Tissue inhibitor of metalloproteinases-3 (TIMP-3) maintains a healthy extracellular matrix by regulating matrix metalloproteinases (MMP), disintegrin–metalloproteinases (ADAM), and disintegrin–metalloproteinases with ThromboSpondin-like motifs (ADAMTS) activity. Currently, there is a need for a comprehensive understanding of the effects of TIMP-3 on the bone quality and integrity. In this study, we examined the mechanical, morphological, and compositional properties of TIMP-3 knock out (Timp-3−/−) mouse bone. We hypothesize that the lack of TIMP-3 plays an important role in maintaining the overall bone integrity. Mechanical properties of humeri, lumbar vertebrae, and femurs from Timp-3−/− mice were determined using 3-point bending, compression, and notched 3-point bending, respectively. Morphological properties of the humeral cortical and trabecular bone and the caudal vertebrae cortical bone were evaluated using micro-computed tomography, while the composition of the femoral cortical and trabecular bone was examined using Fourier transform infrared spectroscopic imaging. Our results revealed that the integrity of the Timp-3−/− bone is compromised due to changes in its composition, structure, and mechanics. Reductions in the yield and ultimate load and stress capacity, and loss in bone fracture toughness were attributed to reduced density and thickness, and increased porosity of cortical bone. Thin trabeculae were dense, highly connected, and closely packed in Timp-3−/− bone. Furthermore, altered cortical and trabecular bone mineralization and increased compositional heterogeneity were found in Timp-3−/− bone, all being indicative of high bone remodeling. In conclusion, this study suggests that the lack of TIMP-3 is detrimental to bone development and maintenance.
Tissue inhibitor of metalloproteinase-3 Bone strength Bone fracture Bone tissue mineral density Bone structure Bone composition
This is a preview of subscription content, log in to check access.
The authors are grateful for the provision of Timp-3−/− mice by Dr. Rama Khokha (Toronto, Canada). This study was funded by the Florida Institute of Technology and partly supported by Arthritis Research UK (ARUK) grant 20039 awarded to GB and by grants awarded to AAP by ARUK (20581) and Biotechnology and Biological Sciences Research Council (BB/J003727/1).
Compliance with Ethical Standards
Conflict of interest
The authors Brendyn Miller, Lyudmila Spevak, Lyudmila Lukashova, Behzad Javaheri, Andrew A. Pitsillides, Adele Boskey, George Bou-Gharios, and Alessandra Carriero declare that they have no conflict of interest.
Human and Animal Rights and Informed Consent
Mice were maintained under standard laboratory conditions and experiments were conducted in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for reporting. Briefly, mice were housed up to 4 per cage in polypropylene cages with wood chip and paper bedding and provided standard rodent maintenance diet and water ad libitum throughout the study. All procedures complied with the UK Animals (Scientific Procedures) Act 1986 and were reviewed and approved by UK Home Office and local ethics committee.
Brew K, Nagase H (2010) The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity. Biochimica et Biophysica Acta (BBA)-Mol Cell Res 1803(1):55–71CrossRefGoogle Scholar
Zhao H et al (2004) Differential inhibition of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3 regulates pro-MMP-2 activation. J Biol Chem 279(10):8592–8601CrossRefPubMedGoogle Scholar
Apte SS, Parks WC (2015) Metalloproteinases: A parade of functions in matrix biology and an outlook for the future. Matrix Biol 44:1–6CrossRefPubMedGoogle Scholar
Maretzky T et al (2009) Characterization of the catalytic activity of the membrane-anchored metalloproteinase ADAM15 in cell-based assays. Biochem J 420(1):105–113CrossRefPubMedGoogle Scholar
Cruz-Munoz W, Kim I, Khokha R (2006) TIMP-3 deficiency in the host, but not in the tumor, enhances tumor growth and angiogenesis. Oncogene 25(4):650–655PubMedGoogle Scholar
Verstappen J, Von den Hoff JW (2006) Tissue inhibitors of metalloproteinases (TIMPs): their biological functions and involvement in oral disease. J Dent Res 85(12):1074–1084CrossRefPubMedGoogle Scholar
Weber BH et al (1994) Mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with Sorsby’s fundus dystrophy. Nat Genet 8(4):352–356CrossRefPubMedGoogle Scholar
