Degradation properties of a biodegradable shape memory elastomer, poly(glycerol dodecanoate), for soft tissue repair.

Development of biodegradable shape memory elastomers (SMEs) is driven by the growing need for materials to address soft tissue pathology using a minimally invasive surgical approach. Composition, chain length and crosslinking of biocompatible polymers like PCL and PLA have been investigated to control mechanical properties, shape recovery and degradation rates. Depending on the primary mechanism of degradation, many of these polymers become considerably stiffer or softer resulting in mechanical properties that are inappropriate to support the regeneration of surrounding soft tissues. Additionally, concerns regarding degradation byproducts or residual organic solvents during synthesis accelerated interest in development of materials from bioavailable monomers. We previously developed a biodegradable SME, poly(glycerol dodecanoate) (PGD), using biologically relevant metabolites and controlled synthesis conditions to tune mechanical properties for soft tissue repair. In this study, we investigate the influence of crosslinking density on the mechanical and thermal properties of PGD during in vitro and in vivo degradation. Results suggest polymer degradation in vivo is predominantly driven by surface erosion, with no significant effects of initial crosslinking density on degradation time under the conditions investigated. Importantly, mechanical integrity is maintained during degradation. Additionally, shifts in melt transitions on thermograms indicate a potential shift in shape memory transition temperatures as the polymers degrade. These findings support the use of PGD for soft tissue repair and warrant further investigation towards tuning the molecular and macromolecular properties of the polymer to tailor degradation rates for specific clinical applications.

Mathematical modelling of glycosaminoglycan production by stem cell aggregates incorporated with growth factor-releasing polymer microspheres.

Systems composed of high density cells incorporated with growth factor-releasing polymer microspheres have recently been shown to promote chondrogenic differentiation and cartilage formation. Within these systems, the effects of spatial and temporal patterning of growth factor release on hyaline cartilage-specific extracellular matrix production have been examined. However, at present, it is unclear which microsphere densities and growth factor delivery profiles are optimal for inducing human mesenchymal stem cell differentiation and glycosaminoglycan production. A mathematical model to describe glycosaminoglycan production as a function of initial microsphere loading and microsphere degradation rate over a period of 3 weeks is presented. Based on predictions generated by this model, it may be feasible to design a bioactive microsphere system with specific spatiotemporal growth factor presentation characteristics to promote glycosaminoglycan production at controllable rates. Copyright © 2014 John Wiley & Sons, Ltd.

Tailoring the physicochemical and shape memory properties of the biodegradable polymer poly(glycerol dodecanoate) via curing conditions.

A major challenge in the repair and regeneration of soft tissue damage occurring as a result of aging, injury, or disease is recapitulating the biomechanical properties of the native tissue. Ideally, a candidate biomaterial for soft tissue engineering applications should be biocompatible, nonlinearly elastic to match soft tissue mechanical behavior, biodegradable to enable tissue remodeling, and tailorable to achieve a range of nonlinear elastic mechanical properties to match specific soft tissues. In addition, for cardiac and other applications, the biomaterial should have shape memory characteristics to facilitate minimally invasive and/or catheter-based delivery. Poly(glycerol dodecanoate) (PGD) is a shape memory material that has nonlinear elastic properties at body temperature and elastic-plastic behavior at room temperature. In this study, we investigated the effects of curing conditions on the nonlinear elastic, shape memory, and biocompatibility properties of PGD. Increased curing and crosslinking resulted in an increase in both the initial stiffness and the nonlinear strain stiffening behavior of PGD. After shape fixation at 60% strain, 100% shape recovery was achieved within 1 min at body temperature for all conditions tested. Polymer curing had no adverse effects on the cellular biocompatibility or non-hemolytic characteristics of PGD, indicating the potential suitability of these formulations for blood-contacting device applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1618-1623, 2017.

Cellular Self-Assembly with Microsphere Incorporation for Growth Factor Delivery Within Engineered Vascular Tissue Rings.

Cellular self-assembly has been used to generate living tissue constructs as an alternative to seeding cells on or within exogenous scaffold materials. However, high cell and extracellular matrix density in self-assembled constructs may impede diffusion of growth factors during engineered tissue culture. In the present study, we assessed the feasibility of incorporating gelatin microspheres within vascular tissue rings during cellular self-assembly to achieve growth factor delivery. To assess microsphere incorporation and distribution within vascular tissue rings, gelatin microspheres were mixed with a suspension of human smooth muscle cells (SMCs) at 0, 0.2, or 0.6 mg per million cells and seeded into agarose wells to form self-assembled cell rings. Microspheres were distributed throughout the rings and were mostly degraded within 14 days in culture. Rings with microspheres were cultured in both SMC growth medium and differentiation medium, with no adverse effects on ring structure or mechanical properties. Incorporated gelatin microspheres loaded with transforming growth factor beta 1 stimulated smooth muscle contractile protein expression in tissue rings. These findings demonstrate that microsphere incorporation can be used as a delivery vehicle for growth factors within self-assembled vascular tissues.

Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification.

Bone tissue engineering via endochondral ossification has been explored by chondrogenically priming cells using soluble mediators for at least 3 weeks to produce a hypertrophic cartilage template. Although recapitulation of endochondral ossification has been achieved, long-term in vitro culture is required for priming cells through repeated supplementation of inductive factors in the media. To address this challenge, a microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Sequential exogenous presentation of soluble transforming growth factor-ß1 (TGF-ß1) and bone morphogenetic protein-2 (BMP-2) at various defined time courses resulted in varying degrees of chondrogenesis and osteogenesis as demonstrated by glycosaminoglycan and calcium content. The time course that best induced endochondral ossification was used to guide the development of the microparticle-based controlled delivery system for TGF-ß1 and BMP-2. Gelatin microparticles capable of relatively rapid release of TGF-ß1 and mineral-coated hydroxyapatite microparticles permitting more sustained release of BMP-2 were then incorporated within hMSC aggregates and cultured for 5 weeks following the predetermined time course for sequential presentation of bioactive signals. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated TGF-ß1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity at week 2 and a greater degree of mineralization by week 5. Staining for types I and II collagen, osteopontin, and osteocalcin revealed the presence of cartilage and bone. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming. Significance: This study demonstrates the regulation of chondrogenesis and osteogenesis with regard to endochondral bone formation in high-density stem cell systems through the controlled presentation of inductive factors from incorporated microparticles. This work lays the foundation for a rapidly implantable tissue engineering system that promotes bone repair via endochondral ossification, a pathway that can delay the need for a functional vascular network and has an intrinsic ability to promote angiogenesis. The modular nature of this system lends well to using different cell types and/or growth factors to induce endochondral bone formation, as well as the production of other tissue types.

Spatially organized differentiation of mesenchymal stem cells within biphasic microparticle-incorporated high cell density osteochondral tissues.

Giving rise to both bone and cartilage during development, bone marrow-derived mesenchymal stem cells (hMSC) have the unique capacity to generate the complex tissues of the osteochondral interface. Utilizing a scaffold-free hMSC system, biphasic osteochondral constructs are incorporated with two types of growth factor-releasing microparticles to enable spatially organized differentiation. Gelatin microspheres (GM) releasing transforming growth factor-ß1 (TGF-ß1) combined with hMSC form the chondrogenic phase. The osteogenic phase contains hMSC only, mineral-coated hydroxyapatite microparticles (MCM), or MCM loaded with bone morphogenetic protein-2 (BMP-2), cultured in medium with or without BMP-2. After 4 weeks, TGF-ß1 release from GM within the cartilage phase promotes formation of a glycosaminoglycan- and type II collagen-rich matrix, and has a local inhibitory effect on osteogenesis. In the osteogenic phase, type X collagen and osteopontin are produced in all conditions. However, calcification occurs on the outer edges of the chondrogenic phase in some constructs cultured in media containing BMP-2, and alkaline phosphatase levels are elevated, indicating that BMP-2 releasing MCM provides better control over region-specific differentiation. The production of complex, stem cell-derived osteochondral tissues via incorporated microparticles could enable earlier implantation, potentially improving outcomes in the treatment of osteochondral defects.

Driving cartilage formation in high-density human adipose-derived stem cell aggregate and sheet constructs without exogenous growth factor delivery.

