We have demonstrated a simple and versatile method for generating a continuously graded, bonelike calcium phosphate coating on a nonwoven mat of electrospun nanofibers. A linear gradient in calcium phosphate content could be achieved across the surface of the nanofiber mat. The gradient had functional consequences with regard to stiffness and biological activity. Specifically, the gradient in mineral content resulted in a gradient in the stiffness of the scaffold and further influenced the activity of mouse pre-osteoblast MC3T3 cells. This new class of nanofiber-based scaffolds can potentially be employed for repairing the tendon-to-bone insertion site via a tissue engineering approach.The attachment of dissimilar materials is a grand challenge because of the high levels of localized stress that develops at such an interface. 1 An effective biological solution to this problem can be found in the attachment of tendon (a compliant, structural "soft tissue") to bone (a stiff, structural "hard tissue"). 2 The natural tendon-to-bone attachment relies on a gradient in structure and composition that translates into a spatial variation of mechanical stiffness. 3 Recent evidence supports the idea that this unusual spatial variation eliminates high levels of stress at the interface, providing effective transfer of mechanical loads from tendon to bone. However, this unique transitional tissue between uninjured tendon and bone is not recreated during tendon-to-bone healing. 4 Surgical reattachment of these two dissimilar biological materials therefore often fails. For example, failure rates for rotator cuff repair (which requires tendon-to-bone healing) have been reported to be as high as 94%. 5 To address this clinical problem, it is critical to develop a new scaffold with a controllable gradation in mineral content along the surface. The gradation in mineral content can result in a spatial variation in the stiffness of the scaffold and thus be potentially used for repairing the tendon-to-bone insertion site via a tissue engineering approach.Nanofibers can be routinely prepared as nonwoven mats by electrospinning from a wide variety of biocompatible and biodegradable polymers (both natural and synthetic), as well as composites containing inorganic materials. 6 Owing to the small feature size, a nonwoven mat derived from electrospun nanofibers typically exhibits a high porosity and large surface area *e-mails: xia@biomed.wustl.edu (for scaffold fabrication and cell culture study), ThomopoulosS@wudosis.wustl.edu (for mechanical property characterization). Supporting Information Available: Detailed descriptions of experimental procedures and one additional figure are provided. These materials are available free of charge via the Internet at http://pubs.acs.org. and can thus mimic the hierarchical structure of extracellular matrix (ECM) critical to cell attachment and nutrient transport. 7 The fibers can also be conveniently functionalized by encapsulation or attachment of bioactive species such as ECM proteins, enzy...

We have demonstrated the fabrication of “aligned-to-random” electrospun nanofiber scaffolds that mimic the structural organization of collagen fibers at the tendon-to-bone insertion site. Tendon fibroblasts cultured on such a scaffold exhibited highly organized and haphazardly oriented morphologies, respectively, on the aligned and random portions.

Muscle forces are essential for skeletal patterning during development. Eliminating muscle forces, e.g., through paralysis, leads to bone and joint deformities. Botulinum toxin (BtxA)-induced paralysis of mouse rotator cuffs throughout postnatal development closely mimics neonatal brachial plexus palsy, a significant clinical condition in infants. In these mice, the tendon-to-bone attachment (i.e., the tendon enthesis) presents defects in mineral accumulation and fibrocartilage formation, presumably impairing the function of the tissue. The objective of the current study was to investigate the functional consequences of muscle unloading using BtxA on the developing supraspinatus tendon enthesis. We found that the maximum endurable load and stiffness of the supraspinatus tendon attachment decreased after four and eight weeks of post-natal BtxA-muscle unloading relative to controls. Tendon cross-sectional area was significantly reduced by BtxA-unloading, suggesting that the reduction of mechanical function resulted in part from geometric changes. However, strength, modulus, and toughness were also decreased in the BtxA-unloaded group compared to controls, indicating a decrease in tissue quality. Polarized-light microscopy and Raman microprobe analysis were used to determine collagen fiber alignment and mineral characteristics, respectively, in the tendon enthesis that might contribute to the reduced biomechanical performance in BtxA-unloaded shoulders. Collagen fiber alignment was significantly reduced in BtxA-unloaded shoulders. The mineral-to-matrix ratio in mineralized fibrocartilage was not affected by loading. However, the crystallographic atomic order of the hydroxylapatite phase (a measure of crystallinity) was reduced and the amount of carbonate (substituting for phosphate) in the hydroxylapatite crystals was increased. Taken together, these micrometer-scale structural and compositional changes partly explain the observed decreases in the mechanical functionality of the tendon enthesis in the absence of muscle loading.

The nanometre-scale structure of collagen and bioapatite within bone establishes bone's physical properties, including strength and toughness. However, the nanostructural organization within bone is not well known and is debated. Widely accepted models hypothesize that apatite mineral ('bioapatite') is present predominantly inside collagen fibrils: in 'gap channels' between abutting collagen molecules, and in 'intermolecular spaces' between adjacent collagen molecules. However, recent studies report evidence of substantial extrafibrillar bioapatite, challenging this hypothesis. We studied the nanostructure of bioapatite and collagen in mouse bones by scanning transmission electron microscopy (STEM) using electron energy loss spectroscopy and high-angle annular dark-field imaging. Additionally, we developed a steric model to estimate the packing density of bioapatite within gap channels. Our steric model and STEM results constrain the fraction of total bioapatite in bone that is distributed within fibrils at less than or equal to 0.42 inside gap channels and less than or equal to 0.28 inside intermolecular overlap regions. Therefore, a significant fraction of bone's bioapatite (greater than or equal to 0.3) must be external to the fibrils. Furthermore, we observe extrafibrillar bioapatite between non-mineralized collagen fibrils, suggesting that initial bioapatite nucleation and growth are not confined to the gap channels as hypothesized in some models. These results have important implications for the mechanics of partially mineralized and developing tissues.

Reattachment of tendon to bone has been a challenge in orthopedic surgery. The disparate mechanical properties of the two tissues make it difficult to achieve direct surgical repair of the tendon-to-bone insertion. Healing after surgical repair typically does not regenerate the natural attachment, a complex tissue that connects tendon and bone across a gradient in both mineral content and cell phenotypes. To facilitate the regeneration of the attachment, our groups have developed a nanofiber-based scaffold with a graded mineral coating to mimic the mineral composition of the native tendon-to-bone insertion. In the present work, we evaluated the ability of this scaffold to induce graded osteogenesis of adipose-derived mesenchymal stem cells (ASCs). Results from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferating cell nuclear antigen staining indicated that cell proliferation was negatively correlated with the mineral content. In contrast, alkaline phosphatase staining, an indicator of osteogenesis, was positively correlated with the mineral content. Likewise, runt-related transcription factor 2 (an early marker of osteoblast differentiation) and osteocalcin (a late marker of osteoblast differentiation) immunostaining were both positively correlated with the mineral content. These results indicate that a gradient in mineral content on the surface of a nanofiber scaffold is capable of inducing graded differentiation of ASCs into osteoblasts for enthesis repair.