Inhibition of TGF‐β signaling by 1D11 antibody treatment increases bone mass and quality in vivo

Transforming growth factor β (TGF‐β) is an abundant bone matrix protein that influences osteoblast and osteoclast interactions to control bone remodeling. As such, TGF‐β represents an obvious pharmacologic target with the potential to regulate both bone formation and resorption to improve bone volume and strength. To investigate the skeletal effect of TGF‐β inhibition in vivo, we used an antibody (1D11) specifically directed at all three isoforms of TGF‐β. Normal mice were treated with 1D11 or control antibody (4 weeks), and cortical and trabecular bone was assessed by micro–computed tomographic (µCT) scanning. Bone volume and cellular distribution were determined by histomorphometric analysis of vertebrae and long bones. Also, whole‐bone strength was assessed biomechanically by three‐point bend testing, and tissue‐level modulus and composition were analyzed by nanoindentation and Raman microspectroscopy, respectively. TGF‐β blockade by 1D11 increased bone mineral density (BMD), trabecular thickness, and bone volume by up to 54%, accompanied by elevated osteoblast numbers and decreased osteoclasts. Biomechanical properties of bone also were enhanced significantly by 1D11 treatment, with increased bending strength and tissue‐level modulus. In addition, Raman microspectroscopy demonstrated that 1D11‐mediated TGF‐β inhibition in the bone environment led to an 11% increase in the mineral‐to‐collagen ratio of trabecular bone. Together these studies demonstrate that neutralizing TGF‐β with 1D11 increases osteoblast numbers while simultaneously decreasing active osteoclasts in the marrow, resulting in a profound increase in bone volume and quality, similar to that seen in parathyroid hormone (PTH)–treated rodent studies. © 2010 American Society for Bone and Mineral Research.     

Osteoclast TGF‐β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

Osteoblast‐mediated bone formation is coupled to osteoclast‐mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age‐related bone loss. Osteoclasts release and activate TGF‐β from the bone matrix. Here we show that osteoclast‐specific inhibition of TGF‐β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF‐β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF‐β receptor signaling. Osteoclasts in aged murine bones had lower TGF‐β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF‐β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF‐β availability with age. Therefore, osteoclast responses to TGF‐β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age‐related bone loss. © 2015 American Society for Bone and Mineral Research.     

Hyperactive Transforming Growth Factor‐β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

Dysregulated transforming growth factor beta (TGF‐β) signaling is associated with a spectrum of osseous defects as seen in Loeys‐Dietz syndrome, Marfan syndrome, and Camurati‐Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF‐β1 signaling pivotally underpins osseous defects in Nf1^flox/?;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF‐β1 levels are fivefold to sixfold increased both in Nf1^flox/?;Col2.3Cre mice and in a cohort of NF1 patients. Nf1‐deficient osteoblasts, the principal source of TGF‐β1 in bone, overexpress TGF‐β1 in a gene dosage–dependent fashion. Moreover, Nf1‐deficient osteoblasts and osteoclasts are hyperresponsive to TGF‐β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21‐Ras–dependent hyperactivation of the canonical TGF‐β1–Smad pathway. Reexpression of the human, full‐length neurofibromin guanosine triphosphatase (GTPase)‐activating protein (GAP)‐related domain (NF1 GRD) in primary Nf1‐deficient osteoblast progenitors, attenuated TGF‐β1 expression levels and reduced Smad phosphorylation in response to TGF‐β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF‐β receptor 1 (TβRI) kinase inhibitor, SD‐208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1^flox/?;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF‐β1 signaling in the pathogenesis of NF1‐associated osteoporosis and pseudarthrosis, thus implicating the TGF‐β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies. © 2013 American Society for Bone and Mineral Research.     

Dysregulated TGF‐β signaling alters bone microstructure in a mouse model of Loeys‐Dietz syndrome

