Free TGF-β1 Research Articles

Integrin αV mediated activation of myofibroblast via mechanoparacrine of transforming growth factor β1 in promoting fibrous scar formation after myocardial infarction

BackgroundMyocardial infarction (MI) dramatically changes the mechanical stress, which is intensified by the fibrotic remodeling. Integrins, especially the αV subunit, mediate mechanical signal and mechanoparacrine of transforming growth factor β1 (TGF-β1) in various organ fibrosis by activating CFs into myofibroblasts (MFBs). We investigated a possible role of integrin αV mediated mechanoparacrine of TGF-β1 in MFBs activation for fibrous reparation in mice with MI. MethodsHeart samples from MI, sham, or MI plus cilengitide (14 mg/kg, specific integrin αV inhibitor) treated mice, underwent functional and morphological assessments by echocardiography, and histochemistry on 7, 14 and 28 days post-surgery. The mechanical and ultrastructural changes of the fibrous scar were further evaluated by atomic mechanics microscope (AFM), immunofluorescence, second harmonic generation (SHG) imaging, polarized light and scanning electron microscope, respectively. Hydroxyproline assay was used for total collagen content, and western blot for protein expression profile examination. Fibroblast bioactivities, including cell shape, number, Smad2/3 signal and expression of extracellular matrix (ECM) related proteins, were further evaluated by microscopic observation and immunofluorescence in polyacrylamide (PA) hydrogel with adjustable stiffness, which was re-explored in fibroblast cultured on stiff matrix after silencing of integrin αV. The content of total and free TGF-β1 was tested by enzyme-linked immunosorbent assay (ELISA) in both infarcted tissue and cell samples. ResultIncreased stiffness with heterogeneity synchronized with integrin αV and alpha smooth muscle actin (α-SMA) positive MFBs accumulation in those less mature fibrous areas. Cilengitide abruptly reduced collagen content and disrupted collagen alignment, which also decreased TGF-β1 bioavailability, Smad2/3 phosphorylation, and α-SMA expression in the fibrous area. Accordingly, fibroblast on stiff but not soft matrix exhibited obvious MFB phenotype, as evidenced by enlarged cell, hyperproliferation, well-developed α-SMA fibers, and elevated ECM related proteins, while silencing of integrin αV almost abolished this switch via attenuating paracrine of TGF-β1 and nuclear translocation of Smad2/3. ConclusionThis study illustrated that increased tissue stiffness activates CFs into MFBs by integrin αV mediated mechanoparacrine of TGF-β1, especially in immature scar area, which ultimately promotes fibrous scar maturation.

Keratose Hydrogel Drives Differentiation of Cardiac Vascular Smooth Muscle Progenitor Cells: Implications in Ischemic Treatment.

Peripheral artery disease (PAD) is a common vascular disorder in the extremity of limbs with limited clinical treatments. Stem cells hold great promise for thetreatment of PAD, but their therapeutic efficiency is limited due to multiple factors, such as poor engraftment and non-optimal selection of cell type. To date, stem cells from a variety of tissue sources have been tested, but little information is available regarding vascular smooth muscle cells (VSMCs) for PAD therapy. The present study examines the effects of keratose (KOS) hydrogels on c-kit+/CD31- cardiac vascular smooth muscle progenitor cell (cVSMPC) differentiation and the therapeutic potential of the resultant VSMCs in a mouse hindlimb ischemic model of PAD. The results demonstrated that KOS but not collagen hydrogel was able to drive the majority of cVSMPCs into functional VSMCs in a defined Knockout serum replacement (SR) medium in the absence of differentiation inducers. This effect could be inhibited by TGF-β1 antagonists. Further, KOS hydrogel increased expression of TGF-β1-associated proteins and modulated the level of free TGF-β1 during differentiation. Finally, transplantation of KOS-driven VSMCs significantly increased blood flow and vascular densities of ischemic hindlimbs. These findings indicate that TGF-β1 signaling is involved in KOS hydrogel-preferred VSMC differentiation and that enhanced blood flow are likely resulted from angiogenesis and/or arteriogenesis induced by transplanted VSMCs.

