Interface Tissue Engineering Research Articles

Shaping the mechanical properties of a gelatin hydrogel interface via amination.

Injuries to musculoskeletal interfaces, such as the tendon-to-bone insertion of the rotator cuff, present significant physiological and clinical challenges for repair due to complex gradients of structure, composition, and cellularity. Advances in interface tissue engineering require stratified biomaterials able to both provide local instructive signals to support multiple tissue phenotypes while also reducing the risk of strain concentrations and failure at the transition between dissimilar materials. Here, we describe adaptation of a thiolated gelatin (Gel-SH) hydrogel via selective amination of carboxylic acid subunits on the gelatin backbone. The magnitude and kinetics of HRP-mediated primary crosslinking and carbodiimide-mediated secondary crosslinking reactions can be tuned through amination and thiolation of carboxylic acid subunits on the gelatin backbone. We also show that a stratified biomaterial comprised of mineralized (bone-mimetic) and non-mineralized (tendon-mimetic) collagen scaffold compartments linked by an aminated Gel-SH hydrogel demonstrate improved mechanical performance and reduced strain concentrations. Together, these results highlight significant mechanical advantages that can be derived from modifying the gelatin macromer via controlled amination and thiolation and suggest an avenue for tuning the mechanical performance of hydrogel interfaces within stratified biomaterials.

Computational fluid dynamics analysis of the fluid environment of 3D printed gradient structure in interfacial tissue engineering

Mass transport properties within three-dimensional (3D) scaffold are essential for tissue regeneration, such as various fluid environmental cues influence mesenchymal stem cells differentiation. Recently, 3D printing has been emerging as a new technology for scaffold fabrication by controlling the scaffold pore geometry to affect cell growth environment. In this study, the flow field within scaffolds in a perfusion system was investigated with uniform structures, single gradient structures and complex gradient structures using computational fluid dynamics (CFD) method. The CFD results from those uniform structures indicate the fluid velocity and fluid shear stress within the scaffold structure increased as the filament diameter increasing, pore width decreasing, pore shape decreased from 90° to 15°, and layer configuration changing from lattice to stagger structure. By assembling those uniform structure as single gradient structures, it is noted that the fluid dynamic characterisation within the scaffold remains the same as the corresponding uniform structures. A complex gradient structure was designed to mimic natural osteochondral tissue by assembly the uniform structures of filament diameter, pore width, pore shape and layer configuration. The results show that the fluid velocity and fluid shear stress within the complex gradient structure distribute gradually increasing and their maximum magnitude were from 1.15 to 3.20 mm/s, and from 12 to 39 mPa, respectively. CFD technique allows the prediction of velocity and fluid shear stress within the designed 3D gradient scaffolds, which would be beneficial for the tissue scaffold development for interfacial tissue engineering in the future.

Anatomical design and production of a novel three-dimensional co-culture system replicating the human flexor digitorum profundus enthesis.

The enthesis, the specialized junction between tendon and bone, is a common site of injury. Although notoriously difficult to repair, advances in interfacial tissue engineering techniques are being developed for restorative function. Most notably are 3D invitro co-culture models, built to recreate the complex heterogeneity of the native enthesis. While cell and matrix properties are often considered, there has been little attention given to native enthesis anatomical morphometrics and replicating these to enhance clinical relevance. This study focuses on the flexor digitorum profundus (FDP) tendon enthesis and, by combining anatomical morphometrics with computer-aided design, demonstrates the design and construction of an accurate and scalable model of the FDP enthesis. Bespoke 3D-printed mould inserts were fabricated based on the size, shape and insertion angle of the FDP enthesis. Then, silicone culture moulds were created, enabling the production of bespoke anatomical culture zones for an invitro FDP enthesis model. The validity of the model has been confirmed using brushite cement scaffolds seeded with osteoblasts (bone) and fibrin hydrogel scaffolds seeded with fibroblasts (tendon) in individual studies with cells from either human or rat origin. This novel approach allows a bespoke anatomical design for enthesis repair and should be applied to future studies in this area.