Loeys‐Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular and skeletal abnormalities resembling Marfan syndrome, including a predisposition for pathologic fracture. LDS is caused by heterozygous mutations in the genes encoding transforming growth factor‐β (TGF‐β) type 1 and type 2 receptors. In this study, we characterized the skeletal phenotype of mice carrying a mutation in the TGF‐β type 2 receptor associated with severe LDS in humans. Cortical bone in LDS mice showed significantly reduced tissue area, bone area, and cortical thickness with increased eccentricity. However, no significant differences in trabecular bone volume were observed. Dynamic histomorphometry performed in calcein‐labeled mice showed decreased mineral apposition rates in cortical and trabecular bone with normal numbers of osteoblasts and osteoclasts. Mechanical testing of femurs by three‐point bending revealed reduced femoral strength and fracture resistance. In vitro, osteoblasts from LDS mice demonstrated increased mineralization with enhanced expression of osteoblast differentiation markers compared with control cells. These changes were associated with impaired TGF‐β1–induced Smad2 and Erk1/2 phosphorylation and upregulated TGF‐β1 ligand mRNA expression, compatible with G357W as a loss‐of‐function mutation in the TGF‐β type 2 receptor. Paradoxically, phosphorylated Smad2/3 in cortical osteocytes measured by immunohistochemistry was increased relative to controls, possibly suggesting the cross‐activation of TGF‐β–related receptors. The skeletal phenotype observed in the LDS mouse closely resembles the principal structural features of bone in humans with LDS and establishes this mouse as a valid in vivo model for further investigation of TGF‐β receptor signaling in bone. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1447–1454, 2015.     

Regulation of RANKL‐induced osteoclastogenesis by TGF‐β through molecular interaction between Smad3 and Traf6

Previous studies have shown that transforming growth factor β (TGF‐β) promotes receptor activator of nuclear factor‐κB ligand (RANKL)–induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF‐β signals were blocked either by a specific inhibitor of TGF‐β type 1 receptor kinase activity, SB431542, or by introducing a dominant‐negative mutant of TGF‐β type 2 receptor, RANKL‐induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c‐Ski markedly suppressed RANKL‐induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6‐TAB1‐TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6‐TAB1‐TAK1 complex formation was not observed when TGF‐β signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6‐TAB1‐TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL‐induced osteoclast differentiation. In summary, TGF‐β is indispensable in RANKL‐induced osteoclastogenesis, and the binding of Smad3 to the TRAF6‐TAB1‐TAK1 complex is crucial for RANKL‐induced osteoclastogenic signaling. © 2011 American Society for Bone and Mineral Research.     

Wnt pathway regulation by demineralized bone is approximated by both BMP‐2 and TGF‐β1 signaling

Allogeneic demineralized bone is used extensively as a clinical graft material because it has osteo/chondroinductive and osteoconductive properties. Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in three‐dimensional collagen cultures, but the initiating mechanisms have not been fully characterized nor has it been shown that bone morphogenetic proteins (BMPs) recapitulate DBP's effects on target cells. Among the many signaling pathways regulated in hDFs by DBP prior to in vitro chondrogenesis, there are changes in Wnts and their receptors that may contribute to DBP actions. This study tests the hypothesis that DBP modulation of Wnt signaling entails both BMP and TGF‐β pathways. We compared the effects of DBP, TGF‐β1, or BMP‐2 on Wnt signaling components in hDFs by Wnt signaling macroarray, RT‐PCR, in situ hybridization, and Western immunoblot analyses. Many effects of DBP on Wnt signaling components were not shared by BMP‐2, and likewise DBP effects on Wnt genes and β‐catenin only partially required the TGF‐β pathway, as shown by selective inhibition of TGF‐β/activin receptor‐like kinase. The analyses revealed that 64% (16/25) of the Wnt signaling components regulated by DBP were regulated similarly by the sum of effects by BMP‐2 and by TGF‐β1. In conclusion, signaling mechanisms of inductive DBP in human dermal fibroblasts involve the modulation of multiple Wnt signals through both BMP and TGF‐β pathways. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 554–560, 2013     

Cartilage degeneration and excessive subchondral bone formation in spontaneous osteoarthritis involves altered TGF‐β signaling

Transforming growth factor‐β (TGF‐β) has been demonstrated as a potential therapeutic target in osteoarthritis. However, beneficial effects of TGF‐β supplement and inhibition have both been reported, suggesting characterization of the spatiotemporal distribution of TGF‐β during the whole time course of osteoarthritis is important. To investigate the activity of TGF‐β in osteoarthritis progression, we collected knee joints from Dunkin–Hartley (DH) guinea pigs at 3, 6, 9, and 12‐month old (n = 8), which develop spontaneous osteoarthritis in a manner extraordinarily similar to humans. Via histology and micro‐computed tomography (CT) analysis, we found that the joints exhibited gradual cartilage degeneration, subchondral plate sclerosis, and elevated bone remodeling during aging. The degenerating cartilage showed a progressive switch of the expression of phosphorylated Smad2/3 to Smad1/5/8, suggesting dual roles of TGF‐β/Smad signaling during chondrocyte terminal differentiation in osteoarthritis progression. In subchondral bone, we found that the locations and age‐related changes of osterix⁺ osteoprogenitors were in parallel with active TGF‐β, which implied the excessive osteogenesis may link to the activity of TGF‐β. Our study, therefore, suggests an association of cartilage degeneration and excessive bone remodeling with altered TGF‐β signaling in osteoarthritis progression of DH guinea pigs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:763–770, 2016.     