Increased circulating TGF-β1 is associated with impairment in NK cell effector functions in metastatic melanoma patients

Transforming growth factor beta (TGF-β) plays a complex role in carcinogenesis. In 30 melanoma patients and 20 healthy controls (HC) we analysed functional and phenotypic characteristics of NK cells by Flow cytometry, gene expression of TGF-β1 in peripheral blood mononuclear cells by qPCR and serum and supernatant level of free TGF-β1 by ELISA. Melanoma patients had significantly higher serum level of circulatingTGF-β1 compared to HC, especially those with metastasis into the central nervous system (subclass M1d) and high LDH serum values. Melanoma patients compared to HC had significantly higher level of TGF-β1 gene in PBMC. TGF-β1 serum values negatively correlate with NK cell activity analysed by CD107a (degranulation marker), IFN-γ, NKG2D, and NKp46 in patients. Study shows the association of high level of TGF-β1 with NK cell inhibition in patients represents the main mechanism of tumour immune evasion. Targeting TGF-β may become an important cancer treatment for improving antitumor immunity.

Increase in Free and Total Plasma TGF-β1 Following Physical Activity.

To evaluate effects of physical activity and food consumption on plasma concentrations of free and total transforming growth factor beta-1 (TGF-β1), beta-2 (TGF-β2), and beta-3 (TGF-β3) in individuals with knee osteoarthritis (OA). Participants (n = 40 in 2 cohorts of 20; mean age 70 years) with radiographic knee OA were admitted overnight for serial blood sampling. Cohorts 1 and 2 assessed the impacts of food intake and activity, respectively, on TGF-β concentrations. Cohort 1 blood draws included 2 hours postprandial the evening of day 1 (T3), fasting before rising on day 2 (T0), nonfasting 1 hour after rising (T1B), and 4 hours after rising (T2). Cohort 2 blood draws included T3, T0, fasting 1 hour after rising and performing activities of daily living (T1A), and nonfasting 2 hours after rising (T1B). By sandwich ELISAs, we quantified plasma free and total TGF-β1 concentrations in all samples, and plasma total TGF-β2 and TGF-β3 in cohort 2. Free TGF-β1 represented a small fraction of the total systemic concentration (mean 0.026%). In cohort 2, free and total TGF-β1 and total TGF-β2 concentration significantly increased in fasting samples collected after an hour (T1A) of activities of daily living (free TGF-β1: P = 0.006; total TGF-β1: P < 0.001; total TGF-β2: P = 0.001). Total TGF-β3 increased nonsignificantly following activity (P = 0.590) and decreased (P = 0.035) after food consumption while resting (T1B). Increased plasma concentrations of TGF-β with physical activity suggests activity should be standardized prior to TGF-β1 analyses.

Transforming growth factor beta variation with physical activity in knee osteoarthritis

