Load-bearing Tissue Engineering Research Articles

Mechanical performance of additively manufactured cobalt-chromium-molybdenum auxetic meta-biomaterial bone scaffolds

Auxetic meta-biomaterials offer unconventional strain behaviour owing to their negative Poisson's ratio (−υ) leading to deformation modes and mechanical properties different to traditional cellular biomaterials. This can lead to favourable outcomes for load-bearing tissue engineering constructs such as bone scaffolds. Emerging early-stage studies have shown the potential of auxetic architecture in increasing cell proliferation and tissue reintegration owing to their −υ. However, research on the development of CoCrMo auxetic meta-biomaterials including bone scaffolds or implants is yet to be reported. In this regard, this paper proposes a potential framework for the development of auxetic meta-biomaterials that can be printed on demand while featuring porosity requirements suitable for load-bearing bone scaffolds. Overall, the performance of five CoCrMo auxetic meta-biomaterial scaffolds characterised under two scenarios for their potential to offer near-zero and high negative Poisson's ratio is demonstrated. Ashby's criterion followed by prototype testing was employed to evaluate the mechanical performance and failure modes of the auxetic meta-biomaterial scaffolds under uniaxial compression. The best performing scaffold architectures are identified through a multi-criteria decision-making procedure combining ‘analytic hierarchy process’ (AHP) and ‘technique for order of preference by similarity to ideal solution’ (TOPSIS). The results found the Poisson's ratio for the meta-biomaterial architectures to be in the range of −0.1 to −0.24 at a porosity range of 73–82%. It was found that the meta-biomaterial scaffold (AX1) that offered the highest auxeticity also showed the highest elastic modulus, yield, and ultimate strength of 1.66 GPa, 56 MPa and 158 MPa, respectively. The study demonstrates that the elastic modulus, yield stress, and Poisson's ratio of auxetic meta-biomaterials are primarily influenced by the underlying meta-cellular architecture followed by relative density offering a secondary influence.

Tribo-mechanical and biological characterization of PEGDA/bioceramics composites fabricated using stereolithography

This paper aims to investigate the novel Poly(ethylene glycol) diacrylate (PEGDA)/bioceramics composites fabricated using stereolithography (SLA) for load-bearing tissue engineering applications. PEGDA hydrogel resins loaded with 1 wt% content of hydroxyapatite (HAP), calcium phosphate (CaP), and graphite (Gr) have been developed and characterized by rheological and sedimentation tests. PEGDA/bioceramics composites were characterized by mechanical, tribological, physical, and biological tests. It was found that PEGDA/HAP composite has a high tensile strength (79.1% improvement) and flexural strength (219.4% improvement), and PEGDA/Gr composite has high compressive strength (150.3% improvement) compared to unfilled PEGDA. The fractography analysis revealed that brittle fracture occurred in PEGDA and PEGDA/bioceramics composites specimens. Besides this, PEGDA and its composites are found to be thermally stable up to the temperature of ~187°C. Among all, PEGDA/Gr composite showed high compressive strength, wear resistance, low friction force, and high wettability. Additionally, PEGDA/Gr composite was found to be non-toxic, supported cellular growth, and showed similar biocompatibility as unfilled PEGDA and other composites. Thus, a comparative study has investigated the suitability and feasibility of PEGDA/bioceramic composite for tissue engineering applications.

Additively manufactured BaTiO3 composite scaffolds: A novel strategy for load bearing bone tissue engineering applications

Piezoelectric ceramics, such as BaTiO3, have gained considerable attention in bone tissue engineering applications thanks to their biocompatibility, ability to sustain a charged surface as well as improve bone cells' adhesion and proliferation. However, the poor processability and brittleness of these materials hinder the fabrication of three-dimensional scaffolds for load bearing tissue engineering applications. For the first time, this study focused on the fabrication and characterisation of BaTiO3 composite scaffolds by using a multi-material 3D printing technology. Polycaprolactone (PCL) was selected and used as dispersion phase for its low melting point, easy processability and wide adoption in bone tissue engineering. The proposed single-step extrusion-based strategy enabled a faster and solvent-free process, where raw materials in powder forms were mechanically mixed and subsequently fed into the 3D printing system for further processing.PCL, PCL/hydroxyapatite and PCL/BaTiO3 composite scaffolds were successfully produced with high level of consistency and an inner architecture made of seamlessly integrated layers. The inclusion of BaTiO3 ceramic particles (10% wt.) significantly improved the mechanical performance of the scaffolds (54 ± 0.5 MPa) compared to PCL/hydroxyapatite scaffolds (40.4 ± 0.1 MPa); moreover, the presence of BaTiO3 increased the dielectric permittivity over the entire frequency spectrum and tested temperatures. Human osteoblasts Saos-2 were seeded on scaffolds and cellular adhesion, proliferation, differentiation and deposition of bone-like extracellular matrix were evaluated. All tested scaffolds (PCL, PCL/hydroxyapatite and PCL/BaTiO3) supported cell growth and viability, preserving the characteristic cellular osteoblastic phenotype morphology, with PCL/BaTiO3 composite scaffolds exhibiting higher mineralisation (ALP activity) and deposited bone-like extracellular matrix (osteocalcin and collagen I).The single-step multi-material additive manufacturing technology used for the fabrication of electroactive PCL/BaTiO3 composite scaffolds holds great promise for sustainability (reduced material waste and manufacturing costs) and it importantly suggests PCL/BaTiO3 scaffolds as promising candidates for load bearing bone tissue engineering applications to solve unmet clinical needs.