An attractive cell source for cartilage tissue engineering, human adipose-derived stem cells (hASCs) can be easily expanded and signaled to differentiate into chondrocytes. This study explores the influence of growth factor distribution and release kinetics on cartilage formation within 3D hASC constructs incorporated with transforming growth factor-ß1 (TGF-ß1)-loaded gelatin microspheres. The amounts of microspheres, TGF-ß1 concentration, and polymer degradation rate were varied within hASC aggregates. Microsphere and TGF-ß1 loading concentrations were identified that resulted in glycosaminoglycan (GAG) production comparable to those of control aggregates cultured in TGF-ß1-containing medium. Self-assembling hASC sheets were then engineered for the production of larger, more clinically relevant constructs. Chondrogenesis was observed in hASC-only sheets cultured with exogenous TGF-ß1 at 3 weeks. Importantly, sheets with incorporated TGF-ß1-loaded microspheres achieved GAG production similar to sheets treated with exogenous TGF-ß1. Cartilage formation was confirmed histologically via observation of cartilage-like morphology and GAG staining. This is the first demonstration of the self-assembly of hASCs into high-density cell sheets capable of forming cartilage in the presence of exogenous TGF-ß1 or with TGF-ß1-releasing microspheres. Microsphere incorporation may bypass the need for extended in vitro culture, potentially enabling hASC sheets to be implanted more rapidly into defects to regenerate cartilage in vivo.

Decellularized tissue and cell-derived extracellular matrices as scaffolds for orthopaedic tissue engineering.

The reconstruction of musculoskeletal defects is a constant challenge for orthopaedic surgeons. Musculoskeletal injuries such as fractures, chondral lesions, infections and tumor debulking can often lead to large tissue voids requiring reconstruction with tissue grafts. Autografts are currently the gold standard in orthopaedic tissue reconstruction; however, there is a limit to the amount of tissue that can be harvested before compromising the donor site. Tissue engineering strategies using allogeneic or xenogeneic decellularized bone, cartilage, skeletal muscle, tendon and ligament have emerged as promising potential alternative treatment. The extracellular matrix provides a natural scaffold for cell attachment, proliferation and differentiation. Decellularization of in vitro cell-derived matrices can also enable the generation of autologous constructs from tissue specific cells or progenitor cells. Although decellularized bone tissue is widely used clinically in orthopaedic applications, the exciting potential of decellularized cartilage, skeletal muscle, tendon and ligament cell-derived matrices has only recently begun to be explored for ultimate translation to the orthopaedic clinic.

High-density cell systems incorporating polymer microspheres as microenvironmental regulators in engineered cartilage tissues.

To address the significant clinical need for tissue-engineered therapies for the repair and regeneration of articular cartilage, many systems have recently been developed using bioactive polymer microspheres as regulators of the chondrogenic microenvironment within high-density cell cultures. In this review, we highlight various densely cellular systems utilizing polymer microspheres as three-dimensional (3D) structural elements within developing engineered cartilage tissue, carriers for cell expansion and delivery, vehicles for spatiotemporally controlled growth factor delivery, and directors of cell behavior via regulation of cell-biomaterial interactions. The diverse systems described herein represent a shift from the more traditional tissue engineering approach of combining cells and growth factors within a biomaterial scaffold, to the design of modular systems that rely on the assembly of cells and bioactive polymer microspheres as building blocks to guide the creation of articular cartilage. Cell-based assembly of 3D microsphere-incorporated structures represents a promising avenue for the future of tissue engineering.

Spatiotemporal regulation of chondrogenic differentiation with controlled delivery of transforming growth factor-ß1 from gelatin microspheres in mesenchymal stem cell aggregates.

The precise spatial and temporal presentation of growth factors is critical for cartilage development, during which tightly controlled patterns of signals direct cell behavior and differentiation. Recently, chondrogenic culture of human mesenchymal stem cells (hMSCs) has been improved through the addition of polymer microspheres capable of releasing growth factors directly to cells within cellular aggregates, eliminating the need for culture in transforming growth factor-ß1 (TGF-ß1)-containing medium. However, the influence of specific patterns of spatiotemporal growth factor presentation on chondrogenesis within microsphere-incorporated cell systems is unclear. In this study, we examined the effects of altering the chondrogenic microenvironment within hMSC aggregates through varying microsphere amount, growth factor concentration per microsphere, and polymer degradation time. Cartilage formation was evaluated in terms of DNA, glycosaminoglycan, and type II collagen in hMSCs from three donors. Chondrogenesis equivalent to or greater than that of aggregates cultured in medium containing TGF-ß1 was achieved in some conditions, with varied differentiation based on the specific conditions of microsphere incorporation. A more spatially distributed delivery of TGF-ß1 from a larger mass of fast-degrading microspheres improved differentiation by comparison with delivery from a smaller mass of microspheres with a higher TGF-ß1 concentration per microsphere, although the total amount of growth factor per aggregate was the same. Results also indicated that the rate and degree of chondrogenesis varied on a donor-to-donor basis. Overall, this study elucidates the effects of varied conditions of TGF-ß1-loaded microsphere incorporation on hMSC chondrogenesis, demonstrating that both spatiotemporal growth factor presentation and donor variability influence chondrogenic differentiation within microsphere-incorporated cellular constructs.