Purpose: Transforming growth factor beta (TGF-β) is a multifunctional cytokine with three different mammalian isoforms. All three isoforms of TGF-β are secreted as inactive latent complexes. Bound to latency-associated peptide, TGF-β is unable to bind to membrane receptors. Therefore, even though TGF-β is constitutively expressed throughout the body, only the free ligand is biologically active and able to modulate the metabolic activity of cartilage. An existing theory likens the latent TGF-β complex to a molecular sensor that monitors the extracellular matrix and responds to perturbations by releasing free TGF-β. In the cartilage, TGF-β1 plays an essential role in regulating cartilage matrix metabolism. The goal of this study was to assess the effects of physical activity on free and total TGF-β isoforms in plasma from individuals with knee osteoarthritis (OA). Methods: To assess the impact of physical activity on biomarker variation, participants (n=20; 65% female; 80% white; mean age 70 years; mean BMI 31.72 kg/m2) were recruited on the basis of symptomatic OA in at least one knee. Knee OA severity was assessed from a postero-anterior semi-flexed radiograph with a SynaFlexer positioning device and quantified by Kellgren Lawrence (K-L) score for each knee. Blood was obtained at 4 different time points (Figure 1). The interval from T0 to T1A was devoted to physical activity including activities of daily living and non-brisk walking for 1 hour, with no more than 10 minutes rest at any one time. All procedures were approved by the Institutional Review Board. Using commercially available ELISA assays, we quantified free and total TGF-β1 (BioLegend) in plasma samples (n=80) and available matched serum (n=79) samples. Total TGF-β2 and TGF-β3 (MSD U-PLEX ELISA) were also quantified in plasma (n=80). The data for TGF-β3 should be interpreted with caution, as 47.5% of samples had concentrations below the LLOD so were imputed as 1/2 LLOD for analyses. To assess variations in TGF-β over this time period, biomarker concentrations for the four time points were normalized to the mean across all four time points for each individual. Significance was assessed by non-parametric Friedman test and Dunn’s post-hoc test. Spearman correlation was used to evaluate associations between non-normalized: free and total plasma TGF-β1; serum and plasma free and total TGF-β1; all total TGF-β isoforms; and free and total TGF-β1 (T1A and change from T0 to T1A) with sum K-L grade. Results: Median plasma concentrations (range pg/ml) were as follows: free TGF-β1: 8.53 (0-40.71); total TGF-β1: 27343.00 (6760.00-81815.00); total TGF-β2: 24.99 (0-170.30); and total TGF-β3: 1.03 (0-3.10). Free plasma TGF-β1 represented 0.032% of total plasma TGF-β1. Free and total TGF-β1, and total TGF-β2, increased significantly after one hour of activity (Figure 2; time point T0 to T1A). The mean fold-change increase with activity was 1.7 for free TGF-β1, 1.4 for total TGF-β1, and 1.5 for total TGF-β2. After food consumption (T1B), TGF-β3 concentrations decreased an average of 62% from T1A, but no significant changes occurred in the other TGF-β isoforms at this time point. In plasma, total TGF-β1 strongly correlated with free TGF-β1 (rs=0.36, p=0.008), total TGF-β2 (rs=0.69, p<0.0001), and total TGF-β3 (rs=0.36, p=0.001). Total TGF-β2 also correlated with Total TGF-β3 (rs=0.29, p=0.01). Serum TGF-β1 concentrations were consistently higher than plasma concentrations (a mean 17.5-fold for free TGF-β1 and a mean 1.6-fold for total TGF-β1). Plasma and matched sera did not correlate for free TGF-β1 (rs=0.09, p=0.43) or total TGF-β1 (rs=0.19, p=0.09). Neither free nor total TGF-β1 were associated with sum K-L grade. Conclusions: This study demonstrates that plasma concentrations of both free and total TGF-β1, and total TGF-β2, all significantly increased with physical activity, suggesting release from a mechanically sensitive reservoir. These results are consistent with known release of TGF-β1 from extracellular matrix of myofibroblasts upon mechanical contraction in vitro. We observed an effective 1.7-fold increase of circulating free (active) TGF-β1 during an hour of activity (from a mean 8.45 to a mean 13.08 pg/ml). Based upon previous published studies, these concentrations may represent a physiologically relevant modulation. These results suggest the need for standardization of activity prior to biofluid sampling intended for TGF-β analyses to assess its impact on musculoskeletal health and disease.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Temporal-controlled bioactive molecules releasing core-shell nano-system for tissue engineering strategies in endodontics

Temporal-controlled release of bioactive molecules is of key importance in the clinical translation of tissue engineering techniques. We engineered a core-shell nano-system (TD-NS) that sequentially released transforming growth factor-β1 (TGF-β1), a chemotactic/proliferating growth factor and dexamethasone (Dex), an osteo/odontogenic agent in a temporal-controlled manner. In stage-1, there was a rapid release of TGF-β1, reaching a concentration of 2 ng/mL of TGF-β1 in 7 days to 14 days, which tapers subsequently. In stage-2, Dex was released linearly from 9 days to 28 days. The TD-NS group showed a significantly higher (P < 0.05) osteo/odontogenic differentiation compared to the control and free TGF-β1 group (Free-TD) that was further corroborated with animal models/histochemical examination. The findings from this study highlighted the potential of temporal-controlled delivery of TGF-β1 and Dex from a single nano-carrier to direct spatial and temporal-control for a cell-free tissue engineering strategy in the treatment of apical periodontitis.

Chitosan-Polylactide/Hyaluronic Acid Complex Microspheres as Carriers for Controlled Release of Bioactive Transforming Growth Factor-β1.

