Fabrication using a rapid prototyping system and in vitro characterization of PEG-PCL-PLA scaffolds for tissue engineering

n the field of tissue engineering new polymers are needed to fabricate scaffolds with specific properties depending on the targeted tissue. This work aimed at designing and developing a 3D scaffold with variable mechanical strength, fully interconnected porous network, controllable hydrophilicity and degradability. For this, a desktop-robot-based melt-extrusion rapid prototyping technique was applied to a novel tri-block co-polymer, namely poly(ethylene glycol)-block-poly(epsi-caprolactone)-block-poly(DL-lactide), PEG-PCL-P(DL)LA. This co-polymer was melted by electrical heating and directly extruded out using computer-controlled rapid prototyping by means of compressed purified air to build porous scaffolds. Various lay-down patterns (0/30/60/90/120/150°, 0/45/90/135°, 0/60/120° and 0/90°) were produced by using appropriate positioning of the robotic control system. Scanning electron microscopy and micro-computed tomography were used to show that 3D scaffold architectures were honeycomb-like with completely interconnected and controlled channel characteristics. Compression tests were performed and the data obtained agreed well with the typical behavior of a porous material undergoing deformation. Preliminary cell response to the as-fabricated scaffolds has been studied with primary human fibroblasts. The results demonstrated the suitability of the process and the cell biocompatibility of the polymer, two important properties among the many required for effective clinical use and efficient tissue-engineering scaffolding.

Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.

Vitrification as a prospect for cryopreservation of tissue-engineered constructs

Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation

Fumaric acid monoethyl ester-functionalized poly(D,L-Lactide)/N-vinyl-2- pyrrolidone Resins for the preparation of tissue engineering scaffolds by stereolithography

Polymer networks were prepared by photocross-linking fumaric acid monoethyl ester (FAME) functionalized, three-armed poly(D,L-lactide) oligomers using Af-vinyl-2-pyrrolidone (NVP) as diluent and comonomer. The use of NVP together with FAME-functionalized oligomers resulted in copolymerization at high rates, and networks with gel contents in excess of 90 were obtained. The hydrophilicity of the poly(D,L-lactide) networks increases with increasing amounts of NVP, networks containing 50 wt of NVP absorbed 40 of water. As the amount of NVP was increased from 30 to 50 wt , the Young's modulus after equilibration in water decreased from 0.8 to 0.2 GPa, as opposed to an increase from 1.5 to 2.1 GPa in the dry state. Mouse preosteoblasts readily adhered and spread onto all prepared networks. Using stereolithography, porous structures with a well-defined gyroid architecture were prepared from these novel materials. This allows the preparation of tissue engineering scaffolds with optimized pore architecture and tunable material properties.

A poly(D,L-lactide) resin for the preparation of tissue engineering scaffolds by stereolithography

Porous polylactide constructs were prepared by stereolithography, for the first time without the use of reactive diluents. Star-shaped poly(D,L-lactide) oligomers with 2, 3 and 6 arms were synthesised, end-functionalised with methacryloyl chloride and photocrosslinked in the presence of ethyl lactate as a non-reactive diluent. The molecular weights of the arms of the macromers were 0.2, 0.6, 1.1 and 5 kg/mol, allowing variation of the crosslink density of the resulting networks. Networks prepared from macromers of which the molecular weight per arm was 0.6 kg/mol or higher had good mechanical properties, similar to linear high molecular weight poly(D,L-lactide). A resin based on a 2-armed poly(D,L-lactide) macromer with a molecular weight of 0.6 kg/mol per arm (75 wt%), ethyl lactate (19 wt%), photo-initiator (6 wt%), inhibitor and dye was prepared. Using this resin, films and computer-designed porous constructs were accurately fabricated by stereolithography. Pre-osteoblasts showed good adherence to these photocrosslinked networks. The proliferation rate on these materials was comparable to that on high molecular weight poly(D,L-lactide) and tissue culture polystyrene.

Mathematically defined tissue engineering scaffold architectures prepared by stereolithography

The technologies employed for the preparation of conventional tissue engineering scaffolds restrict the materials choice and the extent to which the architecture can be designed. Here we show the versatility of stereolithography with respect to materials and freedom of design. Porous scaffolds are designed with computer software and built with either a poly(d,l-lactide)-based resin or a poly(d,l-lactide-co-ε-caprolactone)-based resin. Characterisation of the scaffolds by micro-computed tomography shows excellent reproduction of the designs. The mechanical properties are evaluated in compression, and show good agreement with finite element predictions. The mechanical properties of scaffolds can be controlled by the combination of material and scaffold pore architecture. The presented technology and materials enable an accurate preparation of tissue engineering scaffolds with a large freedom of design, and properties ranging from rigid and strong to highly flexible and elastic.

