Emerging Techniques in Stratified Designs and Continuous Gradients for Tissue Engineering of Interfaces

Interfacial tissue engineering is an emerging branch of regenerative medicine, where engineers are faced with developing methods for the repair of one or many functional tissue systems simultaneously. Early and recent solutions for complex tissue formation have utilized stratified designs, where scaffold formulations are segregated into two or more layers, with discrete changes in physical or chemical properties, mimicking a corresponding number of interfacing tissue types. This method has brought forth promising results, along with a myriad of regenerative techniques. The latest designs, however, are employing “continuous gradients” in properties, where there is no discrete segregation between scaffold layers. This review compares the methods and applications of recent stratified approaches to emerging continuously graded methods

Osteochondral Interface Regeneration of the Rabbit Mandibular Condyle with Bioactive Signal Gradients

PURPOSE Tissue engineering solutions focused on the temporomandibular joint (TMJ) have expanded in number and variety over the past decade to address the treatment of TMJ disorders. The existing literature on approaches for healing small defects in the TMJ condylar cartilage and subchondral bone, however, is sparse. The purpose of this study was thus to evaluate the performance of a novel gradient-based scaffolding approach to regenerate osteochondral defects in the rabbit mandibular condyle. MATERIALS AND METHODS Miniature bioactive plugs for regeneration of small mandibular condylar defects in New Zealand White rabbits were fabricated. The plugs were constructed from poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres with a gradient transition between cartilage-promoting and bone-promoting growth factors. RESULTS At six weeks of healing, results suggested that the implants provided support for the neo-synthesized tissue as evidenced by histology and 9.4T magnetic resonance imaging. CONCLUSION The inclusion of bioactive factors in a gradient-based scaffolding design is a promising new treatment strategy for focal defect repair in the TMJ

Using chondroitin sulfate to improve the viability and biosynthesis of chondrocytes encapsulated in interpenetrating network (IPN) hydrogels of agarose and poly(ethylene glycol) diacrylate

We recently introduced agarose-poly(ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN) hydrogels to cartilage tissue engineering that were able to encapsulate viable cells and provide a significant improvement in mechanical performance relative to its two constituent hydrogels. The goal of the current study was to develop a novel synthesis protocol to incorporate methacrylated chondroitin sulfate (MCS) into the IPN design hypothesized to improve cell viability and biosynthesis. The IPN was formed by encapsulating porcine chondrocytes in agarose, soaking the construct in a solution of 1:10 MCS:PEGDA, which was then photopolymerized to form a copolymer network as the second network. The IPN with incorporated CS (CS-IPN) (~0.5 wt%) resulted in a 4- to 5-fold increase in the compressive elastic modulus relative to either the PEGDA or agarose gels. After 6 weeks of in vitro culture, more than 50% of the encapsulated chondrocytes remained viable within the CS-modified IPN, in contrast to 35% viability observed in the unmodified. At week 6, the CS-IPN had significantly higher normalized GAG contents (347 ± 34 µg/µg) than unmodified IPNs (158 ± 27 µg/µg, P < 0.05). Overall, the approach of incorporating biopolymers such as CS from native tissue may provide favorable micro-environment and beneficial signals to cells to enhance their overall performance in IPNs

Osteochondral Interface Tissue Engineering Using Macroscopic Gradients of Bioactive Signals

Continuous gradients exist at osteochondral interfaces, which may be engineered by applying spatially patterned gradients of biological cues. In the present study, a protein-loaded microsphere-based scaffold fabrication strategy was applied to achieve spatially and temporally controlled delivery of bioactive signals in three-dimensional (3D) tissue engineering scaffolds. Bone morphogenetic protein-2 and transforming growth factor-β1-loaded poly(d,llactic- co-glycolic acid) microspheres were utilized with a gradient scaffold fabrication technology to produce microsphere-based scaffolds containing opposing gradients of these signals. Constructs were then seeded with human bone marrow stromal cells (hBMSCs) or human umbilical cord mesenchymal stromal cells (hUCMSCs), and osteochondral tissue regeneration was assessed in gradient scaffolds and compared to multiple control groups. Following a 6-week cell culture, the gradient scaffolds produced regionalized extracellular matrix, and outperformed the blank control scaffolds in cell number, glycosaminoglycan production, collagen content, alkaline phosphatase activity, and in some instances, gene expression of major osteogenic and chondrogenic markers. These results suggest that engineered signal gradients may be beneficial for osteochondral tissue engineering

Effect of different sintering methods on bioactivity and release of proteins from PLGA microspheres

