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Research Papers

Morphological and Functional Characteristics of Three-Dimensional Engineered Bone-Ligament-Bone Constructs Following Implantation

[+] Author and Article Information
Jinjin Ma

Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109

Kristen Goble

Department of Molecular and Integrative Physiology,University of Michigan, Ann Arbor, MI 48109

Michael Smietana

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109

Tatiana Kostrominova

Department of Anatomy and Cell Biology, Indiana University School of Medicine-Northwest, Gary, IN 46409-1008

Lisa Larkin

Department of Biomedical Engineering, and Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109

Ellen M. Arruda

Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109; Macromolecular Science and Engineering Program, University of Michigan, Ann Arbor, MI 48109arruda@umich.edu

J Biomech Eng 131(10), 101017 (Oct 13, 2009) (9 pages) doi:10.1115/1.4000151 History: Received November 15, 2008; Revised August 04, 2009; Posted September 03, 2009; Published October 13, 2009

The incidence of ligament injury has recently been estimated at 400,000/year. The preferred treatment is reconstruction using an allograft, but outcomes are limited by donor availability, biomechanical incompatibility, and immune rejection. The creation of an engineered ligament in vitro solely from patient bone marrow stromal cells (has the potential to greatly enhance outcomes in knee reconstructions. Our laboratory has developed a scaffoldless method to engineer three-dimensional (3D) ligament and bone constructs from rat bone marrow stem cells in vitro. Coculture of these two engineered constructs results in a 3D bone-ligament-bone (BLB) construct with viable entheses, which was successfully used for medial collateral ligament (MCL) replacement in a rat model. 1 month and 2 month implantations were applied to the engineered BLBs. Implantation of 3D BLBs in a MCL replacement application demonstrated that our in vitro engineered tissues grew and remodeled quickly in vivo to an advanced phenotype and partially restored function of the knee. The explanted 3D BLB ligament region stained positively for type I collagen and elastin and was well vascularized after 1 and 2 months in vivo. Tangent moduli of the ligament portion of the 3D BLB 1 month explants increased by a factor of 2.4 over in vitro controls, to a value equivalent to those observed in 14-day-old neonatal rat MCLs. The 3D BLB 1 month explants also exhibited a functionally graded response that closely matched native MCL inhomogeneity, indicating the constructs functionally adapted in vivo.

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Copyright © 2009 by American Society of Mechanical Engineers
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Figures

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Figure 1

Fabrication, implantation, and explantation of 3D BLB constructs engineered in vitro for medial collateral ligament replacement. (a) BLB construct just prior to implantation; approximately 3 days after detachment of the monolayer, the cells self-organized into a cylinder. Total length of the construct pin to pin=15 mm; diameter=0.47 mm. (b) 3D BLB from image (a) placed inside silicone tubing and secured in replacement of excised MCL. (c) 3D BLB construct 4 weeks following implantation. The presence of the silicone tubing makes it easy to visualize and excise the implanted construct; following 1 month of implantation, the engineered BLB has fused with the bone at the femur and tibia and increased in diameter to 0.53 mm. (d) 3D BLB excised from bone to be used for histology.

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Figure 2

Histological evaluation of the of 3D BLB construct developed in vitro. (a) H&E staining. ((b) and (d)) Collagen 1 immunostaining (red) of the end of the construct. ((c) and (e)) Collagen 1 (red) immunostaining of the middle part of the construct. (f) Elastin immunostaining (red) of the middle part of the construct. DAPI staining (blue) was used to visualize the nuclei.

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Figure 3

H&E staining of areas of native bone/3D BLB construct interfaces at the tibia ((a) and (b)) and femur ((c) and (d)) sides after 2 months of implantation

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Figure 4

H&E staining of the middle part of the 3D BLB construct after 2 months of implantation ((a)–(c)) and native MCL ligament from 21 day old neonatal (d) and from adult (e) rat

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Figure 5

Immunostaining of the middle part of 3D BLB construct after 2 months of implantation ((a) and (b)) and native MCL ligament from 21 day old neonatal (c) and from adult (d) rat with antibodies against collagen 1 (red). DAPI staining (blue) was used to visualize the nuclei.

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Figure 6

Immunostaining of the middle part of 3D BLB construct after 2 months of implantation ((a) and (b)) and native MCL ligament from 7 day old neonatal (c) and from adult (d) rat with antibodies against CD31 (red) to visualize blood vessels. DAPI staining (blue) was used to visualize the nuclei. WGA lectin-fluorescein (green) was used to visualize the general tissue structure.

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Figure 7

Immunostaining of the middle part of 3D BLB construct after 2 months of implantation (a) and native MCL ligament from 7 day old neonatal (c) and adult (e) rat with antibodies against elastin (red). DAPI staining (blue) was used to visualize the nuclei. WGA lectin-fluorescein ((green in (b), (d), and (f)) was used to visualize the general tissue structure in 3D BLB construct (b), 7 day old neonatal (d), and adult (f) rat.

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Figure 8

Nominal stress versus nominal strain cyclic response curves for in vitro 3D BLBs, 3D BLB explants, and 14 day native MCLs. (a) Stress-strain response of the in vitro 3D BLBs shows that the nonlinear cyclic response includes a toe region, strain hardening, and hysteresis. (b) Stress-strain response of 3D BLB explants after 4 weeks as MCL replacement tissues on a stress scale that is (approximately) twice that in (a), indicating an increase in mechanical stiffness of the construct during in vivo implantation. The nonlinear response includes hysteresis and an earlier and more gradual transition to strain hardening than in the in vitro BLBs. (c) Stress-strain response of native 14 day neonatal MCL shows similar nonlinear stress-strain cyclic behavior to that observed in the 3D BLB explants.

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Figure 9

Comparison of tangent stiffness, average diameter, and cross-sectional area of in vitro 3D BLBs, 3D BLB explants, and 14 day native MCLs

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Figure 10

Localized stress versus strain analysis of 3D BLB constructs in vitro (a), 3D BLB explants (b), and native rat MCL (c) with corresponding regions shown in the specimen photos. Developed for 4 weeks in vitro 3D BLB constructs show uniform strain response. After 1 month of implantation, strain responses are localized in 3D BLB explants, showing a functional gradient that is also indicated in native MCL. Regions that are closer to bones are relatively more compliant than the ligament midsections in both 3D BLB explants and native MCLs.

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