Engineered cartilage via self-assembled hMSC sheets with incorporated biodegradable gelatin microspheres releasing transforming growth factor-ß1.

Self-assembling cell sheets have shown great potential for use in cartilage tissue engineering applications, as they provide an advantageous environment for the chondrogenic induction of human mesenchymal stem cells (hMSCs). We have engineered a system of self-assembled, microsphere-incorporated hMSC sheets capable of forming cartilage in the presence of exogenous transforming growth factor ß1 (TGF-ß1) or with TGF-ß1 released from incorporated microspheres. Gelatin microspheres with two different degrees of crosslinking were used to enable different cell-mediated microsphere degradation rates. Biochemical assays, histological and immunohistochemical analyses, and biomechanical testing were performed to determine biochemical composition, structure, and equilibrium modulus in unconfined compression after 3 weeks of culture. The inclusion of microspheres with or without loaded TGF-ß1 significantly increased sheet thickness and compressive equilibrium modulus, and enabled more uniform matrix deposition by comparison to control sheets without microspheres. Sheets incorporated with fast-degrading microspheres containing TGF-ß1 produced significantly more GAG and GAG per DNA than all other groups tested and stained more intensely for type II collagen. These findings demonstrate improved cartilage formation in microsphere-incorporated cell sheets, and describe a tailorable system for the chondrogenic induction of hMSCs without necessitating culture in growth factor-containing medium.

Affinity-based growth factor delivery using biodegradable, photocrosslinked heparin-alginate hydrogels.

Photocrosslinkable biomaterials are promising for tissue engineering applications due to their capacity to be injected and form hydrogels in situ in a minimally invasive manner. Our group recently reported on the development of photocrosslinked alginate hydrogels with controlled biodegradation rates, mechanical properties, and cell adhesive properties. In this study, we present an affinity-based growth factor delivery system by incorporating heparin into photocrosslinkable alginate hydrogels (HP-ALG), which allows for controlled, prolonged release of therapeutic proteins. Heparin modification had minimal effect on the biodegradation profiles, swelling ratios, and elastic moduli of the hydrogels in media. The release profiles of growth factors from this affinity-based platform were sustained for 3weeks with no initial burst release, and the released growth factors retained their biological activity. Implantation of bone morphogenetic protein-2 (BMP-2)-loaded photocrosslinked alginate hydrogels induced moderate bone formation around the implant periphery. Importantly, BMP-2-loaded photocrosslinked HP-ALG hydrogels induced significantly more osteogenesis than BMP-2-loaded photocrosslinked unmodified alginate hydrogels, with 1.9-fold greater peripheral bone formation and 1.3-fold greater calcium content in the BMP-2-loaded photocrosslinked HP-ALG hydrogels compared to the BMP-2-loaded photocrosslinked unmodified alginate hydrogels after 8weeks implantation. This sustained and controllable growth factor delivery system, with independently controllable physical and cell adhesive properties, may provide a powerful modality for a variety of therapeutic applications.

Chondrogenic differentiation of human mesenchymal stem cell aggregates via controlled release of TGF-beta1 from incorporated polymer microspheres.

Aggregate culture is a useful method for inducing chondrogenic differentiation of human mesenchymal stem cells (hMSC) in a three-dimensional in vitro culture environment. Conventional aggregate culture, however, typically requires repeated growth factor supplementation during media changes, which is both expensive and time-intensive. In addition, homogenous cell differentiation is limited by the diffusion of chondrogenic growth factor from the culture medium into the aggregate and peripheral cell consumption of the growth factor. We have engineered a technology to incorporate growth factor-loaded polymer microspheres within hMSC aggregates themselves. Here, we report on the system's capacity to induce chondrogenesis via sustained delivery of transforming growth factor-beta1 (TGF-beta1). Cartilage formation after 3 weeks in the absence of externally supplied growth factor approached that of aggregates cultured by conventional methods. Chondrogenesis in the central region of the aggregates is enabled at TGF-beta1 levels much lower than those required by conventional culture using exogenously supplied TGF-beta1, which is likely a result of the system's ability to overcome limitations of growth factor diffusion from cell culture media surrounding the exterior of the aggregates. Importantly, the inclusion of growth factor-releasing polymer microspheres in hMSC aggregates could enable in vivo chondrogenesis for cartilage tissue engineering applications without extensive in vitro culture.