Achilles tendon (AT) midsubstance injuries may heal suboptimally, especially in athletes. Transforming growth factor–beta 3 (TGF-β3) shows promise because of its recently discovered tendinogenic effects. Using poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles (NPs) may enhance the results by a sustained-release effect.
Hypothesis:
The application of TGF-β3 will enhance AT midsubstance healing, and the NP form will achieve better outcomes.
Study Design:
Controlled laboratory study.
Methods:
A total of 80 rats underwent unilateral AT transection and were divided into 4 groups: (1) control (C); (2) empty chitosan film (Ch); (3) chitosan film containing free TGF-β3 (ChT); and (4) chitosan film containing TGF-β3–loaded NPs (ChN). The animals were sacrificed at 3 and 6 weeks. Tendons were evaluated for morphology (length and cross-sectional area [CSA]), biomechanics (maximum load, stress, stiffness, and elastic modulus), histology, immunohistochemical quantification (types I and III collagen [COL1 and COL3]), and gene expression (COL1A1, COL3A1, scleraxis, and tenomodulin).
Results:
Morphologically, at 3 weeks, ChT (15 ± 2.7 mm) and ChN (15.6 ± 1.6 mm) were shorter than C (17.6 ± 1.8 mm) (P = .019 and = .004, respectively). At 6 weeks, the mean CSA of ChN (10.4 ± 1.9 mm2) was similar to that of intact tendons (6.4 ± 1.1 mm2) (P = .230), while the other groups were larger. Biomechanically, at 3 weeks, ChT (42.8 ± 4.9 N) had a higher maximum load than C (27 ± 9.1 N; P = .004) and Ch (29.2 ± 5.7 N; P = .005). At 6 weeks, ChN (26.9 ± 3.9 MPa) had similar maximum stress when compared with intact tendons (34.1 ± 7.8 MPa) (P = .121); the other groups were significantly lower. Histologically, at 6 weeks, the mean Movin score of ChN (4.5 ± 1.5) was lower than that of ChT (6.3 ± 1.8). Immunohistochemically, ChN had higher COL3 (1.469 ± 0.514) at 3 weeks and lower COL1 (1.129 ± 0.368) at 6 weeks. COL1A1 gene expression was higher in ChT and ChN at 3 weeks, but COL3A1 gene expression was higher in ChN.
Conclusion:
The application of TGF-β3 had a positive effect on AT midsubstance healing, and the sustained-release NP form improved the outcomes, more specifically accelerating the remodeling process.
Clinical Relevance:
This study demonstrated the effectiveness of TGF-β3 on tendon healing on a rat model, which is an important step toward clinical use. The novel method of using PLGA-b-PEG NPs as a drug-delivery system with sustained-release properties had promising results.
AlexisFPridgenEMolnarLKFarokhzadOC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm. 2008;5(4):505-515.
2.
ArdernCLGlasgowPSchneidersA, et al. 2016 Consensus statement on return to sport from the First World Congress in Sports Physical Therapy, Bern. Br J Sports Med. 2016;50(14):853-864.
3.
BarsbyTGuestD. Transforming growth factor beta3 promotes tendon differentiation of equine embryo-derived stem cells. Tissue Eng Part A. 2013;19(19-20):2156-2165.
4.
BeredjiklianPKFavataMCartmellJS, et al. Regenerative versus reparative healing in tendon: a study of biomechanical and histological properties in fetal sheep. Ann Biomed Eng. 2003;31(10):1143-1152.
5.
BerthetEChenCButcherK, et al. Smad3 binds Scleraxis and Mohawk and regulates tendon matrix organization. J Orthop Res. 2013;31(9):1475-1483.
6.
CaldwellJEVossellerJT. Maximizing return to sports after Achilles tendon rupture in athletes. Foot Ankle Clin. 2019;24(3):439-445.
7.
ChailakhyanRKKonEShekhterAB, et al. Autologous bone marrow-derived mesenchymal stem cells provide complete regeneration in a rabbit model of the Achilles tendon bundle rupture. Int Orthop. 2021;45(12):3263-3276.
8.
ChanKMFuSCWongYP, et al. Expression of transforming growth factor beta isoforms and their roles in tendon healing. Wound Repair Regen. 2008;16(3):399-407.
9.
ChoHGaoJKwonGS. PEG-b-PLA micelles and PLGA-b-PEG-b-PLGA sol-gels for drug delivery. J Control Release. 2016;240:191-201.
10.
