Growing Pains: The Need for Engineered Platforms to Study Growth Plate Biology.

Growth plates, or physis, are highly specialized cartilage tissues responsible for longitudinal bone growth in children and adolescents. Chondrocytes that reside in growth plates are organized into three distinct zones essential for proper function. Modeling key features of growth plates may provide an avenue to develop advanced tissue engineering strategies and perspectives for cartilage and bone regenerative medicine applications and a platform to study processes linked to disease progression. In this review, a brief introduction of the growth plates and their role in skeletal development is first provided. Injuries and diseases of the growth plates as well as physiological and pathological mechanisms associated with remodeling and disease progression are discussed. Growth plate biology, namely, its architecture and extracellular matrix organization, resident cell types, and growth factor signaling are then focused. Next, opportunities and challenges for developing 3D biomaterial models to study aspects of growth plate biology and disease in vitro are discussed. Finally, opportunities for increasingly sophisticated in vitro biomaterial models of the growth plate to study spatiotemporal aspects of growth plate remodeling, to investigate multicellular signaling underlying growth plate biology, and to develop platforms that address key roadblocks to in vivo musculoskeletal tissue engineering applications are described.

Stiffness of Nanoparticulate Mineralized Collagen Scaffolds Triggers Osteogenesis via Mechanotransduction and Canonical Wnt Signaling.

The ability of the extracellular matrix (ECM) to instruct progenitor cell differentiation has generated excitement for the development of materials-based regenerative solutions. Described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material capable of inducing in vivo skull regeneration without exogenous growth factors or ex vivo progenitor cell-priming is described previously. Here, the contribution of titrating stiffness to osteogenicity is evaluated by comparing noncrosslinked (NX-MC) and crosslinked (MC) forms of MC-GAG. While both materials are osteogenic, MC demonstrates an increased expression of osteogenic markers and mineralization compared to NX-MC. Both materials are capable of autogenously activating the canonical BMPR signaling pathway with phosphorylation of Smad1/5. However, unlike NX-MC, human mesenchymal stem cells cultured on MC demonstrate significant elevations in the major mechanotransduction mediators YAP and TAZ expression, coincident with ß-catenin activation in the canonical Wnt signaling pathway. Inhibition of YAP/TAZ activation reduces osteogenic expression, mineralization, and ß-catenin activation in MC, with less of an effect on NX-MC. YAP/TAZ inhibition also results in a reciprocal increase in Smad1/5 phosphorylation and BMP2 expression. The results indicate that increasing MC-GAG stiffness induces osteogenic differentiation via the mechanotransduction mediators YAP/TAZ and the canonical Wnt signaling pathway, whereas the canonical BMPR signaling pathway is activated independent of stiffness.

Heterotypic tumor models through freeform printing into photostabilized granular microgels.

The tissue microenvironment contains a complex assortment of multiple cell types, matrices, and vessel structures, which is difficult to reconstruct in vitro. Here, we demonstrate model tumor microenvironments formed through direct writing of vasculature channels and tumor cell aggregates, within a cell-laden microgel matrix. Photocrosslinkable microgels provide control over local and global mechanics, while enabling the integration of virtually any cell type. Direct writing of a Pluronic sacrificial ink into a stromal cell-microgel suspension is used to form vessel structures for endothelialization, followed by printing of melanoma aggregates. Tumor cells migrate into the prototype vessels as a function of spatial location, thereby providing a measure of invasive potential. The integration of perfusable channels with multiple spatially defined cell types provides new avenues for modelling development and disease, with scope for both fundamental research and drug development efforts.

Sequential sequestrations increase the incorporation and retention of multiple growth factors in mineralized collagen scaffolds.

Trauma induced injuries of the mouth, jaw, face, and related structures present unique clinical challenges due to their large size and complex geometry. Growth factor signaling coordinates the behavior of multiple cell types following an injury, and effective coordination of growth factor availability within a biomaterial can be critical for accelerating bone healing. Mineralized collagen scaffolds are a class of degradable biomaterial whose biophysical and compositional parameters can be adjusted to facilitate cell invasion and tissue remodeling. Here we describe the use of modified simulated body fluid treatments to enable sequential sequestration of bone morphogenic protein 2 and vascular endothelial growth factor into mineralized collagen scaffolds for bone repair. We report the capability of these scaffolds to sequester 60-90% of growth factor from solution without additional crosslinking treatments and show high levels of retention for individual (>94%) and multiple growth factors (>88%) that can be layered into the material via sequential sequestration steps. Sequentially sequestering growth factors allows prolonged release of growth factors in vitro (>94%) and suggests the potential to improve healing of large-scale bone injury models in vivo. Future work will utilize this sequestration method to induce cellular activities critical to bone healing such as vessel formation and cell migration.