Regulatory mechanisms and regeneration strategies of the soft–hard tissue interface in the human periodontium

AbstractThe soft‐hard tissue interface of the human periodontium is responsible for periodontal homeostasis and is essential for normal oral activities. This soft‐hard tissue interface is formed by the direct insertion of fibrous ligaments into the bone tissue. It differs from the unique four‐layer structure of the fibrocartilage interface. This interface is formed by a combination of physical, chemical, and biological factors. The physiological functions of this interface are regulated by different signaling pathways. The unique structure of this soft‐hard tissue interface has inspired scientists to construct biomimetic gradient structures. These biomimetic systems include nanofiber scaffolds, cell sheets, and hydrogels. Exploring methods to repair this soft‐hard tissue interface can help solve clinically unresolved problems. The present review examines the structure of the soft‐hard tissue interface of the periodontium and the factors that influence the development of this interface. Relevant regulatory pathways and biomimetic reconstruction methods are also presented to provide ideas for future research on interfacial tissue engineering.

Transforming layered 2D mats into multiphasic 3D nanofiber scaffolds with tailored gradient features for tissue regeneration.

Multiphasic scaffolds with tailored gradient features hold significant promise for tissue regeneration applications. Herein, this work reports the transformation of two-dimensional (2D) layered fiber mats into three dimensional (3D) multiphasic scaffolds using a 'solids-of-revolution' inspired gas-foaming expansion technology. These scaffolds feature precise control over fiber alignment, pore size, and regional structure. Manipulating nanofiber mat layers and Pluronic F127 concentrations allows further customization of pore size and fiber alignment within different scaffold regions. The cellular response to multiphasic scaffolds demonstrates the number of cells migrated and proliferated onto the scaffolds are mainly dependent on the pore size rather than fiber alignment. In vivo subcutaneous implantation of multiphasic scaffolds to rats reveals substantial cell infiltration, neo tissue formation, collagen deposition, and new vessel formation within scaffolds, greatly surpassing the capabilities of traditional nanofiber mats. Histological examination indicates the importance of optimizing pore size and fiber alignment for promotion of cell infiltration and tissue regeneration. Overall, these scaffolds have potential applications in tissue modeling, studying tissue-tissue interactions, interface tissue engineering, and high-throughput screening for optimized tissue regeneration.

Multimodal Three-Dimensional Printing for Micro-Modulation of Scaffold Stiffness Through Machine Learning.

The ability to precisely control a scaffold's microstructure and geometry with light-based three-dimensional (3D) printing has been widely demonstrated. However, the modulation of scaffold's mechanical properties through prescribed printing parameters is still underexplored. This study demonstrates a novel 3D-printing workflow to create a complex, elastomeric scaffold with precision-engineered stiffness control by utilizing machine learning. Various printing parameters, including the exposure time, light intensity, printing infill, laser pump current, and printing speed were modulated to print poly (glycerol sebacate) acrylate (PGSA) scaffolds with mechanical properties ranging from 49.3 ± 3.3 kPa to 2.8 ± 0.3 MPa. This enables flexibility in spatial stiffness modulation in addition to high-resolution scaffold fabrication. Then, a neural network-based machine learning model was developed and validated to optimize printing parameters to yield scaffolds with user-defined stiffness modulation for two different vat photopolymerization methods: a digital light processing (DLP)-based 3D printer was utilized to rapidly fabricate stiffness-modulated scaffolds with features on the hundreds of micron scale and a two-photon polymerization (2PP) 3D printer was utilized to print fine structures on the submicron scale. A novel 3D-printing workflow was designed to utilize both DLP-based and 2PP 3D printers to create multiscale scaffolds with precision-tuned stiffness control over both gross and fine geometric features. The described workflow can be used to fabricate scaffolds for a variety of tissue engineering applications, specifically for interfacial tissue engineering for which adjacent tissues possess heterogeneous mechanical properties (e.g., muscle-tendon).

Silane-modified hydroxyapatite nanoparticles incorporated into polydioxanone/poly(lactide-co-caprolactone) creates a novel toughened nanocomposite with improved material properties and in vivo inflammatory responses.

