Andrea G. Schwartz

Gold nanocages covered by smart polymers for controlled release with near-infrared light

Photosensitive caged compounds have enhanced our ability to address the complexity of biological systems by generating effectors with remarkable spatial/temporal resolutions1-3. The caging effect is typically removed by photolysis with ultraviolet light to liberate the bioactive species. Although this technique has been successfully applied to many biological problems, it suffers from a number of intrinsic drawbacks. For example, it requires dedicated efforts to design and synthesize a precursor compound to the effector. The ultraviolet light may cause damage to biological samples and is only suitable for in vitro studies because of its quick attenuation in tissue4. Here we address these issues by developing a platform based on the photothermal effect of gold nanocages. Gold nanocages represent a class of nanostructures with hollow interiors and porous walls5. They can have strong absorption (for the photothermal effect) in the near-infrared (NIR) while maintaining a compact size. When the surface of a gold nanocage is covered with a smart polymer, the pre-loaded effector can be released in a controllable fashion using a NIR laser. This system works well with various effectors without involving sophiscated syntheses, and is well-suited for in vivo studies due to the high transparency of soft tissue in NIR6.

Quantifying the cellular uptake of antibody-conjugated Au nanocages by two-photon microscopy and inductively coupled plasma mass spectrometry.

Gold nanocages with localized surface plasmon resonance peaks in the near-infrared region exhibited a broad two-photon photoluminescence band extending from 450 to 650 nm when excited by a Ti:sapphire laser at 800 nm. The bright luminescence makes it possible to explore the use of Au nanocages as a new class of optical imaging agents for two-photon microscopy. In this work, we have demonstrated the use of two-photon microscopy as a convenient tool to directly examine the uptake of antibody-conjugated and PEGylated Au nanocages by U87MGwtEGFR cells. We have also correlated the results from two-photon microscopy with the data obtained by inductively coupled plasma mass spectrometry. Combined together, these results indicate that the antibody-conjugated Au nanocages were attached to the surface of the cells through antibody-antigen binding and then internalized into the cells via receptor-mediated endocytosis. The cellular uptake process was dependent on a number of parameters, including incubation time, incubation temperature, size of the Au nanocages, and the number of antibodies immobilized on each nanocage.

Mineral Distributions at the Developing Tendon Enthesis

Tendon attaches to bone across a functionally graded interface, “the enthesis”. A gradient of mineral content is believed to play an important role for dissipation of stress concentrations at mature fibrocartilaginous interfaces. Surgical repair of injured tendon to bone often fails, suggesting that the enthesis does not regenerate in a healing setting. Understanding the development and the micro/nano-meter structure of this unique interface may provide novel insights for the improvement of repair strategies. This study monitored the development of transitional tissue at the murine supraspinatus tendon enthesis, which begins postnatally and is completed by postnatal day 28. The micrometer-scale distribution of mineral across the developing enthesis was studied by X-ray micro-computed tomography and Raman microprobe spectroscopy. Analyzed regions were identified and further studied by histomorphometry. The nanometer-scale distribution of mineral and collagen fibrils at the developing interface was studied using transmission electron microscopy (TEM). A zone (∼20 µm) exhibiting a gradient in mineral relative to collagen was detected at the leading edge of the hard-soft tissue interface as early as postnatal day 7. Nanocharacterization by TEM suggested that this mineral gradient arose from intrinsic surface roughness on the scale of tens of nanometers at the mineralized front. Microcomputed tomography measurements indicated increases in bone mineral density with time. Raman spectroscopy measurements revealed that the mineral-to-collagen ratio on the mineralized side of the interface was constant throughout postnatal development. An increase in the carbonate concentration of the apatite mineral phase over time suggested possible matrix remodeling during postnatal development. Comparison of Raman-based observations of localized mineral content with histomorphological features indicated that development of the graded mineralized interface is linked to endochondral bone formation near the tendon insertion. These conserved and time-varying aspects of interface composition may have important implications for the growth and mechanical stability of the tendon-to-bone attachment throughout development.

Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair.

Outcomes after tendon repair are often unsatisfactory, despite improvements in surgical techniques and rehabilitation methods. Recent studies aimed at enhancing repair have targeted the paucicellular nature of tendon for enhancing repair; however, most approaches for delivering growth factors and cells have not been designed for dense connective tissues such as tendon. Therefore, we developed a scaffold capable of delivering growth factors and cells in a surgically manageable form for tendon repair. Platelet-derived growth factor BB (PDGF-BB), along with adipose-derived mesenchymal stem cells (ASCs), were incorporated into a heparin/fibrin-based delivery system (HBDS). This hydrogel was then layered with an electrospun nanofiber poly(lactic-co-glycolic acid) (PLGA) backbone. The HBDS allowed for the concurrent delivery of PDGF-BB and ASCs in a controlled manner, while the PLGA backbone provided structural integrity for surgical handling and tendon implantation. In vitro studies verified that the cells remained viable, and that sustained growth factor release was achieved. In vivo studies in a large animal tendon model verified that the approach was clinically relevant, and that the cells remained viable in the tendon repair environment. Only a mild immunoresponse was seen at dissection, histologically, and at the mRNA level; fluorescently labeled ASCs and the scaffold were found at the repair site 9days post-operatively; and increased total DNA was observed in ASC-treated tendons. The novel layered scaffold has the potential for improving tendon healing due to its ability to deliver both cells and growth factors simultaneously in a surgically convenient manner.

Production of Ag nanocubes on a scale of 0.1 g per batch by protecting the NaHS-mediated polyol synthesis with argon.