Chitosan(CH)-polylactide(PLA) copolymers containing varied PLA percentages were synthesized using a group-protection method and one of them with solubility in water-based solvents was used to prepare CH-PLA/hyaluronic acid (HA) complex microspheres for the delivery of transforming growth factor-β1 (TGF-β1). An emulsification processing method was developed for producing TGF-β1-loaded CH-PLA/HA microspheres using sodium tripolyphosphate (TPP) as ionic crosslinker and the size of the microspheres was devised to the micron level in order to achieve high encapsulating efficiency. The encapsulating efficiency, swelling property and release administration of the microspheres could be synergistically regulated by PLA component, the applied TPP dose and the incorporated HA amount. In comparison to CH/HA microspheres, the CH-PLA/HA microspheres had greatly reduced TGF-β1 release rates and were able to administrate the TGF-β1 release at controlled rates over a significant longer period of time. The released TGF-β1 was detected to be bioactive when compared to the free TGF-β1. These results suggest that the presently developed CH-PLA/HA complex microspheres have promising potential in delivering TGF-β1 for cartilage repair applications where the applied TGF-β1 amount in the early stage needs to be low whilst the sustained TGF-β1 release at an appropriate dose in the later stage has to be maintained

NOGO-B promotes EMT in lung fibrosis via MMP14 mediates free TGF-beta1 formation.

Idiopathic pulmonary fibrosis (IPF) is a lung disease with an extremely poor prognosis. Epithelial mesenchymal transition (EMT) appearing on the airway epithelial cell plays an essential role in the formation and development of Idiopathic pulmonary fibrosis. In this paper, Bleomycin (BLM)-induced mice model combined with bioinformatics analysis were employed to elucidate the potential mechanism of EMT in pulmonary fibrosis. The obtained results showed that endoplasmic reticulum protein Nogo-b may promote MMP14-mediated proprotein maturation of TGF-β1, accelerating the release of free TGF-β1 in type II airway epithelial cells A549, subsquently, induce the epithelial-mesenchymal transition (EMT) of the cell. In all, the overexpression of Nogo-b play a role in the course of pulmonary fibrosis by influencing the EMT ability of cells.

Effects of a Bioactive Scaffold Containing a Sustained Transforming Growth Factor-β1–releasing Nanoparticle System on the Migration and Differentiation of Stem Cells from the Apical Papilla

IntroductionThis 2-part study hypothesized that a bioactive scaffold containing a sustained transforming growth factor (TGF)-β1–releasing nanoparticle system will promote migration and enhance differentiation of stem cells from the apical papilla (SCAP). The study aimed to develop and characterize a novel modified chitosan-based scaffold containing TGF-β1–releasing chitosan nanoparticles (TGF-β1-CSnp) to enhance migration and differentiation of SCAP. MethodsPart I concerns the synthesis and characterization of a carboxymethyl chitosan–based scaffold and TGF-β1-CSnp. Part II examines the effect of sustained TGF-β1 release from scaffold containing TGF-β1-CSnp on odontogenic differentiation of SCAP. ResultsThe scaffold demonstrated properties conducive to cellular activities. The incorporation of TGF-β1 in CSnp allowed sustained release of TGF-β1, facilitating delivery of a critical concentration of TGF-β1 at the opportune time. TGF-β1 bioactivity was maintained for up to 4 weeks. SCAP showed greater viability, migration, and biomineralization in the presence of TGF-β1-CSnp than in the presence of free TGF-β1. SCAP cultured in TGF-β1-CSnp + scaffold showed significantly higher dentin matrix protein-1 and dentin sialophosphoprotein signals compared with free TGF-β1 + scaffold or CSnp + scaffold. ConclusionsThese experiments highlighted the potential of a carboxymethyl chitosan–based scaffold with growth factor releasing nanoparticles to promote migration and differentiation of SCAP. The results of this study may have direct application to improve current endodontic regenerative protocols.

Comparative study of dermal components and plasma TGF-β1 levels in Slc39a13/Zip13-KO mice.

