Evaluation of a bioengineered ACL matrix’s osteointegration with BMP-2 supplementation

Autoři: Paulos Y. Mengsteab aff001;  Patrick Conroy aff001;  Mary Badon aff001;  Takayoshi Otsuka aff001;  Ho-Man Kan aff001;  Anthony T. Vella aff005;  Lakshmi S. Nair aff001;  Cato T. Laurencin aff001
Působiště autorů: Connecticut Convergence Institute for Translation in Regenerative Engineering, University of Connecticut Health, Farmington, CT, United States of America aff001;  Raymond and Beverly Sackler Center for Biological, Physical and Engineering Sciences, University of Connecticut Health, Farmington, CT, United States of America aff002;  Department of Orthopedic Surgery, University of Connecticut Health, Farmington, CT, United States of America aff003;  Department of Biomedical Engineering, University of Connecticut, Storrs, CT, United States of America aff004;  Department of Immunology, University of Connecticut School of Medicine, Farmington, CT, United States of America aff005;  Department of Materials Science and Engineering, University of Connecticut, Storrs, CT, United States of America aff006;  Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT, United States of America aff007;  Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, CT, United States of America aff008
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227181


A poly (l-lactic) acid bioengineered anterior cruciate ligament (ACL) matrix has previously demonstrated the ability to support tissue regeneration in a rabbit ACL reconstruction model. The matrix was designed for optimal bone and ligament regeneration by developing a matrix with differential pore sizes in its bone and ligament compartments. Building upon past success, we designed a new bioengineered ACL matrix that is easier to install and can be used with endobutton fixation during ACL reconstruction. To achieve this, a new braiding procedure was developed to allow the matrix to be folded in half, making two-limbs, while maintaining its bone and ligament compartments. The osteointegration of the matrix with and without bone morphogenetic protein 2 (BMP-2) supplementation was evaluated in a rabbit ACL reconstruction model. Two doses of BMP-2 were evaluated, 1 and 10 μg, and delivered by saline injection into the bone tunnel at the end of surgery. A fibrous matrix-to-bone interface with occasional Sharpey’s fibers was the primary mode of osteointegration observed. The matrix was also found to support a fibrocartilage matrix-to-bone interface. In some cases, the presence of chondrocyte-like cells was observed at the aperture of the bone tunnel and the center of the matrix within the bone tunnel. Treatment with BMP-2 was associated with a trend towards smaller bone tunnel cross-sectional areas, and 1 μg of BMP-2 was found to significantly enhance osteoid seam width in comparison with no BMP-2 or 10 μg of BMP-2 treatment. Regenerated tissue was well organized within the bioengineered ACL matrix and aligned with the poly (l-lactic) acid fibers. Disorganized tissue was found between the two-limbs of the bioengineered ACL matrix and hypothesized to be due to a lack of structural scaffolding. This study suggests that the bioengineered ACL matrix can undergo similar modes of osteointegration as current autografts and allografts, and that BMP-2 treatment may enhance osteoblastic activity within the bone tunnels.

Klíčová slova:

Anterior cruciate ligament reconstruction – Cell staining – Cytokines – Knees – Rabbits – Tendons – Fibrocartilage – Tibia


1. Ardern CL, Webster KE, Taylor NF, Feller JA. Return to sport following anterior cruciate ligament reconstruction surgery: a systematic review and meta-analysis of the state of play. Br J Sports Med. 2011;45: 596–606. doi: 10.1136/bjsm.2010.076364 21398310

2. Paterno M V., Rauh MJ, Schmitt LC, Ford KR, Hewett TE. Incidence of Second ACL Injuries 2 Years After Primary ACL Reconstruction and Return to Sport. Am J Sports Med. 2014;42: 1567–1573. doi: 10.1177/0363546514530088 24753238

3. Sajovic M, Stropnik D, Skaza K. Long-term Comparison of Semitendinosus and Gracilis Tendon Versus Patellar Tendon Autografts for Anterior Cruciate Ligament Reconstruction: A 17-Year Follow-up of a Randomized Controlled Trial. Am J Sports Med. 2018;46: 1800–1808. doi: 10.1177/0363546518768768 29741911

4. Gulotta L V, Rodeo SA. Biology of autograft and allograft healing in anterior cruciate ligament reconstruction. Clin Sports Med. 2007;26: 509–24. doi: 10.1016/j.csm.2007.06.007 17920950

