This review summarizes the genetic alterations and knockdown approaches published in the literature to assess the role of key proteoglycans and glycoproteins in the structural development, function, and repair of tendon, ligament, and enthesis. The information was collected from (i) genetically altered mice, (ii)
Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit consists of a core protein with one or more covalently attached GAG chain(s) at specific site(s) [
A tendon is a compositionally complex tissue with a predominantly mechanical function: translating muscular contractions into joint movement by transmitting forces from muscle to bone [
(a) Longitudinal section of FDL tendon from a 3-month old mouse. Tendon is composed of parallel collagen fibers and cells aligned (dark blue) in the direction of fibers. Gliding space is shown. (b) The normal structure of fibrocartilaginous entheses. The 4 zones of tissue at the insertion of the human supraspinatus tendon: dense fibrous connective tissue (CT), uncalcified fibrocartilage (UF), calcified fibrocartilage (CF), and bone (B). Reprinted from Benjamin and Ralphs (1998) [
(a) Masson’s trichrome staining of postnatal day 1 patella with patellar and quadriceps tendon in mouse at 4x. (b) Higher magnification (100x) shows patellar tendon composed of parallel collagen fibers (blue) with cells aligned almost parallel to fibers. Enthesis in rectangle (i.e., quadriceps tendon insertion on patella) is shown (a). Higher magnification of enthesis at 20x (c) and at 100x ((d) and (e)). Collagen fibers (blue) show penetration on patella (d) and ending on patella ((e), yellow arrows).
Enthesis (plural, entheses) is the point at which a tendon or ligament or muscle inserts into bone, where the collagen fibers are mineralized and integrated into bone tissue. There are two types: fibrous entheses and fibrocartilaginous entheses. In a fibrous enthesis, the collagenous tendon or ligament directly attaches to the bone, whilst the fibrocartilaginous enthesis displays 4 zones during the transition from tendon/ligament to bone: (i) tendon area displaying longitudinally oriented fibroblasts and a parallel arrangement of collagen fibers, (ii) a fibrocartilaginous region of variable thickness where the structure of the cells changes to chondrocytes, (iii) an abrupt transition from cartilaginous to calcified fibrocartilage—the so-called tidemark, and (iv) bone [
Tendon or ligament and bone display dramatically different mechanical behavior [
Gene knockout mice [
Tendon, enthuses, and ligament defects in response to genetic alterations: role of proteoglycans and glycoproteins.
Genetic mutation, altered variants, and knockdown approach | Tendon, ligament, and enthesis defects or predisposition to injury | Other tissues/organs affected, in brief and human disease | References |
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Fibromodulin-deficient ( |
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Mice deficient in SLRPs ( |
The double homozygote mutant mice |
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Mice lacking both lumican and fibromodulin exhibit knee dysmorphogenesis, and extreme tendon weakness is the cause for joint laxity in mutant. Fmod deficiency alone leads to significant reduction in tendon stiffness but further loss in stiffness is demonstrated by the loss of Lum in a dose-dependent way. A disproportionate increase in small-diameter collagen fibrils in the |
Double homozygote mutants |
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Decorin-deficient mice exhibit abnormal collagen fibrillogenesis in tail tendon and a decrease in collagen-associated proteoglycans. Using TEM analysis, tendon fibrils showed irregular and ragged outlines in cross-sections in |
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Periodontal ligament is adversely affected by decorin deficiency in mouse. Using SEM, |
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Decorin deficiency alters structure and biomechanical properties of tendon during development. Bgn expression increased substantially in Dcn-deficient tendons suggesting a potential functional compensation. FDL tendon of |
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Patellar tendon retrieved from mature |
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Injured |
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The lack of Dse affects the epimerization of glucuronic acid to iduronic acid in glycosaminoglycans (GAGs) and affects the tendon properties. In tail tendon, in |
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The SLRPs Lum, Fmod, and Dcn coordinately regulate the fibrillar organization of collagen in the PDL. These three SLRPs coexpressed with type I collagen in gingival and PDL. |
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Alteration in matrix proteins in tail tendon affects quasilinear viscoelastic properties. Uniaxial tensile stress-relaxation experiments were performed on tail tendon fascicles taken from mice at different developmental age and genotype groups: 8-week |
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Viscoelasticity of tail tendon fascicle is affected by Dcn content but not by collagen alterations. Mechanical properties of tail tendon fascicles were assessed in different genotype and age group of mice: 8-week |