Ehlers-Danlos syndrome (EDS) is a group of disorders caused by abnormalities that are identified in the extracellular matrix. Transforming growth factor-β1 (TGF-β1) plays a crucial role in formation of the extracellular matrix. It has been reported that the loss of function of zinc transporter ZRT/IRT-like protein 13 (ZIP13) causes the spondylocheiro dysplastic form of EDS (SCD-EDS: OMIM 612350), in which dysregulation of the TGF-β1 signaling pathway is observed, although the relationship between the dermis abnormalities and peripheral TGF-β1 level has been unclear. We investigated the characteristics of the dermis of the Zip13-knockout (KO) mouse, an animal model for SCD-EDS. Both the ratio of dermatan sulfate (DS) in glycosaminoglycan (GAG) components and the amount of collagen were decreased, and there were very few collagen fibrils with diameters of more than 150 nm in Zip13-KO mice dermis. We also found that the TGF-β1 level was significantly higher in Zip13-KO mice serum. These results suggest that collagen synthesis and collagen fibril fusion might be impaired in Zip13-KO mice and that the possible decrease of decorin level by reduction of the DS ratio probably caused an increase of free TGF-β1 in Zip13-KO mice. In conclusion, skin fragility due to defective ZIP13 protein may be attributable to impaired extracellular matrix synthesis accompanied by abnormal peripheral TGF-β homeostasis.

Microvesicle-mediated delivery of transforming growth factor β1 siRNA for the suppression of tumor growth in mice

Cell-derived microvesicles (MVs) have been recently shown as an efficient carrier to deliver small RNAs into the target cells. In the present study, we characterized the inhibitory effect of TGF-β1 siRNA delivered by mouse fibroblast L929 cell-derived MVs (L929 MVs) on the growth and metastasis of murine sarcomas 180 cells both in vitro and in vivo. We found that, comparing to the same concentration of free TGF-β1 siRNA, TGF-β1 siRNA delivered by L929 MVs much more efficiently decreased the level of TGF-β1 in the recipient tumor cells. Functionally, MVs containing TGF-β1 siRNA significantly decreased the viability and migration of sarcomas 180 cells and promoted the apoptosis of tumor cells. Co-immunoprecipitation with Argonaute 2 (AGO2) via anti-AGO2 antibody indicated that the majority of TGF-β1 siRNA in the MVs were associated with AGO2 complex. A tumor implantation mouse model further showed that intravenous injection of TGF-β1 siRNA-containing MVs strongly suppressed TGF-β1 expression and TGF-β1 signaling downstream in the implanted tumor cells, and thus inhibited the growth and lung metastases of tumor cells. In conclusion, our results collectively demonstrate that the delivery of therapeutic TGF-β1 siRNA by cell-derived MVs provides an effective strategy to control tumor cell growth and metastasis.

Antenatal glucocorticoids counteract LPS changes in TGF-β pathway and caveolin-1 in ovine fetal lung

Inflammation and antenatal glucocorticoids, the latter given to mothers at risk for preterm birth, affect lung development and may contribute to the development of bronchopulmonary dysplasia (BPD). The effects of the combined exposures on inflammation and antenatal glucocorticoids on transforming growth factor (TGF)-β signaling are unknown. TGF-β and its downstream mediators are implicated in the etiology of BPD. Therefore, we asked whether glucocorticoids altered intra-amniotic lipopolysaccharide (LPS) effects on TGF-β expression, its signaling molecule phosphorylated sma and mothers against decapentaplegic homolog 2 (pSmad2), and the downstream mediators connective tissue growth factor (CTGF) and caveolin-1 (Cav-1). Ovine singleton fetuses were randomized to receive either an intra-amniotic injection of LPS and/or maternal betamethasone (BTM) intramuscularly 7 and/or 14 days before delivery at 120 days gestational age (GA; term = 150 days GA). Saline was used for controls. Protein levels of TGF-β1 and -β2 were measured by ELISA. Smad2 phosphorylation was assessed by immunohistochemistry and Western blot. CTGF and Cav-1 mRNA and protein levels were determined by RT-PCR and Western blot. Free TGF-β1 and -β2 and total TGF-β1 levels were unchanged after LPS and/or BTM exposure, although total TGF-β2 increased in animals exposed to BTM 7 days before LPS. pSmad2 immunostaining increased 7 days after LPS exposure although pSmad2 protein expression did not increase. Similarly, CTGF mRNA and protein levels increased 7 days after LPS exposure as Cav-1 mRNA and protein levels decreased. BTM exposure before LPS prevented CTGF induction and Cav-1 downregulation. This study demonstrated that the intrauterine inflammation-induced TGF-β signaling can be inhibited by antenatal glucocorticoids in fetal lungs.

