensional tissues with different inherent cell characteristics. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 163–175, 2016.     

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.^{1, 2, 3} Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.⁴ An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.     

Fabrication of chitin-chitosan/nano ZrO(2) composite scaffolds for tissue engineering applications

The urge to repair and regenerate natural tissues can now be satisfactorily fulfilled by various tissue engineering approaches. Chitin and chitosan are the most widely accepted biodegradable and biocompatible materials subsequent to cellulose. The incorporation of nano ZrO₂ onto the chitin-chitosan scaffold is thought to enhance osteogenesis. Hence a nanocomposite scaffold was fabricated by lyophilization technique using chitin-chitosan with nano ZrO₂. The prepared nanocomposite scaffolds were characterized using SEM, FTIR, XRD and TGA. In addition, the swelling, degradation, biomineralization, cell viability and cell attachment of the composite scaffolds were also evaluated. The results demonstrated better swelling and controlled degradation in comparison to the control scaffold. Cell viability studies proved the non toxic nature of the nanocomposite scaffolds. Cells were found to be attached to the pore walls and spread uniformly throughout the scaffolds. All these results suggested that the developed nanocomposite scaffolds possess the prerequisites for tissue engineering scaffolds and could be used for various tissue engineering applications.     

Osteochondral tissue engineering: scaffolds, stem cells and applications

Osteochondral tissue engineering has shown an increasing development to provide suitable strategies for the regeneration of damaged cartilage and underlying subchondral bone tissue. For reasons of the limitation in the capacity of articular cartilage to self-repair, it is essential to develop approaches based on suitable scaffolds made of appropriate engineered biomaterials. The combination of biodegradable polymers and bioactive ceramics in a variety of composite structures is promising in this area, whereby the fabrication methods, associated cells and signalling factors determine the success of the strategies. The objective of this review is to present and discuss approaches being proposed in osteochondral tissue engineering, which are focused on the application of various materials forming bilayered composite scaffolds, including polymers and ceramics, discussing the variety of scaffold designs and fabrication methods being developed. Additionally, cell sources and biological protein incorporation methods are discussed, addressing their interaction with scaffolds and highlighting the potential for creating a new generation of bilayered composite scaffolds that can mimic the native interfacial tissue properties, and are able to adapt to the biological environment.     

Laser fabrication of three-dimensional CAD scaffolds from photosensitive gelatin for applications in tissue engineering

In the present work, 3D CAD scaffolds for tissue engineering applications were developed starting from methacrylamide-modified gelatin (GelMOD) using two-photon polymerization (2PP). The scaffolds were cross-linked employing the biocompatible photoinitiator Irgacure 2959. Because gelatin is derived from collagen (i.e., the main constituent of the ECM), the developed materials mimic the cellular microenvironment from a chemical point of view. In addition, by applying the 2PP technique, structural properties of the cellular microenvironment can also be mimicked. Furthermore, in vitro degradation assays indicated that the enzymatic degradation capability of gelatin is preserved for the methacrylamide-modified derivative. An in depth morphological analysis of the 2PP-fabricated scaffolds demonstrated that the parameters of the CAD model are reproduced with great precision, including the ridge-like surface topography on the order of 1.5 μm. The developed scaffolds showed an excellent stability in culture medium. In a final part of the present work, the suitability of the developed scaffolds for tissue engineering applications was verified. The results indicated that the applied materials are suitable to support porcine mesenchymal stem cell adhesion and subsequent proliferation. Upon applying osteogenic stimulation, the seeded cells differentiated into the anticipated lineage. Energy dispersive X-ray (EDX) analysis showed the induced calcification of the scaffolds. The results clearly indicate that 2PP is capable of manufacturing precisely constructed 3D tissue engineering scaffolds using photosensitive polymers as starting material.     

Polyphosphazene/nano-hydroxyapatite composite microsphere scaffolds for bone tissue engineering

The nontoxic, neutral degradation products of amino acid ester polyphosphazenes make them ideal candidates for in vivo orthopedic applications. The quest for new osteocompatible materials for load bearing tissue engineering applications has led us to investigate mechanically competent amino acid ester substituted polyphosphazenes. In this study, we have synthesized three biodegradable polyphosphazenes substituted with side groups, namely, leucine, valine, and phenylalanine ethyl esters. Of these polymers, the phenylalanine ethyl ester substituted polyphosphazene showed the highest glass transition temperature (41.6 degrees C) and, hence, was chosen as a candidate material for forming composite microspheres with 100 nm sized hydroxyapatite (nHAp). The fabricated composite microspheres were sintered into a three-dimensional (3-D) porous scaffold by adopting a dynamic solvent sintering approach. The composite microsphere scaffolds showed compressive moduli of 46-81 MPa with mean pore diameters in the range of 86-145 microm. The 3-D polyphosphazene-nHAp composite microsphere scaffolds showed good osteoblast cell adhesion, proliferation, and alkaline phosphatase expression and are potential suitors for bone tissue engineering applications.     

Piezoelectric poly(vinylidene fluoride) microstructure and poling state in active tissue engineering

Tissue engineering strategies rely on suitable membranes and scaffolds, providing the necessary physicochemical stimuli to specific cells. This review summarizes the main results on piezoelectric polymers, in particular poly(vinylidene fluoride), for muscle and bone cell culture. Further, the relevance of polymer microstructure and surface charge on cell response is demonstrated. Together with the necessary biochemical cues, the proper design of piezoelectric polymers can open the way to novel and more reliable tissue engineering strategies for cells in which electromechanical stimuli are present in their environment.     

Less harmful acidic degradation of poly(lacticco-glycolic acid) bone tissue engineering scaffolds through titania nanoparticle addition

In the last 10 years, biodegradable aliphatic polyesters, such as poly(lactic-co-glycolic acid) (PLGA), have attracted increasing attention for their use as scaffold materials in bone tissue engineering because their degradation products can be removed by natural metabolic pathways. However, one main concern with the use of these specific polymers is that their degradation products reduce local pH, which in turn induces an inflammatory reaction and damages bone cell health at the implant site. Thus, the objective of the present in vitro study was to investigate the degradation behavior of PLGA when added with dispersed titania nanoparticles. The results of this study provided the first evidence that the increased dispersion of nanophase titania in PLGA decreased the harmful change in pH normal for PLGA degradation. Moreover, previous studies have demonstrated that the increased dispersion of titania nanoparticles into PLGA significantly improved osteoblast (bone-forming cell) functions (such as adhesion, collagen synthesis, alkaline phosphatase activity, and calcium-containing minerals deposition). In this manner, nanophase titania-PLGA composites may be promising scaffold materials for more effective orthopedic tissue engineering applications.