A comparative study of tough hydrogen bonding dissipating hydrogels made with different network structures.

Hydrogels are excellent soft materials to interface with biological systems. Precise control and tunability of dissipative properties of gels are particularly interesting in tissue engineering applications. In this work, we produced hydrogels with tunable dissipative properties by photopolymerizing a second polymer within a preformed cross-linked hydrogel network of poly(acrylamide). We explored second networks made with different structures and capacity to hydrogen bond with the first network, namely linear poly(acrylic acid) and branched poly(tannic acid). Gels incorporating a second network made with poly(tannic acid) exhibited excellent stiffness (0.35 ± 0.035 MPa) and toughness (1.64 ± 0.26 MJ m−3) compared to the poly(acrylic acid) counterparts. We also demonstrate a strategy to fabricate hydrogels where the dissipation (loss modulus) can be tuned independently from the elasticity (storage modulus) suitable for cell culture applications. We anticipate that this modular design approach for producing hydrogels will have applications in tailored substrates for cell culture studies and in load bearing tissue engineering applications.

Fabrication of polycaprolactone/β-tricalciumphosphate based nano scaffolds using electrospinning method for biomedical applications

In this study, we have focused on preparation, fabrication and characterization of polycaprolactone and beta tricalcium phosphate (PCL/β-TCP) composite scaffolds that are used for bone tissue engineering applications. The electrospun composites have been characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM). The developed scaffolds are effectively simulated the morphology, mechanical property and bioactivity for load-bearing tissue engineering applications. The bioactivity of the scaffolds has been evaluated with in vitro cell adhesion and growth studies. The results confirm the nontoxic behaviour of the composite biomaterials and developed scaffolds with MG-63 osteoblast-like cell line. The synthesized scaffolds have shown promising bioactivity with the growth as well as proliferation of new bone cells with considerable osteoconductive properties. The nanoscaffolds possess better physical properties and support high cell adhesion suggesting their application in bone tissue engineering field.

Covalently Adaptable Hydrogel Based on Hyaluronic Acid and Poly(γ-glutamic acid) for Potential Load-Bearing Tissue Engineering.

Reversibly cross-linked adaptable hydrogels show great advantages in tissue engineering. The dynamic adaptable networks can overcome three-dimensional (3D) physical restrictions to enable normal cellular functions without hydrogel degradation. However, because of the dynamic reversibility, adaptable hydrogels typically exhibit weak mechanical properties and rapid erosion behaviors. Herein, we develop a facile strategy to prepare stable adaptable hydrogels using dynamic covalent chemistry, stable covalent chemistry, and the interpenetrating polymeric network (IPN) strategy. The developed IPN HA/γ-PGA adaptable hydrogels have stable structures, good mechanical properties, enzymatic degradability, and injectability. Compression test indicates that although containing 95% water, the IPN HA/γ-PGA adaptable hydrogels can suffer more than 85% compressive strain and show a fast shape recovery capacity and good antifatigue ability. Benefitting from the good cytocompatibility of functionalized HA and γ-PGA and the mild preparation process of IPN HA/γ-PGA adaptable hydrogels, NIH 3T3 cells can tolerate the 3D encapsulation process and show high cell viability. Therefore, owing to their desirable properties, the developed HA/γ-PGA adaptable hydrogels have great potential applications for load-bearing tissue engineering.