The interface tissue between bone and soft tissues, such as tendon and ligament (TL), is highly prone to injury. Although different biomaterials have been developed for TL regeneration, few address the challenges of the TL-bone interface. Here, we aim to develop novel hybrid nanocomposites based on poly(p-dioxanone) (PDO), poly(lactide-co-caprolactone) (LCL), and hydroxyapatite (HA) nanoparticles suitable for TL-bone interface repair. Nanocomposites, containing 3-10% of both unmodified and chemically modified hydroxyapatite (mHA) with a silane coupling agent. We then explored biocompatibility through in vitro and in vivo studies using a subcutaneous mouse model. Through different characterisation tests, we found that mHA increases tensile properties, creates rougher surfaces, and reduces crystallinity and hydrophilicity. Morphological observations indicate that mHA nanoparticles are attracted by PDO rather than LCL phase, resulting in a higher degradation rate for mHA group. We found that adding the 5% of nanoparticles gives a balance between the properties. In vitro experiments show that osteoblasts' activities are more affected by increasing the nanoparticle content compared with fibroblasts. Animal studies indicate that both HA and mHA nanoparticles (10%) can reduce the expression of pro-inflammatory cytokines after six weeks of implantation. In summary, this work highlights the potential of PDO/LCL/HA nanocomposites as an excellent biomaterial for TL-bone interface tissue engineering applications.

Microfluidic 3D Printing of Emulsion Ink for Engineering Porous Functionally Graded Materials

AbstractIn the last decade, 3D printing systems have greatly evolved both in terms of processable materials and printing resolutions, becoming a top seed technology for many academic and industrial applications. Nevertheless, manufacturing polymeric materials characterized by a trabecular porosity and functionally graded architecture—where both the local porosity and chemical composition of the matrix change in the 3D space—through additive platforms remains an open technical challenge. In this study, a 3D extrusion printing strategy to tackle this problem is presented. The proposed systems are based on a flow‐focusing microfluidic printing head—to continuously generate oil‐in‐water emulsion inks—and on an agarose fluid–gel used as a temporary support bath for the deposition of the photo‐curable emulsion inks. It is demonstrated that through this strategy one can design a priori and build with high accuracy both discontinuous and continuous functionally graded polymeric foams, where both the density and composition of the materials could be varied independently within arbitrarily complex 3D architectures. This study provides new means for the synthesis of microporous, polymeric FGMs which could find applications ranging from interface tissue engineering to automotive and construction industries.

Regulation and Reconstruction of Cell Phenotype Gradients Along the Tendon‐Bone Interface

AbstractTendon–bone interface is prevalent in the human body. It is divided into four zones: tendon (soft tissue), unmineralized fibrocartilage, mineralized fibrocartilage, and bone (hard tissue). Tendon–bone interface is characterized by a cell phenotype gradient that appears in the different zones. The cell phenotype gradients at the tendon–bone interface are orchestrated by specific intracellular molecular mechanisms, extracellular factors, immune signals, and neurovascular factors. These features have inspired scientists to design systems that mimic natural cell phenotype gradients. These biomimetic systems include the construction of cell sheets, regulation of cellular microenvironments, and the design of gradient functional scaffolds. Exploration of methods to mimic cell phenotype gradients is instructional for future clinical applications in reconstituting the tendon–bone interface. The present review elucidates the gradient composition of the tendon–bone interface. The associated regulatory mechanisms and applications are discussed, with the anticipation of creating a mise en scène for future research in interface tissue engineering.

Rational Design of Soft-Hard Interfaces through Bioinspired Engineering.

Soft-hard tissue interfaces in nature present a diversity of hierarchical transitions in composition and structure to address the challenge of stress concentrations that would otherwise arise at their interface. The translation of these into engineered materials holds promise for improved function of biomedical interfaces. Here, soft-hard tissue interfaces found in the body in health and disease, and the application of the diverse, functionally graded, and hierarchical structures that they present to bioinspired engineering materials are reviewed. A range of such bioinspired engineering materials and associated manufacturing technologies that are on the horizon in interfacial tissue engineering, hydrogel bioadhesion at the interfaces, and healthcare and medical devices are described.