5. Mengsteab PY, Nair LS, Laurencin CT. The past, present and future of ligament regenerative engineering. Regen Med. 2016;11: 871–881. doi: 10.2217/rme-2016-0125 27879170

6. Cooper JA, Sahota JS, Gorum WJ, Carter J, Doty SB, Laurencin CT. Biomimetic tissue-engineered anterior cruciate ligament replacement. Proc Natl Acad Sci U S A. 2007;104: 3049–54. doi: 10.1073/pnas.0608837104 17360607

7. Ma CB, Kawamura S, Deng X-H, Ying L, Schneidkraut J, Hays P, et al. Bone Morphogenetic Proteins-Signaling Plays A Role In Tendon-To-Bone Healing: A Study of rhBMP-2 and Noggin. Am J Sports Med. 2007;35: 597–604. doi: 10.1177/0363546506296312 17218656

8. Song EK, Rowe SM, Chung JY, Moon ES, Lee KB. Failure of Osteointegration of Hamstring Tendon Autograft After Anterior Cruciate Ligament Reconstruction. Arthrosc—J Arthrosc Relat Surg. 2004;20: 424–428. doi: 10.1016/j.arthro.2004.01.005 15067284

9. Rodeo SA, Suzuki K, Deng X, Wozney J, Warren RF. Use of Recombinant Human Bone Morphogenetic Protein-2 to Enhance Tendon Healing in a Bone Tunnel. Am J Sports Med. 1999;27: 476–488. doi: 10.1177/03635465990270041201 10424218

10. Yang HS, La WG, Cho YM, Shin W, Yeo GD, Kim BS. Comparison between heparin-conjugated fibrin and collagen sponge as bone morphogenetic protein-2 carriers for bone regeneration. Exp Mol Med. 2012;44: 350–355. doi: 10.3858/emm.2012.44.5.039 22322342

11. Schützenberger S, Schultz A, Hausner T, Hopf R, Zanoni G, Morton T, et al. The optimal carrier for BMP-2: A comparison of collagen versus fibrin matrix. Arch Orthop Trauma Surg. 2012;132: 1363–1370. doi: 10.1007/s00402-012-1551-2 22660797

12. Li RH, Bouxsein ML, Blake CA, D’Augusta D, Kim H, Li XJ, et al. rhBMP-2 injected in a calcium phosphate paste (α-BSM) accelerates healing in the rabbit ulnar osteotomy model. J Orthop Res. 2003;21: 997–1004. doi: 10.1016/S0736-0266(03)00082-2 14554211

13. Li Y, Li R, Hu J, Song D, Jiang X, Zhu S. Recombinant human bone morphogenetic protein-2 suspended in fibrin glue enhances bone formation during distraction osteogenesis in rabbits. Arch Med Sci. 2016;12: 494–501. doi: 10.5114/aoms.2016.59922 27279839

14. Van Der Stok J, Koolen MKE, De Maat MPM, Amin Yavari S, Alblas J, Patka P, et al. Full regeneration of segmental bone defects using porous titanium implants loaded with BMP-2 containing fibrin gels. Eur Cells Mater. 2015;29: 141–154. doi: 10.22203/eCM.v029a11 25738583

15. Kaipel M, Schützenberger S, Schultz A, Ferguson J, Slezak P, Morton TJ, et al. BMP-2 but not VEGF or PDGF in fibrin matrix supports bone healing in a delayed-union rat model. J Orthop Res. 2012;30: 1563–1569. doi: 10.1002/jor.22132 22508566

16. Murakami N, Saito N, Horiuchi H, Okada T, Nozaki K, Takaoka K. Repair of segmental defects in rabbit humeri with titanium fiber mesh cylinders containing recombinant human bone morphogenetic protein-2 (rhBMP-2) and a synthetic polymer. J Biomed Mater Res. 2002;62: 169–174. doi: 10.1002/jbm.10236 12209936

17. Milano G, Mulas PD, Ziranu F, Piras S, Manunta A, Fabbriciani C. Comparison Between Different Femoral Fixation Devices for ACL Reconstruction With Doubled Hamstring Tendon Graft: A Biomechanical Analysis. Arthrosc J Arthrosc Relat Surg. 2006;22: 660–668. doi: 10.1016/J.ARTHRO.2006.04.082 16762706

