The Mechanical Challenge of Attaching Tendon to Bone
Director, Carroll Laboratories for Orthopedic Surgery
Vice Chair, Basic Science Research, Orthopedic Surgery
Professor of Biomechanics (in Orthopedic Surgery and Biomedical Engineering)
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).
I. Structure-function relationships - Defining the design criteria
(funded by NIH U01 EB016422) 5-12
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.
II. Developmental biology - Defining the roadmap
(funded by NIH R01 AR055580) 10,16-23
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.
III. Development of biomimetic regenerative medicine strategies
(funded by NIH R01 AR060820 through 2016 and R01 AR062947 through 2017) 25-34
The structure-function and developmental biology results described above were used to guide regenerative medicine strategies for tendon-to-bone repair. Scaffolds were synthesized that mimicked the structure and mechanics of the healthy enthesis. Specifically, we developed aligned nanofiber scaffolds with spatial gradients in mineral (Figure 4).25,30 These structural and compositional gradients resulted in a functionally graded mechanical response, recreating the behavior of the natural enthesis. Furthermore, the gradient in mineral content led to spatially graded osteogenesis of mesenchymal stem cells.29 Additional enthesis features such as a variations in fiber orientation, a crimped fiber microstructure, and a cell phenotype gradient were also recreated in nanofiber scaffolds.28,31,32 These materials are currently being combined with mesenchymal stem cells and optimized for in vivo use.33,34
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).
IV. Improving tendon-to-bone healing through regenerative medicine strategies
(funded by NIH R01/R56 AR057836) 41-51
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.
- Thomopoulos, S., G.M. Genin, and V. Birman, eds. Structural Interfaces and Attachments in Biology. 2013, Springer: New York.
- Lu, H.H. and S. Thomopoulos, Functional attachment of soft tissues to bone: development, healing, and tissue engineering. Annu Rev Biomed Eng, 2013. 15: p. 201-26.
- Galatz, L.M., C.M. Ball, S.A. Teefey, W.D. Middleton, and K. Yamaguchi, The outcome and repair integrity of completely arthroscopically repaired large and massive rotator cuff tears. J Bone Joint Surg Am, 2004. 86-A(2): p. 219-24.
- Harryman, D.T., 2nd, L.A. Mack, K.Y. Wang, S.E. Jackins, M.L. Richardson, and F.A. Matsen, 3rd, Repairs of the rotator cuff. Correlation of functional results with integrity of the cuff. Journal of Bone & Joint Surgery, 1991. 73(7): p. 982-9.
- Thomopoulos, S., G.R. Williams, J.A. Gimbel, M. Favata, and L.J. Soslowsky, Variation of biomechanical, structural, and compositional properties along the tendon to bone insertion site. Journal of Orthopaedic Research, 2003. 21(3): p. 413-9.
- Thomopoulos, S., J.P. Marquez, B. Weinberger, V. Birman, and G.M. Genin, Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations. J Biomech, 2006. 39(10): p. 1842-51.
- Genin, G.M., A. Kent, V. Birman, B. Wopenka, J.D. Pasteris, P.J. Marquez, and S. Thomopoulos, Functional grading of mineral and collagen in the attachment of tendon to bone. Biophys J, 2009. 97(4): p. 976-85.
- Liu, Y., V. Birman, C. Chen, S. Thomopoulos, and G.M. Genin, Mechanisms of bimaterial attachment at the interface of tendon to bone. Journal of Engineering Materials and Technology, 2011. 133(011006): p. 281-8.
- Liu, Y.X., S. Thomopoulos, V. Birman, J.S. Li, and G.M. Genin, Bi-material attachment through a compliant interfacial system at the tendon-to-bone insertion site. Mechanics of Materials, 2012. 44: p. 83-92.
- Schwartz, A.G., J.D. Pasteris, G.M. Genin, T.L. Daulton, and S. Thomopoulos, Mineral distributions at the developing tendon enthesis. PLoS One, 2012. 7(11): p. e48630.