ChoiBKimSLinBWuBMLeeM. Cartilaginous extracellular matrix-modified chitosan hydrogels for cartilage tissue engineering. ACS Appl Mater Interfaces. 2014;6(22):20110-20121.
11.
CooperRRMisolS. Tendon and ligament insertion. A light and electron microscopic study. J Bone Joint Surg Am. 1970;52(1):1-20.
12.
da SilvaFSAbreuBJErikssonBIAckermannPW. Complete mid-portion rupture of the rat Achilles tendon leads to remote and time-mismatched changes in uninjured regions. Knee Surg Sports Traumatol Arthrosc. 2021;29(6):1990-1999.
13.
DengSSunZZhangCChenGLiJ. Surgical treatment versus conservative management for acute Achilles tendon rupture: a systematic review and meta-analysis of randomized controlled trials. J Foot Ankle Surg. 2017;56(6):1236-1243.
14.
DymentNAKazemiNAschbacher-SmithLE, et al. The relationships among spatiotemporal collagen gene expression, histology, and biomechanics following full-length injury in the murine patellar tendon. J Orthop Res. 2012;30(1):28-36.
15.
FangCHLinYWSunJSLinFH. The chitosan/tri-calcium phosphate bio-composite bone cement promotes better osteo-integration: an in vitro and in vivo study. J Orthop Surg Res. 2019;14(1):162.
16.
FergusonMWDuncanJBondJ, et al. Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies. Lancet. 2009;373(9671):1264-1274.
17.
GeZTangHChenW, et al. Downregulation of type I collagen expression in the Achilles tendon by dexamethasone: a controlled laboratory study. J Orthop Surg Res. 2020;15(1):70.
18.
GoncalvesAIBerdeckaDRodriguesMT, et al. Evaluation of tenogenic differentiation potential of selected subpopulations of human adipose-derived stem cells. J Tissue Eng Regen Med. 2019;13(12):2204-2217.
19.
HanBJonesIAYangZFangWVangsnessCTJr.Repair of rotator cuff tendon defects in aged rats using a growth factor injectable gel scaffold. Arthroscopy. 2020;36(3):629-637.
20.
HavisEBonninMAEsteves de LimaJ, et al. TGFβ and FGF promote tendon progenitor fate and act downstream of muscle contraction to regulate tendon differentiation during chick limb development. Development. 2016;143(20):3839-3851.
21.
IndinoCD’AmbrosiRUsuelliFG. Biologics in the treatment of Achilles tendon pathologies. Foot Ankle Clin. 2019;24(3):471-493.
22.
JamesRKesturuGBalianGChhabraAB. Tendon: biology, biomechanics, repair, growth factors, and evolving treatment options. J Hand Surg Am. 2008;33(1):102-112.
23.
JandackaDSilvernailJFUchytilJ, et al. Do athletes alter their running mechanics after an Achilles tendon rupture?J Foot Ankle Res. 2017;10:53.
24.
JavidiMMcGowanCPSchieleNRLinDC. Tendons from kangaroo rats are exceptionally strong and tough. Sci Rep. 2019;9(1):8196.
25.
JunejaSCSchwarzEMO’KeefeRJAwadHA. Cellular and molecular factors in flexor tendon repair and adhesions: a histological and gene expression analysis. Connect Tissue Res. 2013;54(3):218-226.
26.
KajiDAHowellKLBalicZHubmacherDHuangAH. Tgfβ signaling is required for tenocyte recruitment and functional neonatal tendon regeneration. Elife. 2020;9:e51779.
27.
KangasJPajalaAOhtonenPLeppilahtiJ. Achilles tendon elongation after rupture repair: a randomized comparison of 2 postoperative regimens. Am J Sports Med. 2007;35(1):59-64.
28.
KeeneDJAlsousouJHarrisonP, et al. Platelet rich plasma injection for acute Achilles tendon rupture: PATH-2 randomised, placebo controlled, superiority trial. BMJ. 2019;367:l6132.
29.
KleinMBYalamanchiNPhamHLongakerMTChangJ. Flexor tendon healing in vitro: effects of TGF-beta on tendon cell collagen production. J Hand Surg Am. 2002;27(4):615-620.
30.
KorntnerSKunkelNLehnerC, et al. A high-glucose diet affects Achilles tendon healing in rats. Sci Rep. 2017;7(1):780.
31.
KovacevicDFoxAJBediA, et al. Calcium-phosphate matrix with or without TGF-β3 improves tendon-bone healing after rotator cuff repair. Am J Sports Med. 2011;39(4):811-819.