18. Madhavarapu S, Rao R, Libring S, Fleisher E, Yankannah Y, Freeman JW. Design and characterization of three-dimensional twist-braid scaffolds for anterior cruciate ligament regeneration. TECHNOLOGY. 2017;5: 98–106. doi: 10.1142/s2339547817500066

19. Erben RG. Embedding of Bone Samples in Methylmethacrylate: An Improved Method Suitable for Bone Histomorphometry, Histochemistry, and Immunohistochemistry. J Histochem Cytochem. 1997;45: 307–313. doi: 10.1177/002215549704500215 9016319

20. Villanueva AR, Mehr LA. Modifications of the Goldner and Gomori one-step trichrome stains for plastic-embedded thin sections of bone. Am J Med Technol. 1977;43: 536–8. Available: http://www.ncbi.nlm.nih.gov/pubmed/69401 69401

21. Thomopoulos S, Genin GM, Galatz LM. The development and morphogenesis of the tendon-to-bone insertion—what development can teach us about healing -. J Musculoskelet Neuronal Interact. 2010;10: 35–45. Available: http://www.ncbi.nlm.nih.gov/pubmed/20190378 20190378

22. Weiler A, Hoffmann RFG, Bail HJ, Rehm O, Südkamp NP. Tendon healing in a bone tunnel. Part II: Histologic analysis after biodegradable interference fit fixation in a model of anterior cruciate ligament reconstruction in sheep. Arthroscopy. 2002;18: 124–135. doi: 10.1053/jars.2002.30657 11830805

23. Fealy S, Rodeo SA, MacGillivray JD, Nixon AJ, Adler RS, Warren RF. Biomechanical evaluation of the relation between number of suture anchors and strength of the bone–tendon interface in a goat rotator cuff model. J Arthrosc Relat Surg. 2004;22: 595–602. doi: 10.1016/j.arthro.2006.03.008 16762696

24. Weimin P, Dan L, Yiyong W, Yunyu H, Li Z. Tendon-to-bone healing using an injectable calcium phosphate cement combined with bone xenograft/BMP composite. Biomaterials. 2013;34: 9926–9936. doi: 10.1016/j.biomaterials.2013.09.018 24075477

25. Ma CB, Kawamura S, Deng X-H, Ling Ying L, Schneidkraut J, Hays P, et al. Bone Morphogenetic Proteins-Signaling Plays a Role in Tendon-to-Bone Healing. Am J Sports Med. 2007;35: 597–604. doi: 10.1177/0363546506296312 17218656

26. Soon MYH, Hassan A, Hui JHP, Goh JCH, Lee EH. An Analysis of Soft Tissue Allograft Anterior Cruciate Ligament Reconstruction in a Rabbit Model. Am J Sports Med. 2007;35: 962–971. doi: 10.1177/0363546507300057 17400750

27. Lim JK, Hui J, Li L, Thambyah A, Goh J, Lee EH. Enhancement of tendon graft osteointegration using mesenchymal stem cells in a rabbit model of anterior cruciate ligament reconstruction. Arthrosc—J Arthrosc Relat Surg. 2004;20: 899–910. doi: 10.1016/S0749-8063(04)00653-X

28. Soon MYH, Hassan A, Hui JHP, Goh JCH, Lee EH. An Analysis of Soft Tissue Allograft Anterior Cruciate Ligament Reconstruction in a Rabbit Model. Am J Sports Med. 2007;35: 962–971. doi: 10.1177/0363546507300057 17400750

29. Dong Y, Zhang Q, Li Y, Jiang J, Chen S, Dong Y, et al. Enhancement of Tendon–Bone Healing for Anterior Cruciate Ligament (ACL) Reconstruction Using Bone Marrow-Derived Mesenchymal Stem Cells Infected with BMP-2. Int J Mol Sci. 2012;13: 13605–13620. doi: 10.3390/ijms131013605 23202970

30. Cheng P, Han P, Zhao C, Zhang S, Wu H, Ni J, et al. High-purity magnesium interference screws promote fibrocartilaginous entheses regeneration in the anterior cruciate ligament reconstruction rabbit model via accumulation of BMP-2 and VEGF. Biomaterials. 2016;81: 14–26. doi: 10.1016/j.biomaterials.2015.12.005 26713681