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In a large multivariate model, glycosaminoglycan (GAG) content is the largest predictor of mechanical properties. Tendon fascicle structure-function relationship was established in transgenic models using multiple regression models. Relative contributions of seven different structural and compositional variables were used in predicting tissue mechanical properties through the use of multiple regression statistical models. Structural, biochemical, and mechanical analyses were performed on tail tendon fascicles from different groups of transgenic mice as shown in the last reference [ |
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Lack of biglycan adversely affects the mechanical property of the healing bone insertion site of the patellar tendon (PT) fibers. For that, the authors compared 12-week-old hemizygous |
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Lack of biglycan compromises the integrity of periodontal tissue. IHC of SLRPs indicated that Bgn is expressed in PDL, alveolar bone (AB), at the AB-PDL, and cementum-PDL attachment sites in WT mice. Histomorphometric analysis of |
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Abnormal collagen fibrils in tendons of Bgn/Fmod-deficient mice lead to gait impairment, ectopic ossification, and osteoarthritis. Collagen fibrils in tendons from Dbl-KO mice are structurally and mechanically altered resulting in unstable joints. The mice progressively develop gait impairment, tendon ectopic ossification (EO), and severe premature OA. Daily forced running of |
Bgn and Fmod double deficient mice exhibit premature OA and predisposition to OA. The Dbl-KO mouse represents a model for spontaneous OA, early onset and rapid progression of OA. | [ | |
Mice deficient in biglycan and fibromodulin ( |
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Mice deficient in biglycan and fibromodulin develop ectopic ossification (EO) in tendon with aging. At the age of 3 months, |
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Collagen fibrillogenesis is altered in tail tendon of |
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Tendons are tailored according to their specific location and function. Mechanical properties of tail tendon fascicles, patellar tendon (PT), and FDL tendon, each differing in their |
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Decorin deficiency protects aged tendons. Multiple properties of patellar tendon (PT) from mature (age, 150 days) and aged mice (age, 570 days) were studied in |
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Decorin and biglycan contribute to tendon’s response to load, in particular with realignment of collagen fibers. Isolated supraspinatus tendon (SSTs) from Bgn- and Dcn-deficient mice, at three different age groups (90 days, 300 days, and 570 days), were tested in tension. Changes in realignment in WT tendons showed a decreased response to load with aging. Tendons at 300 days and 570 days do not realign their collagen fibers until the linear region, a later response than 90-day tendons. This result in WT tendon midsubstance is indicative of a breakdown of the structural organization of tendon over time. The proteoglycan-deficient tendons showed altered mechanical properties with age, predominantly at the insertion site. However, changes in realignment throughout age were not found in the midsubstance of the Bgn-deficient tendons or at the insertion of Dcn-deficient tendons. Lack of these proteoglycans may shield the tendon from deteriorating effects. Changes in mechanical properties did not occur in concert with changes in collagen fiber realignment, suggesting that typical mechanical property measurements alone are not sufficient to describe how structural alterations affect tendon’s response to load. | N/A | [ | |
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Knockdown of |
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MicroRNAs, miR-21, and miR-101 regulate asporin/PLAP-1 expression in PDL cells. Bioinformatic analysis predicted miRNAs that potentially regulate the gene expression of asporin. Dual luciferase reporter assay and qRT-PCR showed the effects of miR-21 and miR-101 on asporin gene expression. The results indicated that miR-21 and miR-101 target asporin to regulate its expression during osteogenic differentiation of PDL cells. | N/A | [ |
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Lack of thrombospondin 2 results in connective tissue abnormalities associated with disordered collagen fibrillogenesis. Tail tendon exhibited abnormalities in |
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Thrombospondin 2 modulates cell-matrix interaction during postnatal development of tendon. Developing hind limb flexor muscle tendon showed abnormalities in fibroblast-collagen fibril interaction in |