CCN5 Reduces Ligamentum Flavum Hypertrophy by Modulating the TGF‐β Pathway

Ligamentum flavum hypertrophy (LFH) is the most important component of lumbar spinal canal stenosis. Although the pathophysiology of LFH has been extensively studied, no method has been proposed to prevent or treat it. Since the transforming growth factor‐β (TGF‐β) pathway is known to be critical in LFH pathology, we investigated whether LFH could be prevented by blocking or modulating the TGF‐β mechanism. Human LF cells were used for the experiments. First, we created TGF‐β receptor 1 (TGFBR1) knock out (KO) cells with CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 biotechnology and treated them with TGF‐β1 to determine the effects of blocking the TGF‐β pathway. Subsequently, we studied the effect of CCN5, which has recently been proposed to modulate the TGF‐β pathway. To assess the predisposition toward fibrosis, α‐smooth muscle actin (αSMA), fibronectin, collagen‐1, collagen‐3, and CCN2 were evaluated with quantitative real‐time polymerase chain reaction, western blotting, and immunocytochemistry. The TGFBR1 KO LF cells were successfully constructed with high KO efficiency. In wild‐type (WT) cells, treatment with TGF‐β1 resulted in the overexpression of the messenger RNA (mRNA) of fibrosis‐related factors. However, in KO cells, the responses to TGF‐β1 stimulation were significantly lower. In addition, CCN5 and TGF‐β1 co‐treatment caused a notable reduction in mRNA expression levels compared with TGF‐β1 stimulation only. The αSMA protein expression increased with TGF‐β1 but decreased with CCN5 treatment. TGF‐β1 induced LF cell transdifferentiation from fibroblasts to myofibroblasts. However, this cell transition dramatically decreased in the presence of CCN5. In conclusion, CCN5 could prevent LFH by modulating the TGF‐β pathway. © 2019 The Authors. Journal of Orthopaedic Research^® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2634–2644, 2019     

Transforming growth factor‐β (TGF‐β) expression is increased in the subsynovial connective tissues of patients with idiopathic carpal tunnel syndrome

Non‐inflammatory fibrosis of the subsynovial connective tissue (SSCT) is a hallmark of carpal tunnel syndrome (CTS). The etiology of this finding and its relationship to the development of CTS remain poorly understood. Recent studies have found that transforming growth factor‐β (TGF‐β) plays a central role in fibrosis. The purpose of this study was to investigate the expression of TGF‐β and connective tissue growth factor (CTGF), a downstream mediator of TGF‐β, in the pathogenesis of CTS. We compared SSCT specimens from 26 idiopathic CTS patients with specimens from 10 human cadaver controls with no previous diagnosis of CTS. Immunohistochemistry was performed to determine levels TGF‐β1, CTGF, collagen 1(Col1) and collagen 3 (Col3) expression. TGF‐β1 (p < 0.01), CTGF (p < 0.01), and Col3 (p < 0.01) were increased in SSCT of CTS patients compared with control tissue. In addition, a strong positive correlation was found between TGF‐β1 and CTGF, (R² = 0.80, p < 0.01) and a moderate positive correlation between Col3 and TGF‐β1 (R² = 0.49, p < 0.01). These finding suggest that there is an increased expression of TGF‐β and CTGF, a TGF‐β regulated protein, and that this TGF‐β activation may be responsible for SSCT fibrosis in CTS patients. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:116–122, 2014.     

Sclerostin,Osteocytes, and Chronic Kidney Disease – Mineral Bone Disorder

Osteocytes respond to kidney damage by increasing production of secreted factors important to bone and mineral metabolism. These circulating proteins include the antianabolic factor, sclerostin, and the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Elevated sclerostin levels correlate with increased FGF23, localized reduction in Wnt/β‐catenin signaling in the skeleton and reduced osteoblast differentiation/activity. Decreased Wnt/β‐catenin signaling occurs regardless of the overall changes in bone formation rates, suggesting that a reduction in the anabolic response may be a common feature of renal bone disorders but additional mechanisms may contribute to the diversity of osteodystrophy phenotypes. Recent preclinical studies support this hypothesis, as treatment with antisclerostin antibodies improved bone quality in the context of low but not high turnover renal osteodystrophy. Sclerostin also appears in the circulation suggesting additional roles outside the skeleton in normal and disease states. In patients with chronic kidney disease (CKD), serum levels are elevated several fold relative to healthy individuals. Emerging data suggest that these changes are associated with increased fracture rates but the relationship between sclerostin and cardiovascular disease is unclear. Additional epidemiologic studies that examine stage specific and patient sub‐populations are needed to assess whether sclerostin elevations influence comorbidities associated with CKD.     

