RESUMEN
The tendon-to-bone junction is a highly specialized tissue which dissipates stress concentrations between mechanically dissimilar tendon and bone. Upon injury, the local heterogeneities across this insertion are not regenerated, leading to poor functional outcomes such as formation of scar tissue at the insertion and re-failure rates exceeding 90%. Although current tissue engineering methods are moving towards the development of spatially-graded biomaterials to begin to address these injuries, significant opportunities remain to engineer the often complex local mechanical behavior of such biomaterials to enhance their bioactivity. Here, we describe the use of three-dimensional printing techniques to create customizable arrays of poly-lactic acid (PLA) fibers that can be incorporated into a collagen scaffold under development for tendon bone junction repair. Notably, we use additive manufacturing concepts to generate arrays of spatially-graded fibers from biodegradable PLA that are incorporated into collagen scaffolds to create a collagen-PLA composite. We demonstrate the ability to tune the mechanical performance of the fiber-scaffold composite at the bulk scale. We also demonstrate the incorporation of spatially-heterogeneous fiber designs to establish non-uniform local mechanical performance of the composite biomaterial under tensile load, a critical element in the design of multi-compartment biomaterials for tendon-to-bone regeneration applications. Together, this work highlights the capacity to use multi-scale composite biomaterials to control local and bulk mechanical properties, and provides key insights into design elements under consideration for mechanically competent, multi-tissue regeneration platforms.
Asunto(s)
Materiales Biocompatibles/química , Colágeno/fisiología , Poliésteres/química , Andamios del Tejido , Regeneración Ósea , Huesos/fisiología , Humanos , Ensayo de Materiales , Impresión Tridimensional , Tendones/fisiología , Resistencia a la Tracción , Ingeniería de TejidosRESUMEN
The tendon-bone junction is a unique, mechanically dynamic, structurally graded anatomical zone, which transmits tensile loads between tendon and bone. Current surgical repair techniques rely on mechanical fixation and can result in high re-failure rates. A new class of collagen biomaterial that contains discrete mineralized and structurally aligned regions linked by a continuous interface to mimic the graded osteotendinous insertion has been recently described. Here the combined influence of graded biomaterial environment and increasing levels of applied strain (0%-20%) on mesenchymal stem cell (MSC) orientation and alignment have been reported. In osteotendinous scaffolds, which contain opposing gradients of mineral content and structural alignment characteristic of the native osteotendinous interface, MSC nuclear, and actin alignment is initially dictated by the local pore architecture, while applied tensile strain enhances cell alignment in the direction of strain. Comparatively, in layered scaffolds that do not contain any structural alignment cues, MSCs are randomly oriented in the unstrained condition, then become oriented in a direction perpendicular to applied strain. These findings provide an initial understanding of how scaffold architecture can provide significant, potentially competitive, feedback influencing MSC orientation under applied strain, and form the basis for future tissue engineering efforts to regenerate the osteotendinous enthesis.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Núcleo Celular/metabolismo , Colágeno/química , Células Madre Mesenquimatosas/metabolismo , Nicho de Células Madre , Estrés Mecánico , Andamios del Tejido/química , Humanos , Células Madre Mesenquimatosas/clasificación , Ingeniería de Tejidos/métodosRESUMEN
Tendon is a highly aligned connective tissue which transmits force from muscle to bone. Each year, people in the US sustain more than 32 million tendon injuries. To mitigate poor functional outcomes due to scar formation, current surgical techniques rely heavily on autografts. Biomaterial platforms and tissue engineering methods offer an alternative approach to address these injuries. Scaffolds incorporating aligned structural features can promote expansion of adult tenocytes and mesenchymal stem cells capable of tenogenic differentiation. However, appropriate balance between scaffold bioactivity and mechanical strength of these constructs remains challenging. The high porosity required to facilitate cell infiltration, nutrient and oxygen biotransport within three-dimensional constructs typically results in insufficient biomechanical strength. Here we describe the use of three-dimensional printing techniques to create customizable arrays of acrylonitrile butadiene styrene (ABS) fibers that can be incorporated into a collagen scaffold under development for tendon repair. Notably, mechanical performance of scaffold-fiber composites (elastic modulus, peak stress, strain at peak stress, and toughness) can be selectively manipulated by varying fiber-reinforcement geometry without affecting the native bioactivity of the collagen scaffold. Further, we report an approach to functionalize ABS fibers with activity-inducing growth factors via sequential oxygen plasma and carbodiimide crosslinking treatments. Together, we report an adaptable approach to control both mechanical strength and presence of biomolecular cues in a manner orthogonal to the architecture of the collagen scaffold itself. STATEMENT OF SIGNIFICANCE: Tendon injuries account for more than 32 million injuries each year in the US alone. Current techniques use allografts to mitigate poor functional outcomes, but are not ideal platforms to induce functional regeneration following injury. Tissue engineering approaches using biomaterial substrates have significant potential for addressing these defects. However, the high porosity required to facilitate cell infiltration and nutrient transport often dictates that the resultant biomaterials has insufficient biomechanical strength. Here we describe the use of three-dimensional printing techniques to generate customizable fiber arrays from ABS polymer that can be incorporated into a collagen scaffold under development for tendon repair applications. Notably, the mechanical performance of the fiber-scaffold composite can be defined by the fiber array independent of the bioactivity of the collagen scaffold design. Further, the fiber array provides a substrate for growth factor delivery to aid healing.