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Spp1 plays role in tendon remodeling after denervation-induced mechanical stress deprivation. Six-week-old males demonstrated mechanical stress deprivation in quadriceps femoris muscle after femoral nerve denervation. Patellar tendon underwent dynamic remodeling as evidenced by collagen fibril degradation. Transient upregulation of SPP1 expression was observed in early phase followed by induction of MMP13 during patellar tendon remodeling in WT animals, the later involved degradation of collagen. MMP-13 gene expression increased 20.7-fold at day 14 after stress deprivation in WT mice and 4.1-fold in mutants. Thus Spp1 plays a crucial role in conveying the effect of denervation-induced mechanical stress deprivation to the tendon fibroblasts to degrade ECM by regulating MMP-13 expression. | N/A | [ | |
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Sparc plays a role in controlling collagen content in PDL and is required for PDL homeostasis. Sparc expression, evaluated at 4 time points of aging, In the highest levels were at the age of 1 month and >18 months and reduced levels at 4 months and 6 months in mouse PDL. Absence of Sparc influenced cellular and fibrillar collagen content in PDL. The greatest differences in cell number and in collagen content between |
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Sparc protects collagen content in PDL and alveolar bone in experimental periodontal disease. Periodontal disease was induced in 4-month WT and |
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Periostin is required for the maintenance of PDL integrity in response to mechanical stress. |
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Periostin is essential for the integrity and function of the PDL during occlusal loading. PDL integrity is required for periodontium structure function. Before tooth eruption, PDL is normal in |
Alveolar bone, cementum, and enamel are affected adversely in |
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Periostin is an ECM protein required for the eruption of incisors in mice. Remodeling of PDL of incisors is defective in |
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Periostin regulates collagen cross-linking in tendon. Periostin-deficient mice show decreased cross-linking in tendon. IHC and immunogold TEM demonstrated that periostin colocalizes with collagen type I in tendon. Using differential scanning calorimetry on samples from 3-month-old mice, lower denaturation temperatures for |
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Complete deficiency of periostin does not appear to affect failure load in intact as well as in healing Achilles tendon, whereas partial deficiency does. Role of periostin was investigated on intact and healing Achilles tendon using mouse deleted in |
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Genetic variants within |
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The GT repeats polymorphism within |
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Tnx deficiency alters properties of force transmission pathways of muscle, the later being the part of myotendinous or myofascial pathways, and directly affects muscle function in |
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N/A | Chondromodulin-I-deficient ( |
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Tenomodulin is a regulator of tenocyte proliferation and is involved in collagen fibril maturation. Tnmd-deficient Achilles tendon exhibits lower cell density at 1 month and 6 months of age. Patellaris and Achilles tendon show reduced cell densities in 2 weeks but not in newborn or at P7 |
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Tenomodulin regulates cell proliferation. Targeting TNMD by RNAi assay (with siRNA against human TNMD exon 5) in human flexor carpi radialis (FCR) cells knocked down all the three human TNMD isoforms. The knockdown of FCR cells showed reduced cell proliferation and upregulated expression of myostatin and scleraxis. | N/A | [ |
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Lack of Cd44 antigen leads to improved healing in injured patellar tendon. The study was conducted in a patellar injury model of 12-week-old |
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Aggrecan turnover is Adamts5-mediated, and lack of Adamts5 results in adverse effects on structure and function of tendon and enthesis. FDL tendon (a single fascicle lacking prominent insertion site) of 12-week-old |
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Lack of Prg4 causes abnormal calcification of tendon and sheath involved in ankle joints leading to precocious joint failure. The morphologic changes in 7-month-old |
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Prg4 plays a role in interfascicular lubrication in tendon. The study involved pulling of a 15 mm long fascicle segment proximally from a distally cut tail of 10–16-week |
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Prg4 expression plays a role in the viscoelastic properties of tail tendon fascicles. Tendon stiffness and viscoelasticity was determined in 10–13-week |
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Congenital camptodactyly (Human case report) | In two sisters with congenital camptodactyly and joint effusions, abnormalities in tendons were restricted to the portion within synovial sheaths implying a disease of the tenosynovium. In areas of chronic involvement, some tendons were replaced by fibrous tissue. Fingers, in the patient with the disease, had hard scarring tissue. | N/A | [ |
