(L-470) Nanofibrillar Collagen Scaffolds with Tunable Biophysical Properties Drive Endothelial Inflammatory Phenotype, Myogenesis and Osteogenesis

Introduction:: Severe musculoskeletal trauma involves injury to multiple tissue components including vasculature, skeletal muscle, and bone, and is associated with poor healing outcomes including reduced musculoskeletal strength, mobility, range of motion, and potentially leads to lifelong disability. Current clinical standards to regenerate musculoskeletal tissues fail primarily due to unsuccessful host-material integration. The need to develop a biologically mimetic material to aid in the integration of biomaterials to musculoskeletal tissues gives rise to extracellular matrix (ECM) based therapeutics that aim to recapitulate the native musculoskeletal niche.



The regeneration of musculoskeletal tissues relies upon the coordinated interaction of cells with the surrounding ECM to drive differentiation and facilitate healing. The main objective of this study is to design a biomaterial scaffold to mimic the biophysical conditions of native musculoskeletal ECM for the regeneration of vasculature, skeletal muscle, and bone. However, the disparate biophysical conditions that are required to optimally regenerate each of the key components of musculoskeletal tissues present an engineering challenge that has limited the application of ECM-based therapeutics. To address this, we have fabricated collagen-based scaffolds with tunable biophysical properties to delineate the material characteristics that are the largest drivers of regeneration.



Previously, our lab has demonstrated a shear-based approach to modulate the topography of collagen scaffolds, producing highly aligned and random nanofibril topography. Here, we plan to use pH to further modulate the biophysical properties of collagen scaffolds. Together, this work will enable the generation of a single material scaffold that can optimally regenerate and repair vasculature, muscle, and bone.


Materials and Methods:: To mimic the range of biophysical properties of musculoskeletal ECM, high-density type I collagen was shear extruded into a buffer of varying pHs (5, 7, 10) to produce nano resolution collagen scaffolds (Fig.1A-C). The combination of shear rate and pH adjustment modulated the biophysical properties of the scaffolds including stiffness, anisotropy/topography (aligned to random fibers), and porosity (Fig.1A-C). To evaluate the ability of each type of scaffold with separate biophysical properties to facilitate vasculogenesis, myogenesis, and osteogenesis, representative cells of each tissue type were cultured on the scaffolds.



Specifically, to assess vasculogenesis, primary endothelial cells (ECs) were cultured on the collagen scaffolds and stained for an inflammatory marker (VCAM-1) and a junctional marker (VE-Cadherin) to assess EC phenotype (Fig.1D-F). EC inflammation was assessed by quantifying VCAM-1 fluorescence, overall cell circularity, and EC membrane thickness.



To evaluate myogenesis, C2C12 skeletal muscle myoblasts were seeded on the scaffolds and differentiated into myotubes for 5 days in low serum media. The myotubes were then stained for myosin heavy chain (MHC), a marker of mature myotubes; the length, anisotropy, orientation, and nuclei count of the resulting myotubes were quantified (Fig.1G-I).



To assess the potential for the scaffolds to drive osteogenesis, MC3T3 pre-osteoblasts were seeded on the scaffolds and differentiated into osteoblasts for 21 days. The osteoblasts were evaluated for an early osteogenic marker at day 7 in alkaline phosphatase (ALP) activity and further evaluated for a late osteogenic marker at day 21 in bone mineralization using Alizarin red staining (Fig.1J&K).


Results, Conclusions, and Discussions:: As shown, pH adjustment (pH 5, 7, 10) modulated the stiffness, porosity, fiber diameter, and anisotropy in the collagen scaffolds (Fig.1A). As pH increases, the stiffness (storage modulus) of the collagen scaffolds significantly increased (p< 0.05) from the pH5 scaffolds (615 ± 283 Pa) to the pH10 scaffolds (1020 ± 238 Pa) (Fig.1B). Contrastingly, an increase in pH demonstrated a decrease in scaffold porosity and anisotropy, and fiber diameter in the collagen scaffolds (Fig.1A). Using our shear-based extrusion method, we are able to create topographically aligned and random nanofibrillar collagen scaffolds of 30-40 nm in diameter (Fig.1C).



Topography played the largest role in determining endothelial cell inflammation. Endothelial cell inflammation was shown to be most improved in the pH7 Aligned scaffold (Fig.1D). The ECs on the aligned scaffolds displayed a more elongated shape whereas in the random scaffolds, the ECs adopted a more cobblestone-like morphology, suggesting a difference in EC phenotype (Fig.1F). Lower levels of the inflammatory marker, VCAM-1, were expressed by ECs on all aligned (highly anisotropic) scaffolds compared to all randomly-oriented scaffolds (p< 0.01) regardless of stiffness (Fig.1E).



In contrast, topography and stiffness both greatly influenced myoblast differentiation. Myotube formation was most efficient on scaffolds with aligned topography and stiffness (pH10 Aligned) (Fig.1G); with an average length of about 700 µm with 21 nuclei/myotube in the pH10 aligned scaffolds compared to an average length of about 30 µm with 6 nuclei/myotube in the pH5 random scaffolds (Fig.1G-I).



Lastly, osteoblasts showed equally improved osteogenesis in all collagen scaffolds compared to tissue culture plastic (TCP) controls regardless of biophysical properties. This was shown with significantly higher (p< 0.05) levels of osteogenic bone mineralization in all collagen scaffolds when compared to TCP (Fig.1J&K).



Taken together, this work demonstrates the importance and influence of biophysical properties on musculoskeletal cell guidance and establishes a framework for the development of single material musculoskeletal constructs for the regeneration of multi-tissue injuries.


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