Measurement of human latent transforming growth factor-β1 using a latency associated protein-reactive ELISA

Human Transforming Growth Factor (TGF)-β1, one of three TGF-β isoforms, is a pleotropic cytokine critical for many physiological and immunological processes. TGF-β1 is secreted in a latent form, linked to Latency Associated Protein (LAP). Analysis of Latent TGF-β1 by TGF-β1 ELISA requires dissociation of TGF-β1 from LAP, e.g. by acidification of samples. The ELISA then measures total TGF-β1, equivalent to dissociated Latent TGF-β1 plus any free TGF-β1 present prior to acidification. Evolutionary conservation of TGF-β1 across mammals also renders TGF-β1 ELISAs reactive with TGF-β1 in bovine serum often used in human cell cultures. To enable a direct analysis of Latent TGF-β1, monoclonal antibodies were made against LAP from human Latent TGF-β1 and used to develop a LAP ELISA detecting Latent TGF-β1. The ELISA did not react with LAP from human Latent TGF-β2 or 3, respectively, nor with Latent TGF-β in bovine serum. EDTA-containing plasma from healthy subjects (n=20) was analyzed by conventional TGF-β1 ELISA and LAP ELISA. By TGF-β1 ELISA, total TGF-β1 were detected in all samples (median 133pM, range 34–348pM); low levels of free TGF-β1 found in 8/20 non-acidified samples showed that >98.5% of the total TGF-β1 derived from Latent TGF-β1. Latent TGF-β1 found in non-acidified samples by LAP ELISA (median 154pM, range 48–403pM) was comparable in molar levels to, and correlated with, total TGF-β1 (rs 0.96, p<0.0001). A similar agreement between the total TGF-β1 and the LAP ELISA was found with citrate- and heparin-containing plasma. The LAP ELISA facilitates analysis of Latent TGF-β1 without sample acidification and is not compromised by the presence of bovine serum in human cell supernatants.

Leptin deficiency prevents the activation of the murine α 2(I) collagen promoter by acetaldehyde

Leptin enhances hepatic fibrosis induced experimentally by various agents. The influence of leptin deficiency on murine α 2(I) collagen promoter activation by acetaldehyde and by TGFβ1 was investigated. Acetaldehyde failed to activate the promoter in ob/ob stellate cells as compared to its activating effect in wild-type cells. By contrast, TGFβ1 increased the activity of the promoter in the ob/ob and wild-type cells. Total cell TGFβ1 and secretion of total and free TGFβ1 were lower in cultured stellate cells from ob/ob mice than wild-type mice. Acetaldehyde increased free cell TGFβ1 and secretion of total and free TGFβ1 in wild-type, but not in ob/ob cells. The lack of activation of the α 2(I) collagen promoter by acetaldehyde in association with a lack of increase in TGFβ1 in response to acetaldehyde in ob/ob stellate cells provides further evidence for a mediating role of TGFβ1 in the actions of acetaldehyde on collagen transcription.

Repairing of rabbit skull defect by dehydrothermally crosslinked collagen sponges incorporating transforming growth factor β1

