Source: http://www.columbiaortho.org/research/thomopoulos-lab
Timestamp: 2019-04-19 16:28:58+00:00

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The attachment of dissimilar materials is a major challenge because of the high levels of localized stress that develop at such interfaces. An effective biologic solution to this problem can be seen at the attachment of tendon (a compliant, structural “soft tissue”) to bone (a stiff, structural “hard tissue”).1,2 The enthesis, a transitional tissue that exists between uninjured tendon and bone (Figure 1), is not recreated during healing, so surgical reattachment of these two dissimilar biologic materials often fails (e.g., recurrent tears after rotator cuff repair range from 20% to 94%, depending on the patient population3,4). To develop successful strategies for tendon-to-bone repair, necessary for rotator cuff repair and anterior cruciate ligament reconstruction, we must first understand the mechanisms by which the healthy attachment transfers load between tendon and bone, and how cells build a functional attachment during development. In order to achieve these goals, we are focusing on: (I) understanding the structure-function relationships that allow for effective load transfer at the healthy enthesis, (II) determining the biophysical and molecular cues that drive the development of the enthesis, (III) developing regenerative medicine strategies motivated by structure-function and developmental biology results, and (IV) applying these strategies to improve tendon-to-bone healing.
Figure 1: Tendons attach to bone across the enthesis, a functionally graded fibrocartilaginous transitional tissue (left: toluidine blue-stained section from an adult rat supraspinatus tendon enthesis).
We have demonstrated that the enthesis is a functionally graded material with regard to its cell phenotypes, extracellular matrix composition, structural organization, and mechanical properties. A number of mechanisms across multiple spatial scales combine to produce a robust mechanical attachment between the two materials (Figure 2). At the nanometer length scale, mineral crystals accumulate on collagen fibrils to stiffen them, but only after a percolated network of mineral forms.7,11,12 The particular arrangement of the mineral crystals relative to the collagen fibril gap channels and outer surfaces dictates their stiffening effects. At the micrometer length scale, the concentration of mineral, the interdigitation of mineralized and unmineralized tissues, and a compliant zone serve to balance strength and toughness of the attachment.7,12-14 For example, although interdigitation leads to a small decrease in attachment strength, it also leads to a dramatic increase in attachment toughness.13 Similarly, a surprising and counter-intuitive compliant zone between tendon and bone, measured experimentally5, serves to reduce stress concentrations at the enthesis and further toughen the attachment.9 At the millimeter length scale, the tendon splays at the attachment to reduce stress concentrations6 and the attachment footprint area scales to normalize stress15. Understanding these mechanisms of load transfer provides the design criteria for effective attachment of tendon to bone.
Figure 2: At the millimeter length scale (left), tendon attaches to bone over a large footprint area. At the micrometer length scale (middle), gradients exist in mineral content and orientation, and tissues interdigitate across wavy interfaces. At the nanometer length scale (right), mineral accumulates in collagen fibril gap spaces and on surfaces to stiffen the fibrils.
The enthesis mineralizes and matures postnatally through a coordinated set of biologic events driven by mechanical loading and genetic cues.24 We have studied the role of muscle loading on enthesis development by paralyzing the rotator cuff muscles at birth in mice.16,18-20,23 A series of experiments using chemical or physical denervation demonstrated that muscle load is necessary for the formation of a functional enthesis. In the absence of load, a cell phenotype gradient failed to form at the developing enthesis, leading to defects in fibrocartilage formation, mineralization, collagen deposition, and a mechanically deficient attachment. Studies of the molecular cues driving enthesis formation led to the identification of a unique population of hedgehog-responsive cells at the developing enthesis (Figure 3).17 Lineage tracing experiments demonstrated that this cell population populates the enthesis through the early post-natal period and controls mineralization of the attachment. Genetic deletion of hedgehog responsiveness in these cells or ablation of these cells led to severe defects in mineralization and attachment mechanical properties. Understanding the biophysical and molecular signals that drive the development of a functional attachment between tendon and bone provides a roadmap for improved healing and for development of tissue engineered replacements.
Figure 3: The fate of hedgehog-active cells at the enthesis at P56 is shown in green, labeled at P7 (left) and P52 (right). These cells build the enthesis, turning off hedgehog activation with maturity (P56). Scale = 100μm, t: tendon and b: bone.
To apply developmental biology results to regenerative medicine, we developed a rotator cuff enthesis injury model that can be performed in neonatal and adult mice. Tendon-to-bone healing in the adult is scar mediated and often results in failure. In contrast, wound healing studies in skin 35-37 and tendon 38-40 show that tissues injured in utero or early postnatally heal via regenerative pathways rather than scar-mediated pathways. Using the enthesis injury model along with lineage tracing approaches, we are probing the necessity of the hedgehog-responsive cell lineage for enthesis regeneration, particularly related to mineralization. Understanding the necessity of enthesis cells from specific lineages for regeneration of a functional enthesis will allow us to put forward new strategies for enhanced tendon-to-bone repair.
Figure 4: (A) Testing of scaffolds with spatial gradients in mineral were performed from slack conditions, and conducted with a strain rate of 0.4 %/sec to achieve quasi-static loading conditions. (B) Locations were selected from the grip-to-grip stress-strain curve. The images were then analysed to demonstrate the effect of mineral content on the strain fields and mechanical properties. (C) Local strain fields were calculated directly. The first principal strain is shown using a heat map. (D) The relationship between modulus and mineral content was approximately linear, with the slope representing the stiffening effect of the mineral (R: Pearson’s correlation coefficient).
Healing of tendon to bone does not reproduce the structural or compositional features of the healthy enthesis 42,44,52 (Figure 5). This results in a mechanically inferior attachment that is prone to rupture. Healing can be improved through the implementation of design criteria from the uninjured attachment (e.g., through surgical manipulation), application of the roadmap defined from developmental biology studies (e.g., through the application of growth factor or cell-based therapies), or application of tissue engineered scaffolds. Prior and ongoing studies are evaluating the role of mechanical loading,42,45,48,53,54 growth factors,47,55 functionally graded scaffolds,25,28-31 and mesenchymal stem cells29,33,34 on tendon-to-bone repair. Results have demonstrated that healing of tendon to bone can be modulated by controlling loading across the repair site and that growth factor- and cell-based therapies hold great promise for regenerating the native enthesis.
Figure 5: The functionally graded transition between tendon and bone is not regenerated during the healing process (healthy attachment is shown on the left, healing attachment is shown on the right; hematoxylin- and eosin-stained images are shown under bright field in the top row and under polarized light in the bottom row).
The four research themes described above inform each other and are expected to lead to clinical therapies for tendon-to-bone repair. Basic science studies will identify the critical features necessary for successful tendon-to-bone repair and inform translational studies on cell- and growth factor-based regeneration for rotator cuff repair and anterior cruciate ligament reconstruction. Of critical importance is recreation of the multiscale mechanical behavior derived from the hierarchical structures at the healthy tendon-to-bone attachment.
Complete list of references can be found here.
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