Biomaterials with stiffness gradient for interface tissue engineering

Interface tissue engineering is a rapidly growing field that aims to develop engineered tissue alternates with the goal of promoting integration between multiple tissue types. Engineering interface tissues is a challenging process, which requires biomaterials with specific composition, stiffness, cell types, and biochemical molecules. Among these, stiffness-controllable substrates have been developed to investigate the effect of stiffness on cell behavior. Especially these substrates with graded stiffness are advantageous since they allow multiple cell types differentiation and the following tissue development. In this review, we highlight various types of manufacturing techniques that can be applied to fabricate scaffolds with stiffness gradient, discuss methods to characterize them, and gradient biomaterials for regulating cellular behavior including attachment, migration, proliferation, and differentiation. We also address fundamentals of interface tissue organization, and stiffness gradient biomaterials for interface tissue regeneration. Potential challenges and future prospects of this emerging field are also included.

3D-printed bi-layered polymer/hydrogel construct for interfacial tissue regeneration in a canine model

ObjectivesThere are complications in applying regenerative strategies at the interface of hard and soft tissues due to the limited designs of constructs that can accommodate different cell types in different sites. The problem originates from the challenges in the adhesion of dissimilar materials, such as polymers and hydrogels, that can be suitable for regenerating different tissues such as bone and soft tissues. This paper presents a design of a new hybrid construct in which a polymer (polycaprolactone (PCL)) membrane firmly adheres to a layer of hydrogen (gelatin). MethodsPCL membranes with defined size and porosity were fabricated using 3D printing. The gelatin layer was attached to the PCL membranes using the aminolysis procedure. We have examined this construct for the application of Guided Bone Regeneration (GBR) as a typical surgical regenerative procedure of the oral cavity at the interface of bone and soft tissue. Complete in vitro and in vivo investigations on canine tibia bone defects have been performed. Histological analyses for fibrosis morphometric and bone morphometric evaluation, as well as bone-fibrosis histological grading and CBCT imaging, were conducted. ResultsChemical and morphological studies of the membrane proved that gelatin was uniformly attached to the aminolyzed PCL membranes. The in vitro and in vivo studies indicated the membrane's biocompatibility, mechanical stability, and barrier function for the GBR application. Furthermore, in vitro study showed that the membranes could improve osteogenesis and the regeneration of bone defects. The results illustrated that the mean bone density in the membrane groups was about three times more than that of the control group. SignificanceThe fabricated 3D-printed hybrid Gelatin/PCL bi-layered membrane can be a good candidate for interfacial tissue engineering and a promising membrane for GBR procedure.

Making connections: using anatomy to guide tissue engineering approaches at the enthesis.

The enthesis demonstrates a distinct highly ordered zonal microanatomy at the osteotendinous/osteoligamentous tissue connection that allows for the smooth transmission of mechanical forces between tissues. Interfacial tissue engineering (ITE), a subset of the interdisciplinary field of tissue engineering, is directed at replicating this complex transitional anatomy of the enthesis in vitro. Yet, the limited understanding of tissue boundaries, gradients and structural relationships at specific anatomical locations hampers the development of novel therapeutic strategies for bespoke enthesis regeneration, thus reducing their direct clinical applicability. This review provides an overview of ITE approaches for repair of the osteotendinous/osteoligamentous junction and highlights the importance of complementary inclusion of direct anatomical research. The cross-disciplinary collaboration across an array of experts, including anatomists, involved in the design, development and utilisation of bioengineered tissues will enhance the properties of such tissues and improve their clinical relevance. More specifically, a detailed anatomical analysis of the region of interest should drive the in vitro design and enable researchers to develop anatomically and clinically relevant tissue-engineered replacement tissues for human implantation. Finally, the present review discusses the challenges and future directions of the ITE field and highlights the importance of anatomically driven tissue engineering as an emerging tool in clinical translational research.

Vertical Extrusion Cryo(bio)printing for Anisotropic Tissue Manufacturing (Adv. Mater. 12/2022)

3D Cryo-Bioprinting A vertical 3D cryo-bioprinting technique is reported by Yu Shrike Zhang and co-workers in article number 2108931, which concurrently allows the user to create freestanding filamentous structures containing interconnected, anisotropic microchannels featuring gradient sizes aligned in the vertical direction, also associated with enhanced mechanical performance. This method is further extended to a multi-material format, finding potential applications in interface tissue engineering, regenerative medicine, drug discovery, and personalized therapeutics.

