Transforming growth factor (TGF)‑β3 has previously been reported to antagonize hepatic fibrosis in vivo and in vitro. The present study aimed to investigate the mechanism underlying the involvement of TGF‑β3 in hepatic fibrosis. Short hairpin (sh)RNA‑cAMP-responsive element binding protein (CREB) 1 and small interfering (si)RNA‑Smad3 were utilized to silence the expression of CREB1 and Smad3 in hepatic stellate cells (HSCs), whereas the vector pRSV‑CREB1 was used to induce CREB1 overexpression in HSCs. Cells were treated with or without exogenous TGF‑β3 or TGF‑β1, and mRNA and protein expression levels were assessed using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Untreated cells served as the control group. Exogenous TGF‑β3 increased Smad7 mRNA and protein expression levels in rat HSCs, and CREB1 and Smad3 appeared to be implicated in the mechanism of Smad7. CREB1 knockdown inhibited the TGF‑β3‑induced upregulation of Smad7, whereas its overexpression potentiated the Smad7 upregulation in HSCs; conversely, CREB1 manipulations had no effect on Smad7 expression under basal conditions. In addition, TGF‑β3‑induced Smad7 upregulation was blocked when the activity of p38, a kinase upstream of CREB1, was inhibited. Furthermore, silencing Smad3 resulted in decreased Smad7 expression under basal conditions and in TGF‑β3‑stimulated cells. Notably, Smad7 expression appeared to also be induced by exogenous TGF‑β1, independent of CREB1. The present study demonstrated that TGF‑β3 increased Smad7 expression in HSCs, whereas CREB1 and Smad3 appeared to participate in the mechanism of induction. Smad3 is the key regulator whereas CREB‑1 acts as a co‑regulator. These results suggested that this mechanism may underlie the antagonizing effects of TGF‑β3 on hepatic fibrosis.

The high socioeconomic impact of osteoporosis and osteoporotic fracture is due to their high mortality, morbidity, and disease-related costs. Nowadays, bone mineral density (BMD) is a comparatively expensive way to diagnose and follow up patients with osteoporosis. Transforming growth factor-β3 (TGF-β3) is a protein categorized into cytokines. Some previous in vitro studies showed TGF-β3 effects on osteocytes and bone formation. Therefore, we conducted this study to find if there is any significant relationship between TGF-β3 and BMD results.

This was an analytical cross-sectional study conducted in 2017. We included individuals who had been referred from their physicians to undergo BMD dual-energy X-ray absorptiometry. Blood samples were taken from 150 participants for measuring TGF-β3 with ELISA method.

Results

The mean ± standard deviation of TGF-β3 serum level was 79 ± 30.8 pg/ml (minimum 41 pg/ml and maximum 210 pg/ml). There was a statistically significant and direct proportional relationship between TGF-β3 and T-score as a marker for the diagnosis and follow-up of osteoporosis and osteoporotic fracture (P = 0.001) (Pearson's correlation = +0.95).

There was a significant relationship between TGF-β3 serum level and BMD. TGF-β3 serum level may be used as a marker for the diagnosis and follow-up of osteoporosis and osteoporotic fracture.

The aim of the current in vitro study was to investigate if tissue surface modification with collagenase and addition of the TGF-β3 can increase the number of cells present in meniscus tears repaired with the use of newly developed tissue adhesives based on isocyanate-terminated block copolymers. Cylindrical explants were harvested from the inner part of bovine menisci. To simulate a full-thickness tear, the central core of the explants was removed and glued back into the defect, with or without incubation in collagenase solution prior to gluing. The repair constructs were then cultured with or without addition of TGF-β3, and assessed for their histological appearance. The histological staining of the constructs confirmed that both developed adhesives were not cytotoxic. After 28 days, meniscus cells were present in direct contact with the glues. The addition of TGF-β3 to the culture medium resulted in the presence of cells that formed a sheath inside the simulated tear and in increased cell numbers at the edges of annulus of the explants. In the group in which the tissue was incubated in collagenase and cultured in medium containing TGF-β3, thicker layers of cells were observed. These results suggest that repairing the torn meniscus with tissue adhesives after pre-treatment of the tissue with collagenase and stimulation with TGF-β3 is a very promising treatment method, especially when treating the inner avascular part of the meniscus. Nevertheless, longer-term in vitro and in vivo studies are needed to confirm the beneficial effects of this combination therapy.