A two-compartment mechanochemical model of the roles of transforming growth factor-beta and tissue tension in dermal wound healing

The repair of dermal tissue is a complex process of interconnected phenomena, where cellular, chemical and mechanical aspects all play a role, both in an autocrine and in a paracrine fashion. Recent experimental results have shown that transforming growth factor-beta (TGF-beta) and tissue mechanics play roles in regulating cell proliferation, differentiation and the production of extracellular materials. We have developed a 1D mathematical model that considers the interaction between the cellular, chemical and mechanical phenomena, allowing the combination of TGF-beta and tissue stress to inform the activation of fibroblasts to myofibroblasts. Additionally, our model incorporates the observed feature of residual stress by considering the changing zero-stress state in the formulation for effective strain. Using this model, we predict that the continued presence of TGF-beta in dermal wounds will produce contractures due to the persistence of myofibroblasts; in contrast, early elimination of TGF-beta significantly reduces the myofibroblast numbers resulting in an increase in wound size. Similar results were obtained by varying the rate at which fibroblasts differentiate to myofibroblasts and by changing the myofibroblast apoptotic rate. Taken together, the implication is that elevated levels of myofibroblasts is the key factor behind wounds healing with excessive contraction, suggesting that clinical strategies which aim to reduce the myofibroblast density may reduce the appearance of contractures.

Development of a 3-dimensional human skin equivalent wound model for investigating novel wound healing therapies

Numerous difficulties are associated with the conduct of preclinical studies related to skin and wound repair. Use of small animal models such as rodents is not optimal because of their physiological differences to human skin and mode of wound healing. Although pigs have previously been used because of their human-like mode of healing, the expense and logistics related to their use also renders them suboptimal. In view of this, alternatives are urgently required to advance the field. The experiments reported herein were aimed at developing and validating a simple, reproducible, three-dimensional ex vivo de-epidermised dermis human skin equivalent wound model for the preclinical evaluation of novel wound therapies. Having established that the human skin equivalent wound model does in fact “heal," we tested the effect of two novel wound healing therapies. We also examined the utility of the model for studies exploring the mechanisms underpinning these therapies. Taken together the data demonstrate that these new models will have wide-spread application for the generation of fundamental new information on wound healing processes and also hold potential in facilitating preclinical optimization of dosage, duration of therapies, and treatment strategies prior to clinical trials.

Engineering an Ecosystem : Taking Cues from Nature’s Paradigm to Build Tissue in the Lab and the Body

This manuscript took a 'top down' approach to understanding survival of inhabitant cells in the ecosystem bone, working from higher to lower length and time scales through the hierarchical ecosystem of bone. Our working hypothesis is that nature “engineered” the skeleton using a 'bottom up' approach,where mechanical properties of cells emerge from their adaptation to their local me-chanical milieu. Cell aggregation and formation of higher order anisotropic struc- ture results in emergent architectures through cell differentiation and extracellular matrix secretion. These emergent properties, including mechanical properties and architecture, result in mechanical adaptation at length scales and longer time scales which are most relevant for the survival of the vertebrate organism [Knothe Tate and von Recum 2009]. We are currently using insights from this approach to har-ness nature’s regeneration potential and to engineer novel mechanoactive materials [Knothe Tate et al. 2007, Knothe Tate et al. 2009]. In addition to potential applications of these exciting insights, these studies may provide important clues to evolution and development of vertebrate animals. For instance, one might ask why mesenchymal stem cells condense at all? There is a putative advantage to self-assembly and cooperation, but this advantage is somewhat outweighed by the need for infrastructural complexity (e.g., circulatory systems comprised of specific differentiated cell types which in turn form conduits and pumps to overcome limitations of mass transport via diffusion, for example; dif-fusion is untenable for multicellular organisms larger than 250 microns in diameter. A better question might be: Why do cells build skeletal tissue? Once cooperatingcells in tissues begin to deplete local sources of food in their aquatic environment, those that have evolved a means to locomote likely have an evolutionary advantage. Once the environment becomes less aquarian and more terrestrial, self-assembled organisms with the ability to move on land might have conferred evolutionary ad-vantages as well. So did the cytoskeleton evolve several length scales, enabling the emergence of skeletal architecture for vertebrate animals? Did the evolutionary advantage of motility over noncompliant terrestrial substrates (walking on land) favor adaptations including emergence of intracellular architecture (changes in the cytoskeleton and upregulation of structural protein manufacture), inter-cellular con- densation, mineralization of tissues, and emergence of higher order architectures?How far does evolutionary Darwinism extend and how can we exploit this knowl- edge to engineer smart materials and architectures on Earth and new, exploratory environments?[Knothe Tate et al. 2008]. We are limited only by our ability to imagine. Ultimately, we aim to understand nature, mimic nature, guide nature and/or exploit nature’s engineering paradigms without engineer-ing ourselves out of existence.