- Alexander, B., T.L. Daulton, G.M. Genin, J. Lipner, J.D. Pasteris, B. Wopenka, and S. Thomopoulos, The nanometre-scale physiology of bone: steric modelling and scanning transmission electron microscopy of collagen-mineral structure. J R Soc Interface, 2012. 9(73): p. 1774-86.
- Liu, Y., S. Thomopoulos, C. Chen, V. Birman, M.J. Buehler, and G.M. Genin, Modelling the mechanics of partially mineralized collagen fibrils, fibres and tissue. J R Soc Interface, 2014. 11(92): p. 20130835.
- Hu, Y., V. Birman, A. Demyier-Black, A.G. Schwartz, S. Thomopoulos, and G.M. Genin, Stochastic interdigitation as a toughening mechanism at the interface between tendon and bone. Biophys J, 2015. 108(2): p. 431-7.
- Wopenka, B., A. Kent, J.D. Pasteris, Y. Yoon, and S. Thomopoulos, 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. Appl Spectrosc, 2008. 62(12): p. 1285-94.
- Deymier-Black, A., J. Pasteris, G. Genin, and S. Thomopoulos, Allometry of the tendon enthesis: mechanisms of load transfer between tendon and bone. Journal of Biomechanical Engineering, 2015. In Press.
- Schwartz, A.G., J.H. Lipner, J.D. Pasteris, G.M. Genin, and S. Thomopoulos, Muscle loading is necessary for the formation of a functional tendon enthesis. Bone, 2013. 55(1): p. 44-51.
- Schwartz, A.G., F. Long, and S. Thomopoulos, Enthesis fibrocartilage cells originate from a population of Hedgehog-responsive cells modulated by the loading environment. Development, 2014. 142(1): p. 196-206.
- Thomopoulos, S., H.M. Kim, S.Y. Rothermich, C. Biederstadt, R. Das, and L.M. Galatz, Decreased muscle loading delays maturation of the tendon enthesis during postnatal development. J Orthop Res, 2007. 25(9): p. 1154-63.
- Kim, H.M., L.M. Galatz, N. Patel, R. Das, and S. Thomopoulos, Recovery potential after postnatal shoulder paralysis. An animal model of neonatal brachial plexus palsy. J Bone Joint Surg Am, 2009. 91(4): p. 879-91.
- Kim, H.M., L.M. Galatz, R. Das, N. Patel, and S. Thomopoulos, Musculoskeletal deformities secondary to neurotomy of the superior trunk of the brachial plexus in neonatal mice. J Orthop Res, 2010. 28(10): p. 1391-8.
- Tatara, A.M., J.H. Lipner, R. Das, H.M. Kim, N. Patel, E. Ntouvali, M.J. Silva, and S. Thomopoulos, The role of muscle loading on bone (Re)modeling at the developing enthesis. PLoS One, 2014. 9(5): p. e97375.
- Potter, R., N. Havlioglu, and S. Thomopoulos, The developing shoulder has a limited capacity to recover after a short duration of neonatal paralysis. J Biomech, 2014. 47(10): p. 2314-20.
- Liu, Y., A.G. Schwartz, V. Birman, S. Thomopoulos, and G.M. Genin, Stress amplification during development of the tendon-to-bone attachment. Biomech Model Mechanobiol, 2014. 13(5): p. 973-83.
- Zelzer, E., E. Blitz, M.L. Killian, and S. Thomopoulos, Tendon-to-bone attachment: from development to maturity. Birth Defects Res C Embryo Today, 2014. 102(1): p. 101-12.
- Li, X., J. Xie, J. Lipner, X. Yuan, S. Thomopoulos, and Y. Xia, Nanofiber scaffolds with gradations in mineral content for mimicking the tendon-to-bone insertion site. Nano Lett, 2009. 9(7): p. 2763-8.
- Liu, W., Y.C. Yeh, J. Lipner, J. Xie, H.W. Sung, S. Thomopoulos, and Y. Xia, Enhancing the stiffness of electrospun nanofiber scaffolds with a controlled surface coating and mineralization. Langmuir : the ACS journal of surfaces and colloids, 2011. 27(15): p. 9088-93.