Stem cells hold great promise for treating cartilage degenerative diseases such as osteoarthritis (OA). The efficacy of stem cell-based therapy for cartilage repair is highly dependent on their interactions with local cells in the joint. This study aims at evaluating the interactions between osteoarthritic chondrocytes (OACs) and adipose-derived stem cells (ADSCs) using three dimensional (3D) biomimetic hydrogels. To examine the effects of cell distribution on such interactions, ADSCs and OACs were co-cultured in 3D using three co-culture models: conditioned medium (CM), bi-layered, and mixed co-culture with varying cell ratios. Furthermore, the effect of transforming growth factor (TGF)-β3 supplementation on ADSC-OAC interactions and the resulting cartilage formation was examined. Outcomes were analyzed using quantitative gene expression, cell proliferation, cartilage matrix production, and histology. TGF-β3 supplementation led to a substantial increase in cartilage matrix depositions in all groups, but had differential effects on OAC-ADSC interactions in different co-culture models. In the absence of TGF-β3, CM or bi-layered co-culture had negligible effects on gene expression or cartilage formation. With TGF-β3 supplementation, CM and bi-layered co-culture inhibited cartilage formation by both ADSCs and OACs. In contrast, a mixed co-culture with moderate OAC ratios (25% and 50%) resulted in synergistic interactions with enhanced cartilage matrix deposition and reduced catabolic marker expression. Our results suggested that the interaction between OACs and ADSCs is highly dependent on cell distribution in 3D and soluble factors, which should be taken into consideration when designing stem cell-based therapy for treating OA patients.

Craniofacial sutures are bone growth fronts that respond and adapt to biomechanical environments. Little is known of the role sutures play in regulating the skull biomechanical environment during patency and fusion conditions, especially how delayed or premature suture fusion will impact skull biomechanics. Tgf-β3 has been shown to prevent or delay suture fusion over the short term in rat skulls, yet the long-term patency or its consequences in treated sutures is not known. It was therefore hypothesized that Tgf-β3 had a long-term impact to prevent suture fusion and thus alter the skull biomechanics. In this study, collagen gels containing 3 ng Tgf-β3 were surgically placed superficial to the posterior interfrontal suture (IFS) and deep to the periosteum in postnatal day 9 (P9) rats. At P9, P24, and P70, biting forces and strains over left parietal bone, posterior IFS, and sagittal suture were measured with masticatory muscles bilaterally stimulated, after which the rats were sacrificed and suture patency analyzed histologically. Results demonstrated that Tgf-β3 treated sutures showed less fusion over time than control groups, and strain patterns in the skulls of the Tgf-β3-treated group were different from that of the control group. Although bite force increased with age, no alterations in bite force were attributable to Tgf-β3 treatment. These findings suggest that the continued presence of patent sutures can affect strain patterns, perhaps when higher bite forces are present as in adult animals.

BACKGROUND

Mesenchymal stem cells (MSCs) are viewed as a having significant potential for tissue engineering and regenerative medicine therapies. Clinical implementation of MSCs, however, demands that their preparation be stable and reproducible. Given that environmental and bioprocessing parameters such as substrate stiffness, seeding densities, culture medium composition, and mechanical loading can result in undirected differentiation of the MSC population, the objective of this study was to systematically investigate how hydrodynamic loading influences the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) towards the osteochondral lineages both in the presence and absence of exogenous, inductive factors.

METHODS

Expanded bovine MSCs were suspended in 2.5 % agarose, cast in a custom mold, and placed into either static or one of two dynamic culture environments consisting of "high" and "low" magnitude shear conditions. Constructs were supplemented with varying concentrations (0, 1, 10, 100 ng/mL) of either TGF-β3 or BMP-2 throughout cultivation with tissue samples being collected following each week of culture.

RESULTS

In the absence of exogenous supplementation, hydrodynamic loading had little effect on cell phenotype at either magnitude of stimulation. When cultures were supplemented with BMP-2 and TGF-β3, MSCs gene expression progressed towards the osteogenic and chondrogenic pathways, respectively. This progression was enhanced by the presence of hydrodynamic loading, particularly under high shear conditions, but may point the chondrogenic cultures down a hypertrophic path toward osteogenesis reminiscent of endochondral ossification if TGF-β3 supplementation is insufficient.

CONCLUSIONS

Moving forward, these results suggest bioprocessing conditions which minimize exposure of chondrogenic cultures to fluid shear stress to avoid undesirable differentiation of the MSC population.

BACKGROUND

The Autologous Chondrocytes Transplantation (ACT) method is being studied for repair of cartilage diseases. As the chondrocytes dedifferentiated during monolayer culture, three-dimensional cultures are suggested to redifferentiate them. The aim of this study was investigation of the effect of TGF-β3 growth factor on chondrocytes in pellet culture system.

METHODS

The chondrocytes were isolated from three human articular cartilages by enzymatic digestion. The cells of the second passage were transferred to pellet culture system. We determined the chondrogenic medium with TGF-β3 as the experimental group and without it as the control group. After 2 weeks, the aggrecan production was investigated using histological and immunohistochemical (IHC) methods.

