Stavros Thomopoulos

Electrospun Nanofibers for Regenerative Medicine

This Progress Report reviews recent progress in applying electrospun nanofibers to the emerging field of regenerative medicine. It begins with a brief introduction to electrospinning and nanofibers, with a focus on issues related to the selection of materials, incorporation of bioactive molecules, degradation characteristics, control of mechanical properties, and facilitation of cell infiltration. Next, a number of approaches to fabricate scaffolds from electrospun nanofibers are discussed, including techniques for controlling the alignment of nanofibers and for producing scaffolds with complex architectures. The article also highlights applications of the nanofiber-based scaffolds in four areas of regenerative medicine that involve nerves, dural tissues, tendons, and the tendon-to-bone insertion site. The Progress Report concludes with perspectives on challenges and future directions for design, fabrication, and utilization of scaffolds based on electrospun nanofibers.

Nanofiber Scaffolds with Gradations in Mineral Content for Mimicking the Tendon-to-Bone Insertion Site

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 preosteoblast 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.

Contribution of extracellular matrix to the mechanical properties of the heart

Extracellular matrix (ECM) components play essential roles in development, remodeling, and signaling in the cardiovascular system. They are also important in determining the mechanics of blood vessels, valves, pericardium, and myocardium. The goal of this brief review is to summarize available information regarding the mechanical contributions of ECM in the myocardium. Fibrillar collagen, elastin, and proteoglycans all play crucial mechanical roles in many tissues in the body generally and in the cardiovascular system specifically. The myocardium contains all three components, but their mechanical contributions are relatively poorly understood. Most studies of ECM contributions to myocardial mechanics have focused on collagen, but quantitative prediction of mechanical properties of the myocardium, or changes in those properties with disease, from measured tissue structure is not yet possible. Circumstantial evidence suggests that the mechanics of cardiac elastin and proteoglycans merit further study. Work in other tissues used a combination of correlation, modification or digestion, and mathematical modeling to establish mechanical roles for specific ECM components; this work can provide guidance for new experiments and modeling studies in myocardium.

Nicotine delays tendon-to-bone healing in a rat shoulder model

BACKGROUND Many studies have shown that nicotine negatively impacts fracture healing and bone fusion processes. However, very little is known about its effect on tendon and ligament healing. The goal of the present study was to evaluate the effect of nicotine on tendon-to-bone healing. METHODS Supraspinatus tendons in both shoulders of seventy-two rats were transected and repaired to the humeral head. Osmotic pumps were implanted subcutaneously, and nicotine or saline solution was delivered for ten, twenty-eight, or fifty-six days. Cell morphology was evaluated with use of histologic sections. Cells were counted, and proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed to assess cellular proliferation. In situ hybridization was performed to measure type-I collagen mRNA expression. Biomechanical and geometric properties were assessed. RESULTS Inflammation persisted longer in the nicotine group than in the saline solution group. Cellular proliferation was higher in the saline solution group than in the nicotine group at the early time-points. Type-I collagen expression was higher in the saline solution group at twenty-eight days. Mechanical properties increased over time in both groups. Maximum stress was significantly lower in the nicotine group than in the saline solution group at ten days. Maximum force was significantly lower in the nicotine group than in the saline solution group at twenty-eight days. Maximum force was significantly higher in the nicotine group than in the saline solution group at fifty-six days. Stiffness was not different between the groups at any time-point. CONCLUSIONS Nicotine caused a delay in tendon-to-bone healing in a rat rotator cuff animal model. Mechanical properties increased over time in both groups, but the properties in the nicotine group lagged behind those in the saline solution group. Chronic inflammation and decreased cell proliferation may partly explain the inferior biomechanical properties in the nicotine group as compared with the saline solution group. CLINICAL RELEVANCE Failure of rotator cuff repair is a major clinical problem. The adverse effect of nicotine on rotator cuff healing noted in this clinically appropriate animal model may be an important clinical consideration.

Functional Attachment of Soft Tissues to Bone: Development, Healing, and Tissue Engineering

Connective tissues such as tendons or ligaments attach to bone across a multitissue interface with spatial gradients in composition, structure, and mechanical properties. These gradients minimize stress concentrations and mediate load transfer between the soft and hard tissues. Given the high incidence of tendon and ligament injuries and the lack of integrative solutions for their repair, interface regeneration remains a significant clinical challenge. This review begins with a description of the developmental processes and the resultant structure-function relationships that translate into the functional grading necessary for stress transfer between soft tissue and bone. It then discusses the interface healing response, with a focus on the influence of mechanical loading and the role of cell-cell interactions. The review continues with a description of current efforts in interface tissue engineering, highlighting key strategies for the regeneration of the soft tissue-to-bone interface, and concludes with a summary of challenges and future directions.