Macromolecule release from poly(d,l-lactide-co-glycolide) (PLGA) microspheres has been well-characterized, and is a popular approach for delivering bioactive signals from tissue-engineered scaffolds. However, the effect of some processing solvents, sterilization, and mineral incorporation (when used in concert) on long-term release and bioactivity has seldom been addressed. Understanding these effects is of significant importance for microsphere-based scaffolds, given that these scaffolds are becoming increasingly more popular, yet growth factor activity following sintering and/or sterilization is heretofore unknown. The current study evaluated the 6-week release of transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-2 from PLGA and PLGA/hydroxyapatite (HAp) microspheres following exposure to ethanol (EtOH), dense phase carbon dioxide (CO2), or ethylene oxide (EtO). EtO was chosen based on its common use in scaffold sterilization, whereas EtOH and CO2 were chosen given their importance in sintering microspheres together to create scaffolds. Release supernatants were then used in an accelerated cell stimulation study with human bone marrow stromal cells (hBMSCs) with monitoring of gene expression for major chondrogenic and osteogenic markers. Results indicated that in microspheres without HAp, EtOH exposure led to the greatest amount of delivery, whilst those treated with CO2 delivered the least growth factor. In contrast, formulations with HAp released almost half as much protein, regardless of EtOH or CO2 exposure. Notably, EtO exposure was not found to significantly affect the amount of protein released. Cell stimulation studies demonstrated that eluted protein samples performed similarly to positive controls in PLGA-only formulations, and ambiguously in PLGA/HAp composites. In conclusion, the use of EtOH, subcritical CO2, and EtO in microsphere-based scaffolds may have only slight adverse effects, and possibly even desirable effects in some cases, on protein availability and bioactivity

Three-dimensional Macroscopic Scaffolds With a Gradient in Stiffness for Functional Regeneration of Interfacial Tissues

A novel approach has been demonstrated to construct biocompatible, macroporous 3-D tissue engineering scaffolds containing a continuous macroscopic gradient in composition that yields a stiffness gradient along the axis of the scaffold. Polymeric microspheres, made of poly(d,l-lactic-co-glycolic acid) (PLGA), and composite microspheres encapsulating a higher stiffness nano-phase material (PLGA encapsulating CaCO3 or TiO2 nanoparticles) were used for the construction of microsphere-based scaffolds. Using controlled infusion of polymeric and composite microspheres, gradient scaffolds displaying an anisotropic macroscopic distribution of CaCO3/TiO2 were fabricated via an ethanol sintering technique. The controllable mechanical characteristics and biocompatible nature of these scaffolds warrants further investigation for interfacial tissue engineering applications

Osteochondral Interface Tissue Engineering using Macroscopic Gradients of Physicochemical Signals

The field of tissue engineering has continually been described as "cells, signals, and scaffolds." The current thesis work describes the evaluation of a continuously-graded microsphere-based scaffold technology for the regeneration of the osteochondral interface where cartilage and bone have been damaged due to degenerative conditions, such as osteoarthritis. This scaffold technology used spatiotemporal release of bioactive factors from a biodegradable polymer for simultaneous differentiation of stem cells into bone and cartilage. The work in the current thesis evaluated the scaffold formulation in vitro, in vivo, and addressed the feasibility of adding osteoconductive materials to the first generation design for enhanced bone regeneration. Lastly, bioactive signal delivery and bioactivity was investigated as a consequence of preparatory methods used to construct the bioactive scaffolds. Results from the body of in vitro studies included stimulation of gene expression, increased biochemical production, and tissue synthesis. Specifically, gradient constructs outperformed control constructs in glycosaminoglycan content, produced twice as much collagen, and were capable of facilitating regional tissue synthesis. In vivo studies demonstrated the feasibility and potential efficacy of bioactive factor inclusion at two different osteochondral interfaces. In the New Zealand White rabbit knee, implants with embedded signals gradients were capable of regenerating more bone tissue than negative controls. When used in a smaller defect site, such as the New Zealand White rabbit mandibular condyle, the bioactive scaffolds were beneficial in regenerating thicker layers of cartilage. Moreover, this thesis has bridged the gradient-based microsphere scaffold design from concept to practice, in addition to opening new areas of investigation for further refinement of the technology

Deficiencies in Traditional Oral Dosage Forms and the Emergence of Controlled-Release Powder Manufacturing

The importance of providing safe and effective delayed- and extended-release oral formulations that can replace products requiring multiple administrations has been continually cited as an area in need of improvement for pharmaceutical companies. Such controlled release challenges become especially critical when they must be adapted for paediatrics, those suffering from dysphagia, or patients with specific dosage administration limitations. More often than not, lack of palatability and taste-masking compound this formulation challenge. Many particulate approaches show promise, but can be fraught with broad particle size distributions, initial drug burst, poor drug entrapment efficiency, low drug loading, and limited scalability. Here, we summarize the key factors that drive formulation development of format-flexible controlled-release oral powders, and the manufacturing aspects involved with some of the foremost marketed products, including next-generation single-step layered powder manufacturing (below)