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Mutations in |
Human disease: CACP; OMIM: 208250. | [ |
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Sequence analysis of |
Human disease: CACP |
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Small leucine-rich proteoglycans belong to the LRR superfamily of proteins. Members may contain up to 38 LRRs. The LRR domain is 20–29 amino acids long with asparagine and leucine residues in conserved positions [
Fibromodulin and lumican are expressed in collagenous connective tissues and play role in establishing tissue integrity. The core protein of both has one LRRNT and 11 LRRs that are sites of protein-protein interactions. The keratan sulfate proteoglycan Lum is a major component of the corneal stroma. It is also widely expressed in the interstitial connective tissue matrices of the skin, tendon, and intestinal submucosa [
Biglycan and Dcn belong to the SLRPs class I subfamily. Both contain 12 LRRs. Biglycan has two attached GAG chains and Dcn has one. The GAG can be either chondroitin sulfate or dermatan sulfate depending on the tissue origin. Biglycan is found in several connective tissues, predominantly in articular cartilages. It is a homodimer and forms a ternary complex with microfibrillar-associated protein 2 and elastin and may be involved in collagen fiber assembly. Biglycan binds to Col I in the gap zone of the fibrils, and Dcn competes for that interaction [
The effects of the two GAG chains present on Bgn are reasonably different from the single GAG of Dcn. The switch in expression levels of these SLRPs during tendon development is demonstrated by the fact that Dcn protein increases gradually with development from P4 to P30, whereas Bgn core protein decreases from P4 to P30 in flexor tendon. The stage in normal tendon development (P30), where Dcn peaked and Bgn decreased to its lowest level, indicates that Dcn persists until thick fibrils are formed [
Decorin deficiency results in aberrant tendon collagen fibril structure. TEM of tail tendon of 7.5-month old mice showing transverse section of collagen fibrils. A larger fraction of fibrils are noncylindrical and structurally aberrant in homozygous mutant (
Remodeling of the PDL of incisors is defective in
Häkkinen and coworkers reported that Dcn plays role in maintaining structure and cellularity in PDL. In Dcn homozygote mutant mice, PDL collagen fibers are wider, random, and with varied small-sized fibrils. The mutation caused hypercellularity in PDL [
It is worth mentioning an enzyme, dermatan sulfate epimerase (Dse), that impacts the property of SLRP containing chondroitin sulfate/dermatan sulfate (CS/DS) by converting glucuronic acid unit to iduronic acid. Chondroitin sulfate is an unbranched polymer chain composed of alternating glucuronic acid and
Teeth in SLRPs-deficient mice
Investigations from Soslowsky laboratory showed the role of Dcn and collagen content on the tendon properties. In the first study, they reported how quasilinear viscoelastic properties of tendon are affected by their content [
By using Bgn-deficient mice [
By using tendons from different loading regions of mutant mice deficient in Bgn and Dcn, Robinson and co-workers were able to demonstrate that tendons are tailored according to their location [
Asporin contains 11 LRRs and 1 LRRNT domain. It is a critical regulator of TGF
Asporin is expressed at higher level in the heart and specifically and predominantly in the PDL. During tooth development, strong expression is seen in the dental follicle, which is the progenitor tissue that forms cementum, alveolar bone, and the PDL [
Aspn knockdown studies showed that it negatively regulates PDL differentiation and mineralization to ensure that the PDL is not ossified, maintains homeostasis of the tooth-supporting system, and also inhibits Bmp2-induced cytodifferentiation of PDL cells by preventing its binding to BmpR1B, resulting in inhibition of Bmp-dependent activation of SMAD proteins. [
Matricellular proteins are extracellular matrix proteins that modulate cell-matrix interactions and cell function and do not seem to have a direct structural role. The family includes thrombospondin-1, thrombospondin-2, osteopontin/Spp1, osteonectin/Sparc, periostin, tenascin C, and tenascin X. Expression of matricellular proteins is usually high during embryogenesis, but nearly absent during normal postnatal life. Interestingly, it reappears in response to injury [
Thrombospondins are secreted, multimeric multidomain glycoproteins that function at the cell surfaces and the ECM and belong to thrombospondin family. They act as regulators of cell interactions in vertebrates. Thrombospondins consist of two subfamilies, A and B [
Thrombospondin 2 is a ligand for CD36 via the TSP-I repeats and delivers an antiangiogenic effect [
Secreted phosphoprotein 1 is expressed in many tissues and cell types and found in body fluids [
Spp1 binds tightly to hydroxyapatite and forms an integral part of the mineralized matrix. It plays a role in cell-matrix interaction [
Sparc regulates the cell growth through interactions with the ECM and cytokines. It binds to calcium, copper, and several types of collagen, albumin, thrombospondin, PDGF, and cell membranes. There are two calcium binding sites: an acidic domain that binds 5 to 8