Mesoporous bioglass/silk composite scaffolds for bone tissue engineering

In the past 20 years, mesoporous materials have been attracted great attention due to their significant feature of large surface area, ordered mesoporous structure, tunable pore size and volume, and well-defined surface property. They have many potential applications, such as catalysis, adsorption/separation, biomedicine, etc. [1]. Recently, the studies of the applications of mesoporous materials have been expanded into the field of biomaterials science. A new class of bioactive glass, referred to as mesoporous bioactive glass (MBG), was first developed in 2004. This material has a highly ordered mesopore channel structure with a pore size ranging from 5–20 nm [1]. Compared to non-mesopore bioactive glass (BG), MBG possesses a more optimal surface area, pore volume and improved in vitro apatite mineralization in simulated body fluids [1,2]. Vallet-Regí et al. has systematically investigated the in vitro apatite formation of different types of mesoporous materials, and they demonstrated that an apatite-like layer can be formed on the surfaces of Mobil Composition of Matters (MCM)-48, hexagonal mesoporous silica (SBA-15), phosphorous-doped MCM-41, bioglass-containing MCM-41 and ordered mesoporous MBG, allowing their use in biomedical engineering for tissue regeneration [2-4]. Chang et al. has found that MBG particles can be used for a bioactive drug-delivery system [5,6]. Our study has shown that MBG powders, when incorporated into a poly (lactide-co-glycolide) (PLGA) film, significantly enhance the apatite-mineralization ability and cell response of PLGA films. compared to BG [7]. These studies suggest that MBG is a very promising bioactive material with respect to bone regeneration. It is known that for bone defect repair, tissue engineering represents an optional method by creating three-dimensional (3D) porous scaffolds which will have more advantages than powders or granules as 3D scaffolds will provide an interconnected macroporous network to allow cell migration, nutrient delivery, bone ingrowth, and eventually vascularization [8]. For this reason, we try to apply MBG for bone tissue engineering by developing MBG scaffolds. However, one of the main disadvantages of MBG scaffolds is their low mechanical strength and high brittleness; the other issue is that they have very quick degradation, which leads to an unstable surface for bone cell growth limiting their applications. Silk fibroin, as a new family of native biomaterials, has been widely studied for bone and cartilage repair applications in the form of pure silk or its composite scaffolds [9-14]. Compared to traditional synthetic polymer materials, such as PLGA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the chief advantage of silk fibroin is its water-soluble nature, which eliminates the need for organic solvents, that tend to be highly cytotoxic in the process of scaffold preparation [15]. Other advantages of silk scaffolds are their excellent mechanical properties, controllable biodegradability and cytocompatibility [15-17]. However, for the purposes of bone tissue engineering, the osteoconductivity of pure silk scaffolds is suboptimal. It is expected that combining MBG with silk to produce MBG/silk composite scaffolds would greatly improve their physiochemical and osteogenic properties for bone tissue engineering application. Therefore, in this chapter, we will introduce the research development of MBG/silk scaffolds for bone tissue engineering.

Mesoporous bioactive glasses as drug delivery and bone tissue regeneration platforms

The use of mesoporous bioactive glasses (MBG) for drug delivery and bone tissue regeneration has grown significantly over the past 5 years. In this review, we highlight the recent advances made in the preparation of MBG particles, spheres, fibers and scaffolds. The advantages of MBG for drug delivery and bone scaffold applications are related to this material’s well-ordered mesopore channel structure, superior bioactivity, and the application for the delivery of both hydrophilic and hydrophobic drugs. A brief forward-looking perspective on the potential clinical applications of MBG in regenerative medicine is also discussed.