Biphasic effects of transforming growth factor β on bone morphogenetic protein–induced osteoblast differentiation

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP‐induced bone formation may lead to the development of more effective BMP‐based therapies. To identify kinases mediating BMP‐induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP‐6‐induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor β (TGF‐β) generally is considered to antagonize BMP‐induced osteoblast differentiation, C2C12 cells transfected with siRNAs targeting TGF‐β receptors displayed reduced BMP‐6‐induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF‐β type I receptor impaired BMP‐induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF‐β further increased expression of osteoblast‐specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF‐β were found to depend on timing and duration of the costimulation. TGF‐β inhibited BMP‐induced activation of a BMP‐Smad‐dependent luciferase reporter, suggesting that the stimulatory effect of TGF‐β is not due to increased BMP‐Smad activity. TGF‐β also inhibited the BMP‐induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF‐β, besides acting as an inhibitor, also can, by dampening the noggin‐mediated negative‐feedback loop, enhance BMP‐induced osteoblast differentiation, which might be beneficial in fracture healing. © 2011 American Society for Bone and Mineral Research.     

Bone Formation Is Coupled to Resorption Via Suppression of Sclerostin Expression by Osteoclasts

Bone formation is coupled to bone resorption throughout life. However, the coupling mechanisms are not fully elucidated. Using Tnfrsf11b‐deficient (OPG^–/–) mice, in which bone formation is clearly coupled to bone resorption, we found here that osteoclasts suppress the expression of sclerostin, a Wnt antagonist, thereby promoting bone formation. Wnt/β‐catenin signals were higher in OPG^–/– and RANKL‐transgenic mice with a low level of sclerostin. Conditioned medium from osteoclast cultures (Ocl‐CM) suppressed sclerostin expression in UMR106 cells and osteocyte cultures. In vitro experiments revealed that osteoclasts secreted leukemia inhibitory factor (LIF) and inhibited sclerostin expression. Anti‐RANKL antibodies, antiresorptive agents, suppressed LIF expression and increased sclerostin expression, thereby reducing bone formation in OPG^–/– mice. Taken together, osteoclast‐derived LIF regulates bone turnover through sclerostin expression. Thus, LIF represents a target for improving the prolonged suppression of bone turnover by antiresorptive agents. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

Expression of transforming growth factor β isoforms and their roles in tendon healing

Transforming growth factor β (TGF‐β) plays active roles in tendon healing. However, the differential effects of TGF‐β isoforms on tendon healing have not been investigated. In cultured tendon fibroblasts, we tested the effects of TGF‐β1, β2, and β3 on the mRNA levels of COL1A1 and COL3A1 by quantitative real‐time polymerase chain reaction. We also investigated the expression of TGF‐β isoforms, TGF‐β receptors, procollagen Type I and Type III in a rat model of tendon healing. We found that TGF‐β3 exhibited the highest potency in stimulating COL1A1 and COL3A1. TGF‐β1 exerted antagonistic effects to TGF‐β2 and β3. All TGF‐β isoforms and procollagen Type I were confined to the edges of the healing tendon at day 28 postinjury. Our results indicated that interaction of TGF‐β isoforms exist in the regulation of collagen synthesis in tendon fibroblasts. Their effects may be further complicated by uneven spatial distribution of TGF‐β and TGF‐β receptors in healing tendons.     