The inclusion of zinc into mineralized collagen scaffolds for craniofacial bone repair applications.

Implant osteoinduction and subsequent osteogenic activity are critical events that need improvement for regenerative healing of large craniofacial bone defects. Here we describe the augmentation of the mineral content of a class of mineralized collagen scaffolds under development for craniomaxillofacial bone regeneration via the inclusion of zinc ions to promote osteogenesis in vitro. Zinc is an essential trace element in skeletal tissue and bone, with soluble zinc being shown to promote osteogenic differentiation of porcine adipose derived stem cells. We report the development of a new class of zinc functionalized scaffolds fabricated by adding zinc sulfate to a mineralized collagen-glycosaminoglycan precursor suspension that was then freeze dried to form a porous biomaterial. We report analysis of zinc functionalized scaffolds via imaging (scanning electron microscopy), mechanical testing (compression), and compositional (X-ray diffraction, inductively coupled plasma mass spectrometry) analyses. Notably, zinc-functionalized scaffolds display morphological changes to the mineral phase and altered elastic modulus without substantially altering the composition of the brushite phase or removing the micro-scale pore morphology of the scaffold. These scaffolds also display zinc release kinetics on the order of days to weeks and promote successful growth and pro-osteogenic capacity of porcine adipose derived stem cells cultured within these zinc scaffolds. Taken together, we believe that zinc functionalized scaffolds provide a unique platform to explore strategies to improve in vivo osteogenesis in craniomaxillofacial bone injuries models. STATEMENT OF SIGNIFICANCE: Craniomaxillofacial bone defects that arise from traumatic, congenital, and post-oncologic origins cannot heal on their own and often require surgical intervention. We have developed a class of mineralized collagen scaffolds that promotes osteogenesis and bone regeneration. Here we describe the inclusion of zinc sulfate into the mineralized collagen scaffold to improve osteogenesis. Zinc functionalized scaffolds demonstrate altered crystallite microstructure but consistent Brushite chemistry, improved mechanics, and promote zinc transporter expression while supporting stem cell viability, osteogenic differentiation, and mineral biosynthesis.

Osteoprotegerin reduces osteoclast resorption activity without affecting osteogenesis on nanoparticulate mineralized collagen scaffolds.

The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration.

Incorporating ß-cyclodextrin into collagen scaffolds to sequester growth factors and modulate mesenchymal stem cell activity.

The development of biomaterials for a range of tissue engineering applications increasingly requires control over the bioavailability of biomolecular cues such as growth factors in order to promote desired cell responses. While efforts have predominantly concentrated on covalently-bound or freely-diffusible incorporation of biomolecules in porous, three-dimensional biomaterials, opportunities exist to exploit transient interactions to concentrate growth factor activity over desired time frames. Here, we report the incorporation of ß-cyclodextrin into a model collagen-GAG scaffold as a means to exploit the passive sequestration and release of growth factors via guest-host interactions to control mesenchymal stem cell differentiation. Collagen-GAG scaffolds that incorporate ß-cyclodextrin show improved sequestration as well as extended retention and release of TGF-ß1. We further show extended retention and release of TGF-ß1 and BMP-2 from ß-cyclodextrin modified scaffolds was sufficient to influence the metabolic activity and proliferation of mesenchymal stem cells as well as differential activation of Smad 2/3 and Smad 1/5/8 pathways associated with differential osteo-chondral differentiation. Further, gene expression analysis showed TGF-ß1 release from ß-cyclodextrin CG scaffolds promoted early chondrogenic-specific differentiation. Ultimately, this work establishes a novel method for the incorporation and display of growth factors within CG scaffolds via supramolecular interactions. Such a design framework offers opportunities to selectively alter the bioavailability of multiple biomolecules within a three-dimensional collagen-GAG scaffold to enhance cell activity for a range of musculoskeletal regenerative medicine applications. STATEMENT OF SIGNIFICANCE: We describe the incorporation of ß-cyclodextrin into a model CG-scaffold under development for musculoskeletal tissue engineering applications. We show ß-cyclodextrin modified scaffolds promote the sequestration of soluble TGF-ß1 and BMP-2 via guest-host interactions, leading to extended retention and release. Further, ß-cyclodextrin modified CG scaffolds promote TGF-ß1 or BMP-2 specific Smad signaling pathway activation associated with divergent osseous versus chondrogenic differentiation pathways in mesenchymal stem cells.