Yielding behaviors of polymeric scaffolds with implications to tissue engineering

Polymers are amongst the main candidate materials for load bearing tissue engineering scaffolds. Their effective and reliable clinical uses necessitate insightful analyses of the nonlinear characteristics, which has so far received little attention in literature. To better understand the failure characteristics of these porous structures, yielding behaviors of scaffolds made of a commercial photopolymer (RGD720) were studied using experiments and finite element method (FEM). The polymer exhibits substantial softening under uniaxial material test, but the extent of such softening behavior in a scaffold structure is compromised or even eliminated due to the internal non-uniformity of material yielding.

A robust super-tough biodegradable elastomer engineered by supramolecular ionic interactions

Alginate-based supramolecular ionic polyurethanes (ASPUs) as mechanically tunable biomaterials with high strength and toughness in both dry and hydrated states are developed under metal-free conditions. The Young's modulus and tensile strength of ASPUs are tuned from 30 to 100 MPa, and 20 to 50 MPa, respectively. Interestingly, the ASPUs exhibit a small hysteresis loop, minimal loss of tensile strength and minimal creep deformation after 100 repetitive cycles which makes them of use for engineering of load-bearing tissues. This is the first report that describes a linear PU can resist a large number of cyclic stresses without significant stretching. These bio-based elastomers engineered by ionic interactions are biocompatible and biodegradable. The ASPUs demonstrate a similar in vivo degradation rate compared to polycaprolactone (PCL). These biomaterials also demonstrate a rapid self-healing and recovery after rupture, and have a linear biodegradation profile. Furthermore, histological examination of subcutaneous transplanted ASPUs after five months reveals low immunological response and low fibrosis.

Microfabrication of Cell-Laden Hydrogels for Engineering Mineralized and Load Bearing Tissues.

Microengineering technologies and advanced biomaterials have extensive applications in the field of regenerative medicine. In this chapter, we review the integration of microfabrication techniques and hydrogel-based biomaterials in the field of dental, bone, and cartilage tissue engineering. We primarily discuss the major features that make hydrogels attractive candidates to mimic extracellular matrix (ECM), and we consider the benefits of three-dimensional (3D) culture systems for tissue engineering applications. We then focus on the fundamental principles of microfabrication techniques including photolithography, soft lithography and bioprinting approaches. Lastly, we summarize recent research on microengineering cell-laden hydrogel constructs for dental, bone and cartilage regeneration, and discuss future applications of microfabrication techniques for load-bearing tissue engineering.

Biomechanics and mechanobiology in functional tissue engineering

The field of tissue engineering continues to expand and mature, and several products are now in clinical use, with numerous other preclinical and clinical studies underway. However, specific challenges still remain in the repair or regeneration of tissues that serve a predominantly biomechanical function. Furthermore, it is now clear that mechanobiological interactions between cells and scaffolds can critically influence cell behavior, even in tissues and organs that do not serve an overt biomechanical role. Over the past decade, the field of “functional tissue engineering” has grown as a subfield of tissue engineering to address the challenges and questions on the role of biomechanics and mechanobiology in tissue engineering. Originally posed as a set of principles and guidelines for engineering of load-bearing tissues, functional tissue engineering has grown to encompass several related areas that have proven to have important implications for tissue repair and regeneration. These topics include measurement and modeling of the in vivo biomechanical environment; quantitative analysis of the mechanical properties of native tissues, scaffolds, and repair tissues; development of rationale criteria for the design and assessment of engineered tissues; investigation of the effects biomechanical factors on native and repair tissues, in vivo and in vitro; and development and application of computational models of tissue growth and remodeling. Here we further expand this paradigm and provide examples of the numerous advances in the field over the past decade. Consideration of these principles in the design process will hopefully improve the safety, efficacy, and overall success of engineered tissue replacements.

Tough nanocomposite double network hydrogels reinforced with clay nanorods through covalent bonding and reversible chain adsorption.

Polymer hydrogels with superior strength and toughness are potential candidate materials for the replacement or engineering of load-bearing tissues. This manuscript reports novel tough nanocomposite hydrogels with an unusual energy dissipation mechanism based on both covalent and physical interactions between clay nanorods and polymer chains. Attapulgite (ATP) nanorods grafted with vinyl groups on the surface served as macro-crosslinkers to copolymerize with 2-acrylamido-2-methylpropane-sulfonic acid (AMPS) to form an initial nanocomposite network, which subsequently hosted the polymerization of acrylamide (AAm) monomers to generate a novel nanocomposite double network (DN) hydrogel. The morphology, swelling behavior and compressive properties of the ATP-grafted DN hydrogels were investigated as a function of ATP content (CATP), in comparison with the ATP-filled DN gels. With a clay content between 0.1 wt% and 1.0 wt%, the nanocomposite hydrogels did not fracture up to a compressive strain of 98%, exhibiting an initial modulus (E) up to 0.36 MPa, a compressive strength higher than 65.7 MPa, and a work to fracture (or fracture energy) higher than 2.6 MJ m-3, in comparison to 0.19 MPa, 18.6 MPa, and 1.1 MJ m-3 for the conventional DN gels. Cyclic loading-unloading tests showed abnormal residual energy dissipation even though the rigid PAMPS network had fractured. Such viscous energy dissipation decayed during cyclic loading, and could be restored depending on time and temperature. This is related to the reversible desorption-re-adsorption of polymer chains from the clay surface. Possible reinforcing and fracture mechanisms are discussed.