RESULTS

The presence of glycosaminoglycans was proved through Toluiden blue staining. Comparison of IHC results using MATLAB software showed that aggrecan in the experimental group was significantly higher than in the control group (P ≤ 0.05).

CONCLUSIONS

The presence of TGF-β3 in the chondrogenic medium could lead to the production of more aggrecan in chondrocytes cultivated in pellet culture system.

Previous studies have demonstrated that transforming growth factor-β3 (TGF-β3) protected liver against fibrosis in vivo and vitro, but its regulation is poorly understood. In addition, the cAMP-responsive element (CRE) in TGF-β3 promoter is recognized as an important regulatory site for TGF-β3 auto-regulation. Thus, we hypothesize that transcription factor CRE-binding protein-1 (CREB-1) regulates the auto-induction of TGF-β3 in hepatic stellate cells (HSCs). We used exogenous TGF-β3 to activate the signal pathway of TGF-β3 auto-regulation in HSCs, results indicated that exogenous TGF-β3 could up-regulate the protein and mRNA expressions of TGF-β3, and provoke the phosphorylation of CREB-1 on Ser-133, besides, it could induce the DNA binding activity of p-CREB-1 and activate TGF-β3 promoter as well. Additionally, we used pGenesil-1.1-shRNA-CREB-1 and pRSV-CREB-1 expression vector to silence and up-regulate CREB-1 gene expression respectively, and the results indicated that inhibition of CREB-1 suppressed exogenous TGF-β3 stimulation of TGF-β3 mRNA and protein expressions in HSCs, whereas up-regulation of CREB-1 induced this stimulation. Our results indicate that exogenous TGF-β3 up-regulates the activity of TGF-β3 promoter by activating CREB-1, then induces the mRNA and protein expressions of TGF-β3. Especially, p-CREB-1 is a critical transcription factor in mediating TGF-β3 auto-induction.

The aim of this study was to investigate cementogenesis and alveolar bone induction during in vivo periodontal tissue regeneration upon implantation of hTGF-β3 in furcation defects of Papio ursinus and to evaluate the feasibility of gene expression studies.

Class II furcation defects (day 0) were prepared in mandibular first and second molars of three P. ursinus and on day 30 implanted with and without 75 μg hTGF-β3 in Matrigel® matrix. On day 0, 30 and 90, cementum and alveolar bone were harvested for gene expression analyses. Coral-derived bioreactors with and without 250 μg hTGF-β3 were implanted in the rectus abdominis to monitor tissue induction.

hTGF-β3 induced cementogenesis with TGF-β3 , Cementum Protein-1 (Cemp1) and Osteocalcin (OC) up-regulation, and down-regulation of BMP-2 and OP-1. Matrigel® matrix specimens showed up-regulation of BMP-2, TGF-β3 , and OC, with down-regulation of OP-1 and Cemp1. hTGF-β3 induced alveolar bone with down-regulation of OP-1, TGF-β3 , OC, and Cemp1. hTGF-β3 bioreactors induced bone at the periphery only. BMP-3, BMP-4, TGF-β1 and TGF-β3 were up-regulated in the adjacent muscle with TGF-β2 down-regulation.

Cementogenesis and osteogenesis by hTGF-β3 entail the expression and up-regulation of TGF-β3 and OC with fine tuning and modulation of BMP-2 and OP-1.

The momentum to compose this Leading Opinion on the synergistic induction of bone formation suddenly arose when a simple question was formulated during a discussion session on how to boost the often limited induction of bone formation seen in clinical contexts. Re-examination of morphological and molecular data available on the rapid induction of bone formation by the recombinant human transforming growth factor-β3 (hTGF-β3) shows that hTGF-β3 replicates the synergistic induction of bone formation as invocated by binary applications of hOP-1:hTGF-β1 at 20:1 by weight when implanted in heterotopic sites of the rectus abdominis muscle of the Chacma baboon, Papio ursinus. The rapid induction of bone formation in primates by hTGF-β3 may stem from bursts of cladistic evolution, now redundant in lower animal species but still activated in primates by relatively high doses of hTGF-β3. Contrary to rodents, lagomorphs and canines, the three mammalian TGF-β isoforms induce rapid and substantial bone formation when implanted in heterotopic rectus abdominis muscle sites of P. ursinus, with unprecedented regeneration of full thickness mandibular defects with rapid mineralization and corticalization. Provocatively, thus providing potential molecular and biological rationales for the apparent redundancy of osteogenic molecular signals in primates, binary applications of recombinant human osteogenic protein-1 (hOP-1) with low doses of hTGF-β1 and -β3, synergize to induce massive ossicles in heterotopic rectus abdominis, orthotopic calvarial and mandibular sites of P. ursinus. The synergistic binary application of homologous but molecularly different soluble molecular signals has indicated that per force several secreted molecular signals are required singly, synchronously and synergistically to induce optimal osteogenesis. The morphological hallmark of the synergistic induction of bone formation is the rapid differentiation of large osteoid seams enveloping haematopoietic bone marrow that forms by day 15 in heterotopic rectus abdominis sites. Synergistic binary applications also induce the morphogenesis of rudimentary embryonic growth plates indicating that the "memory" of developmental events in embryo can be redeployed postnatally by the application of morphogen combinations. Synergistic binary applications or single relatively high doses of hTGF-β3 have shown that hTGF-β3 induces bone by expressing a variety of inductive morphogenetic proteins that result in the rapid induction of bone formation. Tissue induction thus invocated singly by hTGF-β3 recapitulates the synergistic induction of bone formation by binary applications of hTGF-β1 and -β3 isoforms with hOP-1. Both synergistic strategies result in the rapid induction and expansion of the transformed mesenchymal tissue into large corticalized heterotopic ossicles with osteoblast-like cell differentiation at the periphery of the implanted reconstituted specimens with "tissue transfiguration" in vivo. Molecularly, the rapid induction of bone formation by binary applications of hOP-1 and hTGF-β3 or by hTGF-β3 applied singly resides in the up-regulation of selected genes involved in tissue induction and morphogenesis, Osteocalcin, RUNX-2, OP-1, TGF-β1 and -β3 with however the noted lack of TGF-β2 up-regulation.

