Nathan H. Dormer

Continuous Gradients of Material Composition and Growth Factors for Effective Regeneration of the Osteochondral Interface

Most contemporary biomaterial designs for osteochondral regeneration utilize monolithic, biphasic, or even multiphasic constructs. We have introduced a microsphere-based approach to create a continuous gradient in both material composition and encapsulated growth factors. The gradients were fabricated by filling a cylindrical mold with opposing gradients of two different types of poly(D,L-lactic-co-glycolic acid) microspheres. The chondrogenic microspheres were loaded with transforming growth factor-β1, whereas the osteogenic microspheres contained bone morphogenetic protein-2 with or without nanophase hydroxyapatite. The gradient scaffolds (material gradient only, signal gradient only, or material/signal gradient combination) or blank control scaffolds were implanted in 3.5 mm-diameter defects in rabbit knees for 6 or 12 weeks. This is the first in vivo evaluation of these novel gradient scaffolds in the knee. The gross morphology, MRI, and histology indicated that the greatest extent of regeneration was achieved when both signal and material gradients were included together. This combination resulted in complete bone ingrowth, with an overlying cartilage layer with high glycosaminoglycan content, appropriate thickness, and integration with the surrounding cartilage and underlying bone. The results suggest that osteochondral regeneration may benefit from biomaterials that integrate a continuous gradient in both material composition and encapsulated growth factors.

Hierarchically Designed Agarose and Poly(Ethylene Glycol) Interpenetrating Network Hydrogels for Cartilage Tissue Engineering

A new method for encapsulating cells in interpenetrating network (IPN) hydrogels of superior mechanical integrity was developed. In this study, two biocompatible materials-agarose and poly(ethylene glycol) (PEG) diacrylate-were combined to create a new IPN hydrogel with greatly enhanced mechanical performance. Unconfined compression of hydrogel samples revealed that the IPN displayed a fourfold increase in shear modulus relative to a pure PEG-diacrylate network (39.9 vs. 9.9 kPa) and a 4.9-fold increase relative to a pure agarose network (8.2 kPa). PEG and IPN compressive failure strains were found to be 71% ± 17% and 74% ± 17%, respectively, while pure agarose gels failed around 15% strain. Similar mechanical property improvements were seen when IPNs-encapsulated chondrocytes, and LIVE/DEAD cell viability assays demonstrated that cells survived the IPN encapsulation process. The majority of IPN-encapsulated chondrocytes remained viable 1 week postencapsulation, and chondrocytes exhibited glycosaminoglycan synthesis comparable to that of agarose-encapsulated chondrocytes at 3 weeks postencapsulation. The introduction of a new method for encapsulating cells in a hydrogel with enhanced mechanical performance is a promising step toward cartilage defect repair. This method can be applied to fabricate a broad variety of cell-based IPNs by varying monomers and polymers in type and concentration and by adding functional groups such as degradable sequences or cell adhesion groups. Further, this technology may be applicable in other cell-based applications where mechanical integrity of cell-containing hydrogels is of great importance.

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,l-lactic-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.

Osteochondral interface regeneration of the rabbit knee with macroscopic gradients of bioactive signals

To date, most interfacial tissue engineering approaches have used stratified designs, in which there are two or more discrete layers comprising the interface. Continuously graded interfacial designs, where there is no discrete transition from one tissue type to another, are gaining attention as an alternative to stratified designs. Given that osteochondral regeneration holds the potential to enhance cartilage regeneration by leveraging the healing capacity of the underlying bone, we endeavored to introduce a continuously-graded approach to osteochondral regeneration. The purpose of this study was thus to evaluate the performance of a novel gradient-based scaffolding approach to regenerate osteochondral defects in the New Zealand White rabbit femoral condyle. Bioactive plugs were constructed from poly(D,L-lactic-co-glycolic acid) microspheres with a continuous gradient transition between cartilage-promoting and bone-promoting growth factors. At 6 and 12 weeks of healing, results suggested that the implants provided support for the neo-synthesized tissue, and the gradient in bioactive signaling may have been beneficial for bone and cartilage regeneration compared to the blank control implant, as evidenced by histology. In addition, the effects of preseeding gradient scaffolds with umbilical cord mesenchymal stromal cells (UCMSCs) from the Whartons jelly of New Zealand White rabbits were evaluated. Results indicated that there may be regenerative benefits to prelocalizing UCMSCs within scaffold interiors. The inclusion of bioactive factors in a gradient-based scaffolding design is a promising new treatment strategy for defect repair in the femoral condyle.

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 Rabbit Mandibular Condyle With Bioactive Signal Gradients

PURPOSE Tissue engineering solutions focused on the temporomandibular joint (TMJ) have expanded in number and variety during the past decade to address the treatment of TMJ disorders. The existing data on approaches for healing small defects in the TMJ condylar cartilage and subchondral bone, however, are sparse. The purpose of the present 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) microspheres with a gradient transition between cartilage-promoting and bone-promoting growth factors. RESULTS At 6 weeks of healing, the results suggested that the implants provided support for the neosynthesized tissue as evidenced by the histologic and 9.4 T magnetic resonance imaging findings. 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.

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 CaCO(3) or TiO(2) 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 CaCO(3)/TiO(2) 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.

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, while 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.

Monodispersed Microencapsulation Technology

Microencapsulation technologies that produce uniform microspheres or microcapsules have tremendous potential for the food and flavors industry because of the predictability, reproducibility, and quality control that are inherent to these technologies. Discrete control of microsphere size or microcapsule shell material or thickness provides opportunities to launch differentiated products in the future. In this chapter, we have highlighted prominent existing technologies that have provided or may provide ways to encapsulate flavors and nutraceuticals in monodisperse microparticulate vehicles. Techniques reviewed include microfluidics, electrohydrodynamic spraying, jet cutting process, rotary disc atomization, vibratory process, flow focusing, and vibratory processes combined with a carrier stream. These processes differ from each other in degree of complexity, ability to handle the precursor materials, capability of producing particles of certain size ranges, scalability, and application-specific cost effectiveness. We have also made an attempt to highlight the major advantages and drawbacks of these technologies with specific examples of their use in the food and flavors industry.

Evaluation of a transtympanic delivery system in Mus musculus for extended release steroids

Objective: The current investigation evaluated a novel extended release delivery system for treating inner ear diseases. The platform technology consists of a film forming agent (FFA) and microsphere component to localize and extend drug delivery within the ear. Study design: Studies evaluated dissolution kinetics of microspheres with multiple encapsulates, testing of a variety of FFAs, and ability to localize to the round window membrane in mice in vivo. Setting: Studies were completed at Orbis Biosciences and The University of Kansas Medical Center. Subjects: In conjunction with in vitro characterization, an infrared dye‐containing microsphere formulation was evaluated for round window membrane (RWM) localization and general tolerability in C57/BL6 Mus musculus for 35 days. Methods: In vitro characterization was performed using upright diffusion cells on cellulose acetate membranes, with drug content quantified by high performance liquid chromatography. Mus musculus dosing of infrared dye‐containing microspheres was performed under anesthesia with a 27 GA needle and 2.0 &mgr;L injection volume Results: In vitro dissolution demonstrates the ability of the FFA with microsphere platform to release steroids, proteins, peptides, and nucleic acids for at least one month, while necroscopy shows the ability of the FFA with dye‐loaded microspheres to remain localized to Mus musculus RWM for the same period of time, with favorable tolerability. Conclusions: Combining FFA and microsphere for localized drug delivery may enable cost‐effective, extended release local delivery to the inner ear of new and existing small molecules, proteins, peptides, and nucleic acids.