Oriented/dual-gradient in structure and mechanics chitosan hydrogel bio-films based on stretching for guiding cell orientation

Various anisotropic scaffolds have been fabricated to significantly assist cell alignment or orientation to mimic the natural oriented tissues. To pursuit facile preparation and further exploration, the oriented hydrogel film was manufactured here through stretching-drying method by using high tensile strength chitosan hydrogel film, which could be effectively prepared after at least 3 h drying for 65% strain or 4 h drying for 35% strain. Furthermore, the chitosan hydrogel film with gradient nanofibrous structure and mechanical property was prepared via stretching-inclining-drying way which was newly proposed on the base of stretching-drying method. The gradient hydrogel film not only achieved random-to-aligned hierarchical nanofibrous structure, but also engendered obvious gradient mechanical performance, presenting the variable tensile modulus with a range from about 1 to 22 MPa after 8 h drying. This kind of random-to-aligned gradient structure can even adjust cell directions from random to orientation. Chitosan gradient hydrogel film shows a great promise in inducing cell migration, orientation and differentiation, especially as a powerful bio-scaffold for interface tissue engineering and regenerative medicine .

Vertical Extrusion Cryo(bio)printing for Anisotropic Tissue Manufacturing.

Due to the poor mechanical properties of many hydrogel bioinks, conventional 3D extrusion bioprinting is usually conducted based on the X-Y plane, where the deposited layers are stacked in the Z-direction with or without the support of prior layers. Herein, a technique is reported, taking advantage of a cryoprotective bioink to enable direct extrusion bioprinting in the vertical direction in the presence of cells, using a freezing plate with precise temperature control. Of interest, vertical 3D cryo-bioprinting concurrently allows the user to create freestanding filamentous constructs containing interconnected, anisotropic microchannels featuring gradient sizes aligned in the vertical direction, also associated with enhanced mechanical performances. Skeletal myoblasts within the 3D-cryo-bioprinted hydrogel constructs show enhanced cell viability, spreading, and alignment, compared to the same cells in the standard hydrogel constructs. This method is further extended to a multimaterial format, finding potential applications in interface tissue engineering, such as creation of the muscle-tendon unit and the muscle-microvascular unit. The unique vertical 3D cryo-bioprinting technique presented here suggests improvements in robustness and versatility to engineer certain tissue types especially those anisotropic in nature, and may extend broad utilities in tissue engineering, regenerative medicine, drug discovery, and personalized therapeutics.

Fabrication of bioinspired grid-crimp micropatterns by melt electrospinning writing for bone–ligament interface study

Many strategies have been adopted to engineer bone–ligament interface, which is of great value to both the tissue regeneration and the mechanism understanding underlying interface regeneration. However, how to recapitulate the complexity and heterogeneity of the native bone–ligament interface including the structural, cellular and mechanical gradients is still challenging. In this work, a bioinspired grid-crimp micropattern fabricated by melt electrospinning writing (MEW) was proposed to mimic the native structure of bone–ligament interface. The printing strategy of crimped fiber micropattern was developed and the processing parameters were optimized, which were used to mimic the crimp structure of the collagen fibrils in ligament. The guidance effect of the crimp angle and fiber spacing on the orientation of fibroblasts was studied, and both of them showed different levels of cell alignment effect. MEW grid micropatterns with different fiber spacings were fabricated as bone region. Both the alkaline phosphatase activity and calcium mineralization results demonstrated the higher osteoinductive ability of the MEW grid structures, especially for that with smaller fiber spacing. The combined grid-crimp micropatterns were applied for the co-culture of fibroblasts and osteoblasts. The results showed that more cells were observed to migrate into the in-between interface region for the pattern with smaller fiber spacing, suggested the faster migration speed of cells. Finally, a cylindrical triphasic scaffold was successfully generated by rolling the grid-crimp micropatterns up, showing both structural and mechanical similarity to the native bone–ligament interface. In summary, the proposed strategy is reliable to fabricate grid-crimp triphasic micropatterns with controllable structural parameters to mimic the native bone-to-ligament structure, and the generated 3D scaffold shows great potential for the further bone–ligament interface tissue engineering.