Hybrid printing of mechanically and biologically improved constructs for cartilage tissue engineering applications

Bioprinting is an emerging technique used to fabricate viable, 3D tissue constructs through the precise deposition of cells and hydrogels in a layer-by-layer fashion. Despite the ability to mimic the native properties of tissue, printed 3D constructs that are composed of naturally-derived biomaterials still lack structural integrity and adequate mechanical properties for use in vivo, thus limiting their development for use in load-bearing tissue engineering applications, such as cartilage. Fabrication of viable constructs using a novel multi-head deposition system provides the ability to combine synthetic polymers, which have higher mechanical strength than natural materials, with the favorable environment for cell growth provided by traditional naturally-derived hydrogels. However, the complexity and high cost associated with constructing the required robotic system hamper the widespread application of this approach. Moreover, the scaffolds fabricated by these robotic systems often lack flexibility, which further restrict their applications. To address these limitations, advanced fabrication techniques are necessary to generate complex constructs with controlled architectures and adequate mechanical properties. In this study, we describe the construction of a hybrid inkjet printing/electrospinning system that can be used to fabricate viable tissues for cartilage tissue engineering applications. Electrospinning of polycaprolactone fibers was alternated with inkjet printing of rabbit elastic chondrocytes suspended in a fibrin–collagen hydrogel in order to fabricate a five-layer tissue construct of 1 mm thickness. The chondrocytes survived within the printed hybrid construct with more than 80% viability one week after printing. In addition, the cells proliferated and maintained their basic biological properties within the printed layered constructs. Furthermore, the fabricated constructs formed cartilage-like tissues both in vitro and in vivo as evidenced by the deposition of type II collagen and glycosaminoglycans. Moreover, the printed hybrid scaffolds demonstrated enhanced mechanical properties compared to printed alginate or fibrin–collagen gels alone. This study demonstrates the feasibility of constructing a hybrid inkjet printing system using off-the-shelf components to produce cartilage constructs with improved biological and mechanical properties.

Fabrication and characterization of triple nanobioceramic composite foam

In this study, triple nanobioceramic composite foam consisting of bioactive glass (BG), forsterite, and hydroxyapatite (HA) with highly porous structure and high mechanical strength was successfully fabricated via gelcasting method. The BG and forsterite were added to HA as second-phase ceramics to enhance simultaneously its bioactivity, biodegradability, and mechanical properties. The prepared foams have a crystallite size in the range 30–52 nm (while the crystallite size of initial HA and forsterite powders is about 22 nm), pore size in the range 100–300 µm, and total and interconnected porosity upto 87% and 81%, respectively. The compressive strength measurements showed that the foams have an excellent compressive strength (2.19–3.68 MPa), which is appropriate for load-bearing tissue engineering applications and is close to the compressive strength of spongy bone. In vitro tests proved the good bioactivity and resorbability of the foams. The resulting composite foams have similar chemical composition to the mineral phase of bone and it is possible to optimize the ratio of BG phase to ceramic phases for achieving appropriate mechanical strength, bioactivity, and biodegradability level needed for different applications.

A Roadmap for the Design of Bioreactors in Mechanobiological Research and Engineering of Load-Bearing Tissues