32.
LeeJSBaekSDVenkatesanJ, et al. In vivo study of chitosan-natural nano hydroxyapatite scaffolds for bone tissue regeneration. Int J Biol Macromol. 2014;67:360-366.
33.
LehnerCGehwolfRHirzingerC, et al. Bupivacaine induces short-term alterations and impairment in rat tendons. Am J Sports Med. 2013;41(6):1411-1418.
34.
LemmeNJLiNYDeFrodaSFKleinerJOwensBD. Epidemiology of Achilles tendon ruptures in the United States: athletic and nonathletic injuries from 2012 to 2016. Orthop J Sports Med. 2018;6(11):2325967118808238.
35.
MajewskiMOchsnerPELiuFFluckigerREvansCH. Accelerated healing of the rat Achilles tendon in response to autologous conditioned serum. Am J Sports Med. 2009;37(11):2117-2125.
36.
ManningCNKimHMSakiyama-ElbertS, et al. Sustained delivery of transforming growth factor beta three enhances tendon-to-bone healing in a rat model. J Orthop Res. 2011;29(7):1099-1105.
37.
MorilleMVan-ThanhTGarricX, et al. New PLGA-P188-PLGA matrix enhances TGF- β3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells. J Control Release. 2013;170(1):99-110.
38.
MovinTGadAReinholtFPRolfC. Tendon pathology in long-standing achillodynia. Biopsy findings in 40 patients. Acta Orthop Scand. 1997;68(2):170-175.
39.
MullerSADurselenLHeisterbachPEvansCMajewskiM. Effect of a simple collagen type I sponge for Achilles tendon repair in a rat model. Am J Sports Med. 2016;44(8):1998-2004.
40.
MullerSAEvansCHHeisterbachPEMajewskiM. The role of the paratenon in Achilles tendon healing: a study in rats. Am J Sports Med. 2018;46(5):1214-1219.
41.
MullerSAQuirkNPMuller-LebschiJA, et al. Response of the injured tendon to growth factors in the presence or absence of the paratenon. Am J Sports Med. 2019;47(2):462-467.
42.
MuxikaAEtxabideAUrangaJGuerreroPde la CabaK. Chitosan as a bioactive polymer: processing, properties and applications. Int J Biol Macromol. 2017;105(Pt 2):1358-1368.
43.
OkamotoNKushidaTOeK, et al. Treating Achilles tendon rupture in rats with bone-marrow-cell transplantation therapy. J Bone Joint Surg Am. 2010;92(17):2776-2784.
44.
OmachiTSakaiTHiraiwaH, et al. Expression of tenocyte lineage-related factors in regenerated tissue at sites of tendon defect. J Orthop Sci. 2015;20(2):380-389.
45.
OshiroWLouJXingXTuYManskePR. Flexor tendon healing in the rat: a histologic and gene expression study. J Hand Surg Am. 2003;28(5):814-823.
46.
ParkJSYangHJWooDG, et al. Chondrogenic differentiation of mesenchymal stem cells embedded in a scaffold by long-term release of TGF-β 3 complexed with chondroitin sulfate. J Biomed Mater Res A. 2010;92(2):806-816.
47.
PennJWGrobbelaarAORolfeKJ. The role of the TGF-β family in wound healing, burns and scarring: a review. Int J Burns Trauma. 2012;2(1):18-28.
48.
Perucca OrfeiCViganoMPearsonJR, et al. In vitro induction of tendon-specific markers in tendon cells, adipose- and bone marrow-derived stem cells is dependent on TGFβ3, BMP-12 and ascorbic acid stimulation. Int J Mol Sci. 2019;20(1):149.
49.
PoniatowskiLAWojdasiewiczPGasikRSzukiewiczD. Transforming growth factor beta family: insight into the role of growth factors in regulation of fracture healing biology and potential clinical applications. Mediators Inflamm. 2015;2015:137823.
50.
PryceBAWatsonSSMurchisonND, et al. Recruitment and maintenance of tendon progenitors by TGFbeta signaling are essential for tendon formation. Development. 2009;136(8):1351-1361.
51.
RavindranSRoamJLNguyenPK, et al. Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-β3. Biomaterials. 2011;32(33):8436-8445.
52.
RhimJWHongSIParkHMNgPK. Preparation and characterization of chitosan-based nanocomposite films with antimicrobial activity. J Agric Food Chem. 2006;54(16):5814-5822.