Nanomaterial‐Based Prosthetic Limbs for Disability Mobility Assistance: A Review of Recent Advances

The emergence of new hybrid nanomaterial has enabled prosthetic devices to have more performance and significantly improved the quality of life of the disabled. Due to the biosensing properties of prosthetic limbs made of nanomaterials, a large number of nanocomposites have been designed, developed, and evaluated for various prosthetic limbs, such as e‐skin, e‐skin’s neurotactility sensing, human prosthetic interface tissue engineering, bones, and biosensors. Nano‐based materials are also considered to be the new generation of scientific and technological materials for the preparation of various prosthetic devices for the disabled, which can effectively improve the sense of use of the disabled and achieve functional diversity. The study described various nanomaterials for prosthetic devices, and introduced some basic components of nanocomposites; their applications are in three areas, such as bone, skin, and nerve, and evaluated and summarized the advantages of these applications. The results show that (1) carbon‐based nanomaterials as neural materials have been studied most deeply. Due to that strong stability of the carbon‐based material and the simple transmission mechanism, the cost can be controlled in manufacturing the artificial limb. Materials with human‐computer interaction function are the research focus in the future. (2) Skin nanomaterials are mainly composite materials, generally containing metal‐ and carbon‐based materials. Ionic gels, ionic liquids, hydrogels, and elastomers have become the focus of attention due to the sensitivity, multimodal, and memory properties of their materials. (3) Outstanding nanomaterials for bone are fibrous materials, metallic synthetic materials, and composite materials, with extremely high hardness, weight, and toughness. Of the skeletal materials, the choice of prosthetic socket material is the most important and is typically based on fiber laminate composites. Some of these materials make sensors for durability and performance that can be used for large‐scale clinical testing.

Biomechanical Aspects of Osteochondral Regeneration: Implications and Strategies for Three-Dimensional Bioprinting.

Osteoarthritis is among the most prevalent of musculoskeletal disorders in the world that causes joint pain, deformity, and limited range of movement. The resulting osteochondral defect can significantly decrease the patient's quality of life, but current treatment options have not demonstrated the capacity to fully regenerate the entire osteochondral microenvironment. Structurally, the osteochondral unit is a composite system composed of three layers-articular cartilage, calcified cartilage, and subchondral bone. Collectively these distinct layers contribute to the distinct biomechanical properties that maintain the health and aid in load transfer during joint articulation. The purpose of this review was to examine the role of the osteochondral interface in tissue engineering. Topics of discussion include the biomechanics of the osteochondral unit and an overview of various strategies for osteochondral interface tissue engineering, with a specific focus on three-dimensional bioprinting. The goal of this review was to elucidate the importance of the osteochondral interface and overview some strategies of developing an interface layer within tissue engineered scaffolds. Impact Statement This review provides an overview of interface tissue engineering for osteochondral regeneration. It offers a detailed investigation into the biomechanics of the osteochondral unit as it relates to tissue engineering, and highlights the strategies that have been utilized to develop the osteochondral interface within tissue engineering scaffolds.

Research progress of interfacial tissue engineering in rotator cuff repair

To summarize the research progress of interfacial tissue engineering in rotator cuff repair. The recent literature at home and abroad concerning interfacial tissue engineering in rotator cuff repair was analysed and summarized. Interfacial tissue engineering is to reconstruct complex and hierarchical interfacial tissues through a variety of methods to repair or regenerate damaged joints of different tissues. Interfacial tissue engineering in rotator cuff repair mainly includes seed cells, growth factors, biomaterials, oxygen concentration, and mechanical stimulation. The best strategy for rotator cuff healing and regeneration requires not only the use of biomaterials with gradient changes, but also the combination of seed cells, growth factors, and specific culture conditions (such as oxygen concentration and mechanical stimulation). However, the clinical transformation of the relevant treatment is still a very slow process.