In the field of tissue engineering, a bioreactor is a valuable instrument that mimics a physiological environment to maintain live tissues in vitro. Although bioreactors are conceptually relatively simple, the vast majority of current bioreactors (commercial and custom-built) are not fully adapted to current research needs. Designing the optimal bioreactor requires a very thorough approach to a series of steps in the product development process. These four basic steps are: (1) identifying the needs and technical requirements, (2) defining and evaluating the related concepts, (3) designing the apparatus and drawing up the blueprints, and (4) building and validating the apparatus. Furthermore, the design has to be adapted to the specific purpose of the research and how the tissues will be used. In the emerging field of bioreactor research, roadmaps are needed to assist tissue engineering researchers as they embark on this process. The necessary multidisciplinary expertise covering micromechanical design, mechatronics, viscoelasticity, tissue culture, and human ergonomics is not necessarily available to all research teams. Therefore, the challenge of adapting and conducting each step in the product development process is significant. This paper details our proposal for a roadmap to accompany researchers in identifying their needs and technical requirements: step one in the product development process. Our roadmap proposal is set up in two phases. Phase 1 is based on the analysis of the bioreactor use cycle and phase 2 is based on the analysis of one specific and critical step in the use cycle: conducting stimulation and characterization protocols with the bioreactor. A meticulous approach to these two phases minimizes the risk of forgetting important requirements and strengthens the probability of acquiring or designing a high performance bioreactor.

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

The in vitro production of highly organized collagen fibrils by corneal keratocytes in a three-dimensional scaffold-free culture system presents a unique opportunity for the direct observation of organized matrix formation. The objective of this investigation was to develop such a culture system in a glass substrate (for optical accessibility) and to directly examine the effect of reducing serum and/or increasing insulin on the stratification and secretion of aligned matrix by fourth- to fifth-passage bovine corneal stromal keratocytes. Medium concentrations of 0%, 1%, or 10% fetal bovine serum and 0% or 1% insulin-transferrin-selenium were investigated. High-resolution differential interference contrast microscopy, quick-freeze/deep-etch, and conventional transmission electron microscopy were used to monitor the evolution, morphology, and ultrastructure of the cell-matrix constructs. In a medium containing 1% each of serum and insulin-transferrin-selenium, stromal cells stratified and secreted abundant and locally aligned matrix, generating the thickest cell-matrix constructs (allowing handling with forceps). The results of this study have the potential to significantly advance the field of developmental functional engineering of load-bearing tissues by (i) elucidating cues that modulate in vitro cell secretion of organized matrix and (ii) establishing an optically accessible cell culture system for investigating the mechanism of cell secretion of aligned collagen fibrils.

Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus

Successful engineering of load-bearing tissues requires recapitulation of their complex mechanical functions. Given the intimate relationship between function and form, biomimetic materials that replicate anatomic form are of great interest for tissue engineering applications. However, for complex tissues such as the annulus fibrosus, scaffolds have failed to capture their multi-scale structural hierarchy. Consequently, engineered tissues have yet to reach functional equivalence with their native counterparts. Here we present a novel strategy for annulus fibrosus tissue engineering that replicates this hierarchy with anisotropic nanofibrous laminates seeded with mesenchymal stem cells. These scaffolds directed the deposition of organized, collagen-rich extracellular matrix that mimicked the angle-ply, multi-lamellar architecture and achieved mechanical parity with native tissue after 10 weeks of in vitro culture. Further, we identified a novel role for inter-lamellar shearing in reinforcing the tensile response of biologic laminates, a mechanism that has not previously been considered for these tissues.

Morphology, mechanical characterization and in vivo neo-vascularization of chitosan particle aggregated scaffolds architectures

The present study intended to evaluate the performance of chitosan-based scaffolds produced by a particle aggregation method aimed to be used in tissue engineering applications addressing key issues such as morphological characteristics, mechanical performance and in vivo behaviour. It is claimed that the particle aggregation methodology may present several advantages, such as combine simultaneously a high interconnectivity with high mechanical properties that are both critical for an in vivo successful application. In order to evaluate these properties, micro-Computed Tomography (micro-CT) and Dynamical Mechanical Analysis (DMA) were applied. The herein proposed scaffolds present an interesting morphology as assessed by micro-CT that generally seems to be adequate for the proposed applications. At a mechanical level, DMA has shown that chitosan scaffolds have an elastic behaviour under dynamic compression solicitation, being simultaneously mechanically stable in the wet state and exhibiting a storage modulus of 4.21 ± 1.04 MPa at 1 Hz frequency. Furthermore, chitosan scaffolds were evaluated in vivo using a rat muscle-pockets model for different implantation periods (1, 2 and 12 weeks). The histological and immunohistochemistry results have demonstrated that chitosan scaffolds can provide the required in vivo functionality. In addition, the scaffolds interconnectivity has been shown to be favourable to the connective tissues ingrowth into the scaffolds and to promote the neo-vascularization even in early stages of implantation. It is concluded that the proposed chitosan scaffolds produced by particle aggregation method are suitable alternatives, being simultaneously mechanical stable and in vivo biofunctional that might be used in load-bearing tissue engineering applications, including bone and cartilage regeneration.

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.