Synergistic effect of low‐intensity pulsed ultrasound on growth factor stimulation of nucleus pulposus cells

Low‐intensity pulsed ultrasound (LIPUS) has been reported to stimulate the activity of various cells. We have reported that the capacity of human intervertebral nucleus pulposus cell line to synthesize proteoglycan (PG) was increased by exposure to LIPUS, and postulated that one of the mechanisms underlying this response was an increase in expression of the transforming growth factor‐β type I receptor gene (TGFβR1). Therefore, the present study was conducted to assess the synergistic effect of LIPUS and TGF‐β on nucleus pulposus cells harvested from canines. The cells were cultured under four different sets of conditions: control group (Group A), LIPUS group (Group B), TGF‐β1 group (Group C), and LIPUS + TGF‐β1 group (Group D). They were evaluated by measuring cell proliferation, PG synthesis, PG content, gene expression of TGFβR1, and TGF‐β1 concentration. There were no significant differences in proliferation during culture. However, PG synthesis and endogenous TGF‐β1 production increased and demonstrated a synergistic effect between LIPUS and TGF‐β. Because LIPUS is safe and noninvasive, the results of the present study suggest that it would be a promising new therapy for prevention of intervertebral disc degeneration, which is said to be one of the primary causes of low back pain. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1574–1581, 2007     

Active TGF‐β1 Expression in kidney transplantation: a comparative study of Cyclosporin‐A (CyA) and tacrolimus (FK506)

Abstract Chronic rejection is a major cause of graft dysfunction following kidney transplantation. This fibroproliferative disease may be promoted by overproduction of transforming growth factor beta (TGF‐β). Previous studies have suggested that cyclosporin‐A (CyA) might increase production of this growth factor. The current study was designed to measure the expression of TGF‐β in renal transplant biopsies from patients immunosuppressed with either CyA or tacrolimus. Paraffin‐embedded renal biopsies were sectioned, dewaxed and incubated with primary antibody against active TGF‐β₁ antibody. After washing, the sections were treated with secondary antibody conjugated with fluorescein isothiocyanate (FITC). In each case the sections were assessed by semi‐quantitative scanning laser confocal microscopy. Biopsies from patients receiving CyA expressed significantly more active TGF‐β₁ than biopsies from patients receiving tacrolimus (P < 0.0001, Mann‐Whitney test). The increased level of active TGF‐β₁ expression in renal biopsies of patients receiving CyA may indicate a mechanism of chronic rejection.     

Up‐regulation of expression of selected genes in human bone cells with specific capacitively coupled electric fields

The objective of the described experiments was to determine the electrical parameters that lead to optimal expression of a number of bone‐related genes in cultured human bone cells exposed to a capacitively coupled electric field. Human calvarial osteoblasts were grown in modified plastic Cooper dishes in which the cells could be exposed to various capacitively coupled electric fields. The optimal duration of stimulation and optimal duration of response to the electrical field, and the optimal amplitude, frequency and duty cycle were all determined for each of the genes analyzed. Results indicated that a capacitively coupled electric field of 60 kHz, 20 mV/cm, 50% duty cycle for 2 h duration per day significantly up‐regulated mRNA expression of a number of transforming growth factor (TGF)‐β family genes (bone morphogenetic proteins (BMP)‐2 and ‐4, TGF‐β1, ‐ β2 and ‐β3) as well as fibroblast growth factor (FGF)‐2, osteocalcin (BGP) and alkaline phosphatase (ALP). Protein levels of BMP‐2 and ‐4, and TGF‐β1 and ‐ β2 were also elevated. The clinical relevance of these findings in the context of a noninvasive treatment modality for delayed union and nonunion fracture healing is discussed. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:894–903, 2014.     

Localization of small leucine‐rich proteoglycans and transforming growth factor‐β in human oral mucosal wound healing

Wound healing in oral mucosa is fast and results in little scar formation as compared with skin. The biological mechanisms underlying this property are poorly understood but may provide valuable information about the factors that promote wound regeneration. Small leucine‐rich proteoglycans (SLRPs) decorin, biglycan, fibromodulin and lumican are extracellular matrix molecules that regulate collagen fibrillogenesis, inhibit transforming growth factor‐β (TGF‐β) activity and reduce scarring. In the present study, we analyzed accumulation of SLRPs and TGF‐β during non‐scarring human oral mucosal wound healing. Biopsies were collected from healthy volunteers from unwounded tissue and from standardized experimental wounds 3–60 days postwounding. Localization of SLRPs, TGF‐β1 and TGF‐β3 was analyzed by immunohistochemical staining and quantitated by image analysis. Double immunostaining was used to study localization of SLRPs or active TGF‐β in distinct cells. Decorin, biglycan, fibromodulin, and TGF‐β isoforms showed significantly increased accumulation in the wound extracellular matrix and distinct wound cells while the abundance of lumican in the extracellular matrix was strongly reduced during wound healing. Localization and abundance of fibromodulin, lumican, and TGF‐β isoforms was also spatiotemporally regulated in the wound epithelium. The findings suggest that SLRPs regulate wound reepithelialization and connective tissue regeneration during oral mucosal wound healing.