- Liu, W., S. Thomopoulos, and Y. Xia, Electrospun nanofibers for regenerative medicine. Advanced Healthcare Materials, 2012. 1(1): p. 10-25.
- Liu, W., Y. Zhang, S. Thomopoulos, and Y. Xia, Generation of controllable gradients in cell density. Angew Chem Int Ed Engl, 2013. 52(1): p. 429-32.
- Liu, W., J. Lipner, J. Xie, C.N. Manning, S. Thomopoulos, and Y. Xia, Nanofiber scaffolds with gradients in mineral content for spatial control of osteogenesis. ACS Appl Mater Interfaces, 2014. 6(4): p. 2842-9.
- Lipner, J., W. Liu, Y. Liu, J. Boyle, G.M. Genin, Y. Xia, and S. Thomopoulos, The mechanics of PLGA nanofiber scaffolds with biomimetic gradients in mineral for tendon-to-bone repair. J Mech Behav Biomed Mater, 2014. 40: p. 59-68.
- Xie, J., X. Li, J. Lipner, C.N. Manning, A.G. Schwartz, S. Thomopoulos, and Y. Xia, "Aligned-to-random" nanofiber scaffolds for mimicking the structure of the tendon-to-bone insertion site. Nanoscale, 2010. 2(6): p. 923-6.
- Liu, W., J. Lipner, C.H. Moran, L. Feng, X. Li, S. Thomopoulos, and Y. Xia, Generation of electrospun nanofibers with controllable degrees of crimping through a simple, plasticizer-based treatment. Adv Mater, 2015. 27(16): p. 2583-8.
- Shen, H., R.H. Gelberman, M.J. Silva, S.E. Sakiyama-Elbert, and S. Thomopoulos, BMP12 induces tenogenic differentiation of adipose-derived stromal cells. PLoS One, 2013. 8(10): p. e77613.
- Manning, C.N., A.G. Schwartz, W. Liu, J. Xie, N. Havlioglu, S.E. Sakiyama-Elbert, M.J. Silva, Y. Xia, R.H. Gelberman, and S. Thomopoulos, Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair. Acta biomaterialia, 2013. 9(6): p. 6905-14.
- Martin, P., Wound healing--aiming for perfect skin regeneration. Science, 1997. 276(5309): p. 75-81.
- Ballas, C.B. and J.M. Davidson, Delayed wound healing in aged rats is associated with increased collagen gel remodeling and contraction by skin fibroblasts, not with differences in apoptotic or myofibroblast cell populations. Wound Repair Regeneration, 2001. 9(3): p. 223-37.
- Ferguson, M.W. and S. O'Kane, Scar-free healing: from embryonic mechanisms to adult therapeutic intervention. Philos Trans R Soc Lond B Biol Sci, 2004. 359(1445): p. 839-50.
- Beredjiklian, P.K., M. Favata, J.S. Cartmell, C.L. Flanagan, T.M. Crombleholme, and L.J. Soslowsky, Regenerative versus reparative healing in tendon: a study of biomechanical and histological properties in fetal sheep. Ann Biomed Eng, 2003. 31(10): p. 1143-52.
- Ansorge, H.L., S. Adams, A.F. Jawad, D.E. Birk, and L.J. Soslowsky, Mechanical property changes during neonatal development and healing using a multiple regression model. J Biomech, 2012. 45(7): p. 1288-92.
- Ansorge, H.L., J.E. Hsu, L. Edelstein, S. Adams, D.E. Birk, and L.J. Soslowsky, Recapitulation of the Achilles tendon mechanical properties during neonatal development: a study of differential healing during two stages of development in a mouse model. J Orthop Res, 2012. 30(3): p. 448-56.
- Thomopoulos, S., G. Hattersley, V. Rosen, M. Mertens, L. Galatz, G.R. Williams, and L.J. Soslowsky, The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study. Journal of Orthopaedic Research, 2002. 20(3): p. 454-63.