31. Hashimoto Y, Yoshida G, Toyoda H, Takaoka K. Generation of tendon-to-bone interface “enthesis” with use of recombinant BMP-2 in a rabbit model. J Orthop Res. 2007;25: 1415–24. doi: 10.1002/jor.20447 17557323

32. Pan W, Wei Y, Zhou L, Li D. Comparative in vivo study of injectable biomaterials combined with BMP for enhancing tendon graft osteointegration for anterior cruciate ligament reconstruction. J Orthop Res. 2011;29: 1015–1021. doi: 10.1002/jor.21351 21308754

33. Kim H-J, Kang S-W, Lim H-C, Han S-B, Lee J-S, Prasad L, et al. The Role of Transforming Growth Factor-β and Bone Morphogenetic Protein with Fibrin Glue in Healing of Bone-Tendon Junction Injury. Connect Tissue Res. 2007;48: 309–315. doi: 10.1080/03008200701692610 18075817

34. Guidoin MF, Marois Y, Bejui J, Poddevin N, King MW, Guidoin R. Analysis of retrieved polymer fiber based replacements for the ACL. Biomaterials. 2000;21: 2461–2474. doi: 10.1016/s0142-9612(00)00114-9 11055294

35. Hearle JWS. Atlas of fibre fracture and damage to textiles. 2nd ed. 2006.

36. Walsh WR, Bertollo N, Arciero RA, Stanton RA, Poggie RA. Long-term In-vivo Evaluation Of A Resorbable PLLA Scaffold For Regeneration Of The ACL. Orthop J Sport Med. 2015;3. doi: 10.1177/2325967115S00033

37. Miura K, Woo SL-Y, Brinkley R, Fu Y-C, Noorani S. Effects of Knee Flexion Angles for Graft Fixation on Force Distribution in Double-Bundle Anterior Cruciate Ligament Grafts. Am J Sports Med. 2006;34: 577–585. doi: 10.1177/0363546505281814 16282574

38. Mansour JM, Wentorf FA, Degoede KM. In Vivo Kinematics of the Rabbit Knee in Unstable Models of Osteoarthrosis. Ann Biomed Eng. 1998;26: 353–360. doi: 10.1114/1.133 9570218

39. Fleming B, Beynnon BD, Johnson RJ, McLeod WD, Pope MH. Isometric versus tension measurements. A comparison for the reconstruction of the anterior cruciate ligament. Am J Sports Med. 1993;21: 82–88. doi: 10.1177/036354659302100115 8427374

40. Carson E, Anisko E, Restrepo C, Panariello R, O’Brien S, Warren R. Revision Anterior Cruciate Ligament Reconstruction–Etiology of Failures and Clinical Results. J Knee Surg. 2010;17: 127–132. doi: 10.1055/s-0030-1248210 15366266

41. Nicholas SJ, D’Amato MJ, Mullaney MJ, Tyler TF, Kolstad K, McHugh MP. A Prospectively Randomized Double-Blind Study on the Effect of Initial Graft Tension on Knee Stability after Anterior Cruciate Ligament Reconstruction. Am J Sports Med. 2004;32: 1881–1886. doi: 10.1177/0363546504265924 15572316

42. Numazaki H, Tohyama H, Nakano H, Kikuchi S, Yasuda K. The Effect of Initial Graft Tension in Anterior Cruciate Ligament Reconstruction on the Mechanical Behaviors of the Femur-Graft-Tibia Complex during Cyclic Loading. Am J Sports Med. 2002;30: 800–805. doi: 10.1177/03635465020300060801 12435644

43. Kim SG, Kurosawa H, Sakuraba K, Ikeda H, Takazawa S. The effect of initial graft tension on postoperative clinical outcome in anterior cruciate ligament reconstruction with semitendinosus tendon. Arch Orthop Trauma Surg. 2006;126: 260–264. doi: 10.1007/s00402-005-0045-x 16193302

44. Yoshiya S, Andrish JT, Manley MT, Bauer TW. Graft tension in anterior cruciate ligament reconstruction. An in vivo study in dogs. Am J Sports Med. 1987;15: 464–470. doi: 10.1177/036354658701500506 3674269

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