Sahebjam S, Khokha R, Mort JS (2007) Increased collagen and aggrecan degradation with age in the joints of Timp3−/− mice. Arthritis Rheumatism 56(3):905–909CrossRefPubMedGoogle Scholar
Mohammed FF et al (2004) Abnormal TNF activity in Timp3−/− mice leads to chronic hepatic inflammation and failure of liver regeneration. Nat Genet 36(9):969–977CrossRefPubMedGoogle Scholar
Janssen A et al (2008) Abnormal vessel formation in the choroid of mice lacking tissue inhibitor of metalloprotease-3. Investig Ophthalmol Vis Sci 49(7):pp. 2812–2822CrossRefGoogle Scholar
Gill SE et al (2003) A null mutation for Tissue Inhibitor of Metalloproteinases-3 (Timp-3) impairs murine bronchiole branching morphogenesis. Dev Biol 261(2):313–323CrossRefPubMedGoogle Scholar
Federici M et al (2005) Timp3 deficiency in insulin receptor–haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-α. J Clin Invest 115(12):3494–3505CrossRefPubMedPubMedCentralGoogle Scholar
Javaheri B et al (2016) Deficiency and also transgenic overexpression of Timp-3 both lead to compromised bone mass and architecture in vivo. PLoS ONE 11(8):e0159657CrossRefPubMedPubMedCentralGoogle Scholar
Carriero A et al (2014) A methodology for the investigation of toughness and crack propagation in mouse bone. J Mech Behav Biomed Mater 39:38–47CrossRefPubMedGoogle Scholar
Bouxsein ML et al (2010) Guidelines for assessment of bone microstructure in rodents using micro–computed tomography. J Bone Miner Res 25(7):1468–1486CrossRefPubMedGoogle Scholar
Carriero A et al (2014) Altered lacunar and vascular porosity in osteogenesis imperfecta mouse bone as revealed by synchrotron tomography contributes to bone fragility. Bone 61:116–124CrossRefPubMedGoogle Scholar
Paschalis EP et al (2015) Fourier transform Infrared spectroscopic characterization of mineralizing type I collagen enzymatic trivalent cross-links. Calcif Tissue Int 96(1):18–29CrossRefPubMedGoogle Scholar
Martin EL et al (2007) Lung mechanics in the TIMP3 null mouse and its response to mechanical ventilation. Exp Lung Res 33(2):99–113CrossRefPubMedGoogle Scholar
Hammoud L et al (2011) Deficiency in TIMP-3 increases cardiac rupture and mortality post-myocardial infarction via EGFR signaling: beneficial effects of cetuximab. Basic Res Cardiol 106(3):459–471CrossRefPubMedGoogle Scholar
Bruni-Cardoso A et al (2010) Osteoclast-derived matrix metalloproteinase-9 directly affects angiogenesis in the prostate tumor–bone microenvironment. Mol Cancer Res 8(4):459–470CrossRefPubMedPubMedCentralGoogle Scholar
Ural A, Vashishth D (2007) Effects of intracortical porosity on fracture toughness in aging human bone: a μCT-based cohesive finite element study. J Biomech Eng 129(5):625–631CrossRefPubMedGoogle Scholar
Shigdel R et al (2015) Bone turnover markers are associated with higher cortical porosity, thinner cortices, and larger size of the proximal femur and non-vertebral fractures. Bone 81:1–6CrossRefPubMedGoogle Scholar
Bell K et al (1999) Intracapsular hip fracture: increased cortical remodeling in the thinned and porous anterior region of the femoral neck. Osteoporos Int 10(3):248–257CrossRefPubMedGoogle Scholar
Isaksson H et al (2010) Infrared spectroscopy indicates altered bone turnover and remodeling activity in renal osteodystrophy. J Bone Miner Res 25(6):1360–1366CrossRefPubMedGoogle Scholar
Spevak L et al (2013) Fourier transform infrared spectroscopic imaging parameters describing acid phosphate substitution in biologic hydroxyapatite. Calcif Tissue Int 92(5):418–428CrossRefPubMedPubMedCentralGoogle Scholar
Donnelly E et al (2012) Reduced cortical bone compositional heterogeneity with bisphosphonate treatment in postmenopausal women with intertrochanteric and subtrochanteric fractures. J Bone Miner Res 27(3):672–678CrossRefPubMedPubMedCentralGoogle Scholar
Marisa ME et al (2016) Paracrystalline disorder from phosphate ion orientation and substitution in synthetic bone mineral. Inorg Chem 55(23):12290–12298CrossRefPubMedGoogle Scholar
Ohuchi E et al (1997) Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem 272(4):2446–2451CrossRefPubMedGoogle Scholar
Inada M et al (2004) Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci U S A 101(49):17192–17197CrossRefPubMedPubMedCentralGoogle Scholar
Garnero P et al (2003) The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation. J Bone Miner Res 18(5):859–867CrossRefPubMedGoogle Scholar
Holmbeck K et al (1999) MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 99(1):81–92CrossRefPubMedGoogle Scholar