53.
RibeiroJCVVieiraRSMeloIMAraujoVMALimaV. Versatility of chitosan-based biomaterials and their use as scaffolds for tissue regeneration. ScientificWorldJournal. 2017;2017:8639898.
54.
RothSPSchubertSScheibeP, et al. Growth factor-mediated tenogenic induction of multipotent mesenchymal stromal cells is altered by the microenvironment of tendon matrix. Cell Transplant. 2018;27(10):1434-1450.
55.
RothrauffBBYangGTuanRS. Tissue-specific bioactivity of soluble tendon-derived and cartilage-derived extracellular matrices on adult mesenchymal stem cells. Stem Cell Res Ther. 2017;8(1):133.
56.
SantosTCDRescignanoNBoffL, et al. Manufacture and characterization of chitosan/PLGA nanoparticles nanocomposite buccal films. Carbohydr Polym. 2017;173:638-644.
57.
SchneiderCARasbandWSEliceiriKW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-675.
58.
ShahMForemanDMFergusonMW. Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci. 1995;108 (pt 3):985-1002.
59.
SuchakAABostickGPBeaupreLADurandDCJomhaNM. The influence of early weight-bearing compared with non-weight-bearing after surgical repair of the Achilles tendon. J Bone Joint Surg Am. 2008;90(9):1876-1883.
60.
TadaDBSinghSNageshaD, et al. Chitosan film containing poly(D, L-lactic-co-glycolic acid) nanoparticles: a platform for localized dual-drug release. Pharm Res. 2010;27(8):1738-1745.
61.
TempferHKaser-EichbergerALehnerC, et al. Bevacizumab improves Achilles tendon repair in a rat model. Cell Physiol Biochem. 2018;46(3):1148-1158.
62.
ThomopoulosSParksWCRifkinDBDerwinKA. Mechanisms of tendon injury and repair. J Orthop Res. 2015;33(6):832-839.
63.
Tirado-RodriguezBOrtegaESegura-MedinaPHuerta-YepezS. TGF- β: an important mediator of allergic disease and a molecule with dual activity in cancer development. J Immunol Res. 2014;2014:318481.
64.
TrofaDPMillerJCJangES, et al. Professional athletes’ return to play and performance after operative repair of an Achilles tendon rupture. Am J Sports Med. 2017;45(12):2864-2871.
65.
WangDPunCCMHuangS, et al. Tendon-derived extracellular matrix induces mesenchymal stem cell tenogenesis via an integrin/transforming growth factor-β crosstalk-mediated mechanism. FASEB J. 2020;34(6):8172-8186.
66.
WangWMengQLiQ, et al. Chitosan derivatives and their application in biomedicine. Int J Mol Sci. 2020;21(2):487.
67.
Yabanoglu-CiftciSBaysalIErikciAArıcaBUcarG. Transforming growth factor-β3 (TGF-β3) loaded PLGA-b-PEG nanoparticles: efficacy in preventing cardiac fibrosis induced by TGF-β1. J Drug Delivery Sci Technol. 2018;48:223-234.
68.
YanWXuXXuQ, et al. An injectable hydrogel scaffold with kartogenin-encapsulated nanoparticles for porcine cartilage regeneration: a 12-month follow-up study. Am J Sports Med. 2020;48(13):3233-3244.
69.
YangGRothrauffBBLinHYuSTuanRS. Tendon-derived extracellular matrix enhances transforming growth factor-β3-induced tenogenic differentiation of human adipose-derived stem cells. Tissue Eng Part A. 2017;23(3-4):166-176.
70.
YangQQShaoYXZhangLZZhouYL. Therapeutic strategies for flexor tendon healing by nanoparticle-mediated co-delivery of bFGF and VEGFA genes. Colloids Surf B Biointerfaces. 2018;164:165-176.
71.
ZhangYYuJZhangJHuaY. Simvastatin with PRP promotes chondrogenesis of bone marrow stem cells in vitro and wounded rat Achilles tendon-bone interface healing in vivo. Am J Sports Med. 2019;47(3):729-739.
72.
ZhengDDanYYangSH, et al. Controlled chondrogenesis from adipose-derived stem cells by recombinant transforming growth factor-β3 fusion protein in peptide scaffolds. Acta Biomater. 2015;11:191-203.
73.
ZouJMoXShiZ, et al. A prospective study of platelet-rich plasma as biological augmentation for acute Achilles tendon rupture repair. Biomed Res Int. 2016;2016:9364170.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