Asunto(s)
Colágeno/farmacología , Polímeros/química , Impresión Tridimensional , Andamios del Tejido/química , Acrilonitrilo/química , Tejido Adiposo/citología , Animales , Bovinos , Recuento de Células , Glicosaminoglicanos/farmacología , Proteínas Inmovilizadas/farmacología , Ensayo de Materiales , Factor de Crecimiento Derivado de Plaquetas/farmacología , Albúmina Sérica Bovina/metabolismo , Tiburones , Células Madre/citología , Células Madre/efectos de los fármacos , Sus scrofaRESUMEN
Biomaterial designs are increasingly incorporating multiple instructive signals to induce a desired cell response. However, many approaches do not allow orthogonal manipulation of immobilized growth factor signals and matrix stiffness. Further, few methods support patterning of biomolecular signals across a biomaterial in a spatially-selective manner. Here, we report a sequential approach employing carbodiimide crosslinking and benzophenone photoimmobilization chemistries to orthogonally modify the stiffness and immobilized growth factor content of a model collagen-GAG (CG) biomaterial. We subsequently examined the singular and combined effects of bone morphogenetic protein (BMP-2), platelet derived growth factor (PDGF-BB), and CG membrane stiffness on the bioactivity and osteogenic/adipogenic lineage-specific gene expression of adipose derived stem cells, an increasingly popular cell source for regenerative medicine studies. We found that the stiffest substrates direct osteogenic lineage commitment of ASCs regardless of the presence or absence of growth factors, while softer substrates require biochemical cues to direct cell fate. We subsequently describe the use of this approach to create overlapping patterns of growth factors across a single substrate. These results highlight the need for versatile approaches to selectively manipulate the biomaterial microenvironment to identify synergies between biochemical and mechanical cues for a range of regenerative medicine applications.
Asunto(s)
Adipocitos/citología , Diferenciación Celular , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/química , Proteína Morfogenética Ósea 2/química , Proliferación Celular , Células Cultivadas , Colágeno/química , Glicosaminoglicanos/química , Factor de Crecimiento Derivado de Plaquetas/química , Porcinos , Andamios del Tejido/químicaRESUMEN
Orthopedic tissue engineering requires biomaterials with robust mechanics as well as adequate porosity and permeability to support cell motility, proliferation, and new extracellular matrix (ECM) synthesis. While collagen-glycosaminoglycan (CG) scaffolds have been developed for a range of tissue engineering applications, they exhibit poor mechanical properties. Building on previous work in our lab that described composite CG biomaterials containing a porous scaffold core and nonporous CG membrane shell inspired by mechanically efficient core-shell composites in nature, this study explores an approach to improve cellular infiltration and metabolic health within these core-shell composites. We use indentation analyses to demonstrate that CG membranes, while less permeable than porous CG scaffolds, show similar permeability to dense materials such as small intestine submucosa (SIS). We also describe a simple method to fabricate CG membranes with organized arrays of microscale perforations. We demonstrate that perforated membranes support improved tenocyte migration into CG scaffolds, and that migration is enhanced by platelet-derived growth factor BB-mediated chemotaxis. CG core-shell composites fabricated with perforated membranes display scaffold-membrane integration with significantly improved tensile properties compared to scaffolds without membrane shells. Finally, we show that perforated membrane-scaffold composites support sustained tenocyte metabolic activity as well as improved cell infiltration and reduced expression of hypoxia-inducible factor 1α compared to composites with nonperforated membranes. These results will guide the design of improved biomaterials for tendon repair that are mechanically competent while also supporting infiltration of exogenous cells and other extrinsic mediators of wound healing.