Au nanocages synthesized from Ag nanocubes via the galvanic replacement reaction are finding widespread use in a range of applications because of their tunable optical properties. Most of these applications require the use of nanocages with a uniform size and in large quantities. This requirement translates into a demand for scaling up the production of Ag nanocubes with uniform, well-controlled sizes. Here we report such a method based on the modification of NaHS-mediated polyol synthesis with argon protection for fast reduction, which allows for the production of Ag nanocubes on a scale of 0.1 g per batch. The Ag nanocubes had an edge length tunable from 25 to 45 nm together with a size variation within +/-5 nm. The use of argon protection was the key to the success of this scale-up synthesis, suggesting the importance of controlling oxidative etching during synthesis.

Muscle loading is necessary for the formation of a functional tendon enthesis

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 not significantly reduced by BtxA-unloading, while, strength, modulus, and toughness were 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.

Enthesis fibrocartilage cells originate from a population of Hedgehog-responsive cells modulated by the loading environment

Tendon attaches to bone across a specialized tissue called the enthesis. This tissue modulates the transfer of muscle forces between two materials, i.e. tendon and bone, with vastly different mechanical properties. The enthesis for many tendons consists of a mineralized graded fibrocartilage that develops postnatally, concurrent with epiphyseal mineralization. Although it is well described that the mineralization and development of functional maturity requires muscle loading, the biological factors that modulate enthesis development are poorly understood. By genetically demarcating cells expressing Gli1 in response to Hedgehog (Hh) signaling, we discovered a unique population of Hh-responsive cells in the developing murine enthesis that were distinct from tendon fibroblasts and epiphyseal chondrocytes. Lineage-tracing experiments revealed that the Gli1 lineage cells that originate in utero eventually populate the entire mature enthesis. Muscle paralysis increased the number of Hh-responsive cells in the enthesis, demonstrating that responsiveness to Hh is modulated in part by muscle loading. Ablation of the Hh-responsive cells during the first week of postnatal development resulted in a loss of mineralized fibrocartilage, with very little tissue remodeling 5 weeks after cell ablation. Conditional deletion of smoothened, a molecule necessary for responsiveness to Ihh, from the developing tendon and enthesis altered the differentiation of enthesis progenitor cells, resulting in significantly reduced fibrocartilage mineralization and decreased biomechanical function. Taken together, these results demonstrate that Hh signaling within developing enthesis fibrocartilage cells is required for enthesis formation. Summary: The formation of a functional mineralized tendon enthesis, which links tendons to bones, involves Hedgehog-responsive cells and is modulated by muscle loading.

GDF5 PROGENITORS GIVE RISE TO FIBROCARTILAGE CELLS THAT MINERALIZE VIA HEDGEHOG SIGNALING TO FORM THE ZONAL ENTHESIS

The sequence of events that leads to the formation of a functionally graded enthesis is not clearly defined. The current study demonstrates that clonal expansion of Gdf5 progenitors contributes to linear growth of the enthesis. Prior to mineralization, Col1+ cells in the enthesis appose Col2+ cells of the underlying primary cartilage. At the onset of enthesis mineralization, cells at the base of the enthesis express alkaline phosphatase, Indian hedgehog, and ColX as they mineralize. The mineralization front then extends towards the tendon midsubstance as cells above the front become encapsulated in mineralized fibrocartilage over time. The hedgehog (Hh) pathway regulates this process, as Hh-responsive Gli1+ cells within the developing enthesis mature from unmineralized to mineralized fibrochondrocytes in response to activated signaling. Hh signaling is required for mineralization, as tissue-specific deletion of its obligate transducer Smoothened in the developing tendon and enthesis cells leads to significant reductions in the apposition of mineralized fibrocartilage. Together, these findings provide a spatiotemporal map of events - from expansion of the embryonic progenitor pool to synthesis of the collagen template and finally mineralization of this template - that leads to the formation of the mature zonal enthesis. These results can inform future tendon-to-bone repair strategies to create a mechanically functional enthesis in which tendon collagen fibers are anchored to bone through mineralized fibrocartilage.

Stochastic Interdigitation as a Toughening Mechanism at the Interface between Tendon and Bone

Reattachment and healing of tendon to bone poses a persistent clinical challenge and often results in poor outcomes, in part because the mechanisms that imbue the uninjured tendon-to-bone attachment with toughness are not known. One feature of typical tendon-to-bone surgical repairs is direct attachment of tendon to smooth bone. The native tendon-to-bone attachment, however, presents a rough mineralized interface that might serve an important role in stress transfer between tendon and bone. In this study, we examined the effects of interfacial roughness and interdigital stochasticity on the strength and toughness of a bimaterial interface. Closed form linear approximations of the amplification of stresses at the rough interface were derived and applied in a two-dimensional unit-cell model. Results demonstrated that roughness may serve to increase the toughness of the tendon-to-bone insertion site at the expense of its strength. Results further suggested that the natural tendon-to-bone attachment presents roughness for which the gain in toughness outweighs the loss in strength. More generally, our results suggest a pathway for stochasticity to improve surgical reattachment strategies and structural engineering attachments.

The role of mechanobiology in the attachment of tendon to bone

The attachment of dissimilar materials is a major engineering challenge due to stress concentrations that arise at the interface of the disparate materials. An effective biologic solution to this problem can be seen at the attachment of the relatively compliant tendon to the relatively stiff and brittle bone. In this chapter, we review the functionally graded tissue that exists between tendon and bone. The focus of the chapter is the role of mechanobiology in the development, maintenance, and healing of the tendon-to-bone attachment; the role of mechanical stress on fetal and postnatal development and on tendon-to-bone during healing is discussed. A better understanding of mechanobiology at the insertion may help guide rehabilitation strategies and tissue engineering protocols for enhancing tendon-to-bone healing.