- Thomopoulos, S., G.R. Williams, and L.J. Soslowsky, Tendon to bone healing: differences in biomechanical, structural, and compositional properties due to a range of activity levels. Journal of Biomechanical Engineering, 2003. 125(1): p. 106-13.
- Galatz, L.M., S.Y. Rothermich, M. Zaegel, M.J. Silva, N. Havlioglu, and S. Thomopoulos, Delayed repair of tendon to bone injuries leads to decreased biomechanical properties and bone loss. J Orthop Res, 2005. 23(6): p. 1441-7.
- Galatz, L.M., L.J. Sandell, S.Y. Rothermich, R. Das, A. Mastny, N. Havlioglu, M.J. Silva, and S. Thomopoulos, Characteristics of the rat supraspinatus tendon during tendon-to-bone healing after acute injury. J Orthop Res, 2006. 24(3): p. 541-50.
- Galatz, L.M., N. Charlton, R. Das, H.M. Kim, N. Havlioglu, and S. Thomopoulos, Complete removal of load is detrimental to rotator cuff healing. Journal of Shoulder and Elbow Surgery, 2009. 18(5): p. 669-675.
- Kim, H.M., L.M. Galatz, R. Das, N. Havlioglu, S.Y. Rothermich, and S. Thomopoulos, The role of transforming growth factor beta isoforms in tendon-to-bone healing. Connect Tissue Res, 2011. Epub.
- Manning, C.N., H.M. Kim, S. Sakiyama-Elbert, L.M. Galatz, N. Havlioglu, and S. Thomopoulos, Sustained delivery of transforming growth factor beta three enhances tendon-to-bone healing in a rat model. J Orthop Res, 2011.
- Killian, M.L., L. Cavinatto, L.M. Galatz, and S. Thomopoulos, The role of mechanobiology in tendon healing. J Shoulder Elbow Surg, 2012. 21(2): p. 228-37.
- Kim, H.M., L.M. Galatz, C. Lim, N. Havlioglu, and S. Thomopoulos, The effect of tear size and nerve injury on rotator cuff muscle fatty degeneration in a rodent animal model. J Shoulder Elbow Surg, 2012. 21(7): p. 847-58.
- Killian, M.L., C.T. Lim, S. Thomopoulos, N. Charlton, H.M. Kim, and L.M. Galatz, The effect of unloading on gene expression of healthy and injured rotator cuffs. J Orthop Res, 2013. 31(8): p. 1240-8.
- Killian, M.L., L. Cavinatto, S.A. Shah, E.J. Sato, S.R. Ward, N. Havlioglu, L.M. Galatz, and S. Thomopoulos, The effects of chronic unloading and gap formation on tendon-to-bone healing in a rat model of massive rotator cuff tears. J Orthop Res, 2014. 32(3): p. 439-47.
- Silva, M.J., S. Thomopoulos, N. Kusano, M.A. Zaegel, F.L. Harwood, H. Matsuzaki, N. Havlioglu, T.T. Dovan, D. Amiel, and R.H. Gelberman, Early healing of flexor tendon insertion site injuries: Tunnel repair is mechanically and histologically inferior to surface repair in a canine model. J Orthop Res, 2006. 24(5): p. 990-1000.
- Gimbel, J.A., J.P. Van Kleunen, G.R. Williams, S. Thomopoulos, and L.J. Soslowsky, Long durations of immobilization in the rat result in enhanced mechanical properties of the healing supraspinatus tendon insertion site. J Biomech Eng, 2007. 129(3): p. 400-4.
- Thomopoulos, S., E. Zampiakis, R. Das, M.J. Silva, and R.H. Gelberman, The effect of muscle loading on flexor tendon-to-bone healing in a canine model. J Orthop Res, 2008. 26(12): p. 1611-7.
- Thomopoulos, S., H.M. Kim, M.J. Silva, E. Ntouvali, C.N. Manning, R. Potter, H. Seeherman, and R.H. Gelberman, Effect of bone morphogenetic protein 2 on tendon-to-bone healing in a canine flexor tendon model. J Orthop Res, 2012. 30(11): p. 1702-9.