Collagen sponges of various biodegradabilities were prepared by dehydrothermal crosslinking at 140 °C for different time periods. When the collagen sponges were radioiodinated and implanted subcutaneously into the back of mice, the radioactivity remaining at the implanted site decreased with time; the longer the time of dehydrothermal crosslinking, the slower the radioactivity decrement. The radioactivity following the subcutaneous implantation of collagen sponges incorporating 125I-labeled transforming growth factor (TGF)-β1 also decreased with time. The time profile of both the radioactivity remainings was in good accordance to each other, irrespective of the crosslinking time. This indicates that the TGF-β1 incorporated in the sponges was released as a result of sponge biodegradation. Potential of collagen sponges incorporating 0.1 μg of TGF-β1 in repairing the defect of rabbit skulls was evaluated in a stress-unloaded state. Bone repairing was induced by application of the collagen sponges incorporating 0.1 μg of TGF-β1 whereas that of free TGF-β1 at the same dose and TGF-β1-free, empty collagen sponges were ineffective. The bone defect was histologically closed by the bone tissue newly formed 6 weeks after application. Bone mineral density (BMD) analysis revealed that the collagen sponge incorporating TGF-β1 enhanced the BMD value at the bone defect to a significantly great extent compared with other agents. A maximum enhancement of BMD was observed for the collagen sponge incorporating TGF-β1 which was prepared by dehydrothermal crosslinking for 6 h. It was concluded that the TGF-β1 incorporated in the collagen sponge was released in a biologically active form as a result of sponge biodegradation, resulting in enhanced bone repairing at the skull defect. It is possible that for too slowly degraded sponges, the remaining physically impairs the bone repairing at the skull defect. Induction of bone repairing would not be achieved through a rapid release of TGF-β1 from too fast-degraded sponge.

Repair of full-thickness cartilage defects using liposomal transforming growth factor-β1

AbstractTransforming growth factor-β1 (TGFβ1) is a wellknown, potent growth factor implicated in both in vitro and in vivo chondrogenesis. Liposomes have been employed as a drug delivery system to promote the efficient use of drugs. The objective of this study was to demonstrate that a single injection of liposomal TGFβ1 has an accelerating effect on the repair of an articular cartilage defect. Full-thickness articular cartilage defects were prepared on the patellar grooves of the femurs in knee joints of Japanese white rabbits. One week after surgery, various reagents including liposomal TGFβ1, free TGFβ1, and phosphate-buffered saline were injected into the operated knee joints. At 3 weeks after surgery the specimens obtained from the lesions were evaluated histologically, and the glycosaminoglycan content was quantified. Histological examination revealed that the defects were filled with thicker fibrous cartilage and showed more intense metachromatic staining in the liposomal TGFβ1 group than in the other groups. The glycosaminoglycan content of the repair tissue was also significantly higher in the liposomal TGFβ1 group than in the other groups. This study indicated that the intraarticular injection of liposomal TGFβ1 could accelerate the early-stage repair of full-thickness articular cartilage defects.

Promoted bone healing at a rabbit skull gap between autologous bone fragment and the surrounding intact bone with biodegradable microspheres containing transforming growth factor-beta1.

This study is a trial to promote repairing of the rabbit skull bone gap between an autologous bone flap and the intact bone with biodegradable gelatin microspheres containing transforming growth factor-beta1 (TGF-beta1). A 10-mm diameter bone defect was prepared in rabbit skulls by drilling out a bone flap of 6 mm in diameter. After a surrounding gap defect of 2 mm was created and treated with 0.5 microg of free TGF-beta1 and gelatin microspheres containing 0.5 microg of free TGF-beta1, the circular autologous bone flap was placed in the center. Significant bone healing at the gap defect was observed 3 weeks after implantation of the TGF-beta1-containing gelatin microspheres. The bone mineral density (BMD) was significantly higher than that of other experimental groups. On the contrary, when applied with free TGF-beta1, a fibrous tissue initially infiltrated into the gap defect, resulting in impairing bone healing. The tissue response was similar to that at the defect implanted with empty gelatin microspheres and TGF-beta1-free phosphate-buffered saline solution alone. There was more space in the gap-filling bone in the 16-week view than the 3-week view. It is possible that this was an intermediate step along the way toward normal healing and formation of cancellous bone. We conclude that gelatin microspheres containing TGF-beta1 show promise as an agent to promote bone regeneration of subcritical size defects between surgically positioned autologous bone flaps and surrounding host bone.