Sustained delivery of transforming growth factor beta three enhances tendon-to-bone healing in a rat model

Despite advances in surgical technique, rotator cuff repairs are plagued by a high rate of failure. This failure rate is in part due to poor tendon‐to‐bone healing; rather than regeneration of a fibrocartilaginous attachment, the repair is filled with disorganized fibrovascular (scar) tissue. Transforming growth factor beta 3 (TGF‐β3) has been implicated in fetal development and scarless fetal healing and, thus, exogenous addition of TGF‐β3 may enhance tendon‐to‐bone healing. We hypothesized that: TGF‐β3 could be released in a controlled manner using a heparin/fibrin‐based delivery system (HBDS); and delivery of TGF‐β3 at the healing tendon‐to‐bone insertion would lead to improvements in biomechanical properties compared to untreated controls. After demonstrating that the release kinetics of TGF‐β3 could be controlled using a HBDS in vitro, matrices were incorporated at the repaired supraspinatus tendon‐to‐bone insertions of rats. Animals were sacrificed at 14–56 days. Repaired insertions were assessed using histology (for inflammation, vascularity, and cell proliferation) and biomechanics (for structural and mechanical properties). TGF‐β3 treatment in vivo accelerated the healing process, with increases in inflammation, cellularity, vascularity, and cell proliferation at the early timepoints. Moreover, sustained delivery of TGF‐β3 to the healing tendon‐to‐bone insertion led to significant improvements in structural properties at 28 days and in material properties at 56 days compared to controls. We concluded that TGF‐β3 delivered at a sustained rate using a HBDS enhanced tendon‐to‐bone healing in a rat model.

The role of mechanobiology in tendon healing.

Mechanical cues affect tendon healing, homeostasis, and development in a variety of settings. Alterations in the mechanical environment are known to result in changes in the expression of extracellular matrix proteins, growth factors, transcription factors, and cytokines that can alter tendon structure and cell viability. Loss of muscle force in utero or in the immediate postnatal period delays tendon and enthesis development. The response of healing tendons to mechanical load varies depending on anatomic location. Flexor tendons require motion to prevent adhesion formation, yet excessive force results in gap formation and subsequent weakening of the repair. Excessive motion in the setting of anterior cruciate ligament reconstruction causes accumulation of macrophages, which are detrimental to tendon graft healing. Complete removal of load is detrimental to rotator cuff healing; yet, large forces are also harmful. Controlled loading can enhance healing in most settings; however, a fine balance must be reached between loads that are too low (leading to a catabolic state) and too high (leading to microdamage). This review will summarize existing knowledge of the mechanobiology of tendon development, homeostasis, and healing.

The Development of Structural and Mechanical Anisotropy in Fibroblast Populated Collagen Gels

An in vitro model system was developed to study structure-function relationships and the development of structural and mechanical anisotropy in collagenous tissues. Fibroblast-populated collagen gels were constrained either biaxially or uniaxially. Gel remodeling, biaxial mechanical properties, and collagen orientation were determined after 72 h of culture. Collagen gels contracted spontaneously in the unconstrained direction, uniaxial mechanical constraints produced structural anisotropy, and this structural anisotropy was associated with mechanical anisotropy. Cardiac and tendon fibroblasts were compared to test the hypothesis that tendon fibroblasts should generate greater anisotropy in vitro. However, no differences were seen in either structure or mechanics of collagen gels populated with these two cell types, or between fibroblast populated gels and acellular gels. This study demonstrates our ability to control and measure the development of structural and mechanical anisotropy due to imposed mechanical constraints in a fibroblast-populated collagen gel model system. While imposed constraints were required for the development of anisotropy in this system, active remodeling of the gel by fibroblasts was not. This model system will provide a basis for investigating structure-function relationships in engineered constructs and for studying mechanisms underlying the development of anisotropy in collagenous tissues.

The Tendon-to-Bone Transition of the Rotator Cuff: A Preliminary Raman Spectroscopic Study Documenting the Gradual Mineralization across the Insertion in Rat Tissue Samples

We applied Raman spectroscopy to monitor the distribution of mineral and the degree of mineralization across the tendon-bone insertion site in the shoulders of five rats. We acquired Raman spectra from 100 to 4000 Δcm−1 on individual 1 μm points across the 120 μm wide transition zone of each tissue sample and identified all the peaks detected in pure tendon and in pure bone, as well as in the transition zone. The intensity of the 960 Δcm−1 P–O stretch for apatite (normalized to either the 2940 Δcm−1 C–H stretch or the 1003 Δcm−1 C–C stretch for collagen) was used as an indicator of the abundance of mineral. We relate the observed histological morphology in the tissue thin section with the observed Raman peaks for both the organic component (mostly collagen) and the inorganic component (a carbonated form of the mineral apatite) and discuss spectroscopic issues related to peak deconvolution and quantification of overlapping Raman peaks. We show that the mineral-to-collagen ratio at the insertion site increases linearly (R2 = 0.8 for five samples) over the distance of 120 μm from tendon to bone, rather than abruptly, as previously inferred from histological observations. In addition, narrowing of the 960 Δcm−1 band across the traverse indicates that the crystalline ordering within the apatite increases concomitantly with the degree of mineralization. This finding of mineral gradation has important clinical implications and may explain why the uninjured tendon-to-bone connection of the rotator cuff can sustain very high loads without failure. Our finding is also consistent with recent mechanical models and calculations developed to better understand the materials properties of this unusually strong interface.

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.