Collagen in the PDL has highest turnover rate in the body [
Periostin has enhanced expression in periosteum and PDL. Periostin mRNA was shown to be upregulated at the sites under tension in bone and periodontal tissue remodeling after mechanical stress during experimental tooth movement [
Periostin-deficient mice generated by Rios et al. [
Tenascins are a family of ECM proteins that evolved in early chordates [
A large number of Achilles tendon injuries are associated with participation in sports [
Tenascin X belongs to the tenascin family and contains 19 EGF-like domains, one fibrinogen C-terminal domain and 32 fibronectin type-III domains. Tenascin X mediates interactions between cells and the ECM. TNX is a large 450 kDa ECM protein expressed in a variety of tissues including skin, joints, and blood vessels. Deficiency of TNX causes a recessive form of EDS characterized by joint hypermobility, skin fragility, and hyperextensible skin. Skin of TNX deficient patients shows abnormal elastic fibers and reduced collagen deposition. TNX is homotrimer and interacts with type I, III, and V collagens and tropoelastin via its 29th fibronectin type-III domain [
Tenomodulin is a member of a family of type II transmembrane glycoproteins. Tnmd transcripts have been found in hypovascular tissues such as tendons and ligaments but the biological activity of Tnmd has not yet been fully explored. Tnmd has been suggested to play a role in tendon development. The
Tenomodulin-deficient mice showed reduced cell numbers in adult tendons and a decrease in tenocyte proliferation at newborn stage indicating the role of Tnmd in tenocyte proliferation. In addition, the altered structure of adult collagen fibrils suggests an involvement of Tnmd in postnatal tendon maturation, though the angiogenesis was unchanged in tendons in Dbl-KO mice for Tnmd and ChM-I [
CD44 is a single-pass type I membrane glycoprotein, also called hyaluronate receptor, and is a key mediator during normal wound healing, inflammation, and fibrotic healing process. The CD44 glycoproteins are members of hyaluronate receptor family of cell adhesion molecules. CD44 contains one extracellular lectin-like LINK domain that is responsible for hyaluronan binding. The major ligand is hyaluronate that is an abundant extracellular polysaccharide found in mammalian ECM, but CD44 has many varied functions depending on the extracellular structure of the protein, which can be produced in a myriad of isoforms. The wide range of functional proteins is produced from a single gene by both alternative splicing and post-translational modifications [
Hyaluronic acid is abundant in tendons [
The ADAMTSs are a group of complex proteases found both in mammals and invertebrates. The complete human family has
Aggrecan is most abundant in regions of tendon that experience mechanical compression and at enthesis [
Proteoglycan 4
Different forms varying of Prg4 in molecular weight have been observed. Such forms are possibly due to different levels of glycosylation and protein cleavage. Prg4 contains two hemopexin-like domains and two somatomedin-B domains (Q9JM99 at
Prg4/lubricin-deficient mice demonstrated abnormal calcification of tendon sheaths of tibialis anterior that surround the ankle joint. Absence of Prg4
Understanding the mechanism of molecules in test tube is the first key step. Functional approach then can be better understood using the
Spondyloarthritis (SpA) refers to a group of HLA-B27-positive associated rheumatic diseases that share clinical and genetic features [
A number of genes in this review have addressed the importance of proteoglycans and glycoproteins in joints in response to alteration in ligaments/tendon and hence via entheses. Abnormalities in entheses can lead to enthesitis. Inflammation at the entheses, the sites of attachment of tendon, ligament, fascia, or joint capsule to bone, is the distinguishing pathological feature of AS and the other SpA [
Most commonly affected appendicular entheseal sites in spondyloarthritis (Reprinted (with minor modification) from Eshed et al., 2007 [
Excellent review has been published on a large number of gene variants involved in SpA and AS [
Bone morphogenetic protein
Bone morphogenetic protein receptor, type II
Collagen, type III, alpha 1
Camptodactyly-arthropathy coxa vara-pericarditis syndrome
Double knockout
Embryonic day
Extracellular matrix
Ehlers-Danlos syndrome
Flexor digitorum longus tendon
Glycosaminoglycan
Immunohistochemistry
Insertion, intragenic deletion
In situ hybridization
Location
Leucine-rich repeat
LRR containing N-terminal domain
Mutation
Osteoarthritis
Postnatal day
Platelet-derived growth factor
Periodontal ligament
Scanning electron microscopy
Small leucine-rich proteoglycan
Synonym
Targeted (knockout)
Targeted (knockin)
Targeted (reporter)
Transmission electron microscopy
Transforming growth factor beta
Targeted mutation 1
Wild type (control mouse)
Phenotype not available, that is, phenotype not studied by the authors in that reference paper.
The authors declare that they have no conflict of interests.
Thanks are due to Professors Dr. Archibald Perkins and Prof. Dr. Regis O’Keefe for providing the authors with the best training in tendon and arthritis research.