Bone regeneration by transforming growth factor β1 released from a biodegradable hydrogel

This paper describes the sustained release of transforming growth factor β1 (TGF-β1) from a biodegradable hydrogel based on polyion complexation for the enhancement of bone regeneration activity. Basic TGF-β1 was adsorbed onto the biodegradable hydrogel of acidic gelatin with an isoelectric point of 5.0 by an electrostatic interaction. The TGF-β1 could not be adsorbed onto basic gelatin. When acidic gelatin hydrogels incorporating 125I-labeled TGF-β1 were implanted into the back subcutis of mice, the radioactivity decreased with time and the in vivo retention of TGF-β1 was prolonged with a decrease in the water content of hydrogels. The higher the water content of hydrogels, the faster their biodegradation. The in vivo retention of TGF-β1 correlated well with that of gelatin hydrogels, indicating that TGF-β1 was released from the gelatin hydrogel as a result of hydrogel biodegradation. The ability of TGF-β1-incorporated into acidic gelatin hydrogels to induce bone regeneration was evaluated in a rabbit calvarial defect model. Eight weeks after treatment, the gelatin hydrogels with water contents of 90 and 95 wt% induced significantly high bone regeneration compared with those with lower and higher water contents and free TGF-β1. This indicates that the sustained release of TGF-β1 from the hydrogel with suitable in vivo degradability is necessary to effectively enhance its osteoinductive function. Rapid hydrogel degradation will result in a retention time of TGF-β1 which is too short to induce bone regeneration. It is possible that the slow degradation of the hydrogel physically blocked TGF-β1-induced bone regeneration at the skull defect. It can be concluded that the gelatin hydrogel is a promising matrix of TGF-β1 release to induce skull bone regeneration.

Synergistic effect of gelatin microspheres incorporating TGF-β1 and a physical barrier for fibrous tissue infiltration on skull bone formation

The objective of this study is to examine whether or not bone formation at a skull bone defect induced by gelatin microspheres incorporating transforming growth factor (TGF)-β1 is promoted by prevention of fibrous tissues into the defect. The 6-mm diameter bone defect of rabbit skulls was applied with gelatin microspheres incorporating TGF-β1 or free TGF-β1 and physically covered by a barrier membrane. When the bone formation at the defect was assessed 6 weeks postoperatively, combinational application of gelatin microspheres incorporating 0.1 μg of TGF-β1 with the barrier membrane induced bone formation at the skull defect, in marked contrast to that of 0.1 μg of free TGF-β1 and empty gelatin microspheres. Complete defect closure was histologically observed by the newly formed bone tissue. Without the barrier membrane, gelatin microspheres incorporating TGF-β1 were less effective in inducing bone formation, whereas free TGF-β1 and empty gelatin microspheres were ineffective. The skull defect was occupied by fibrous tissue infiltrated in place of bone tissue. The bone mineral density at the skull defect applied with gelatin microspheres incorporating TGF-β1 plus the membrane was significantly higher than that of gelatin microspheres incorporating TGF-β1 alone. The present data indicated that physical protection from the soft tissue infiltration enabled gelatin microspheres incorporating TGF-β1 to synergistically enhance the osteoinductive ability at the skull defect.

ＴＧＦ‐β１含有吸収性ゼラチンハイドロゲルの頭蓋骨再生能

Hydrogels with water contents of 85, 90, 95, and 98 wt% were prepared through chemical crosslinking of acidic gelatin with varied concentrations of glutaraldehyde. In vivo release test of the gelatin hydrogels incorporating 125I-labeled transforming growth factor (TGF)-β1 revealed that the period of TGF-β1 release in the mouse subcutis prolonged with the decreased hydrogel water content. When the skull defect of rabbits (6 mm diameter) was treated by the TGF-β1-incorporating gelatin hydrogels, significant bone regeneration at the defect was observed at TGF-β1 doses 0.1μg or higher per defect, in marked contrast to free TGF-β1. The most efficious bone regeneration was induced by the hydrogels with water contents of 90 and 95 wt%. This water content dependance is ascribed to balance of TGF-β1 release and hydrogel degradation. Shorter TGF-β1 release from fastest degraded hydrogel (98 wt%) will not be enough to induce its osteogenic effect, while the slowest degradation of hydrogel (85 wt%) will physically impair bone regeneration at the skull defect. It is concluded that our biodegradable gelatin hydrogel functioned not only as a release matrix of TGF-β1 but also as a barrier for making space to prevent ingrowth of soft tissues to the bone defect.