The development of engineered tissues has progressed over the past 20 years from in vitro characterization to in vivo implementation. For musculoskeletal tissue engineering in particular, the emphasis of many of these studies was to select conditions that maximized functional and compositional gains in vitro. However, the transition from the favorable in vitro culture environment to a less favorable in vivo environment has proven difficult, and, in many cases, engineered tissues do not retain their preimplantation phenotype after even short periods in vivo. Our laboratory recently developed disc-like angle-ply structures (DAPS), an engineered intervertebral disc for total disc replacement. In this study, we tested six different preculture media formulations (three serum-containing and three chemically defined, with varying doses of transforming growth factor β3 [TGF-β3] and varying strategies to introduce serum) for their ability to preserve DAPS composition and metabolic activity during the transition from in vitro culture to in vivo implantation in a subcutaneous athymic rat model. We assayed implants before and after implantation to determine collagen content, glycosaminoglycan (GAG) content, metabolic activity, and magnetic resonance imaging (MRI) characteristics. A chemically defined media condition that incorporated TGF-β3 promoted the deposition of GAG and collagen in DAPS in vitro, the maintenance of accumulated matrix in vivo, and minimal changes in the metabolic activity of cells within the construct. Preculture in serum-containing media (with or without TGF-β3) was not compatible with DAPS maturation, particularly in the nucleus pulposus (NP) region. All groups showed increased collagen production after implantation. These findings define a favorable preculture strategy for the translation of engineered discs seeded with disc cells.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a progressive decline in lung function which can be attributed to excessive scarring, inflammation and airway remodelling. Mannose-6-phosphate (M6P) is a strong inhibitor of fibrosis and its administration has been associated with beneficial effects in tendon repair surgery as well as nerve repair after injury. Given this promising therapeutic approach we developed an improved analogue of M6P, namely PXS64, and explored its anti-fibrotic effects in vitro. Normal human lung fibroblasts (NHLF) and human lung fibroblast 19 cells (HF19) were exposed to active recombinant human TGF-β1 to induce increases in fibrotic markers. rhTGF-β1 increased constitutive protein levels of fibronectin and collagen in the NHLF cells, whereas HF19 cells showed increased levels of fibronectin, collagen as well as αSMA (alpha smooth muscle actin). PXS64 demonstrated a robust inhibitory effect on all proteins analysed. IPF patient fibroblasts treated with PXS64 presented an improved phenotype in terms of their morphological appearance, as well as a decrease in fibrotic markers (collagen, CTGF, TGF-β3, tenascin C, αSMA and THBS1). To explore the cell signalling pathways involved in the anti-fibrotic effects of PXS64, proteomics analysis with iTRAQ labelling was performed and the data demonstrated a specific antagonistic effect on the TGF-β1 pathway. This study shows that PXS64 effectively inhibits the production of extracellular matrix, as well as myofibroblast differentiation during fibrosis. These results suggest that PXS64 influences tissue remodelling by inhibiting TGF-β1 signalling in NHLF and HF19 cell lines, as well as in IPF patient fibroblasts. Thus PXS64 is a potential candidate for preclinical application in pulmonary fibrosis.

Repair of hyaline cartilage remains a huge challenge in clinic because of the avascular and aneural characteristics and the paucity of endogenous repair cells. Recently, tissue engineering technique, possessing unique capacity of repairing large tissue defects, avoiding donor complications and two-stage invasive surgical procedures, has been developed a promising therapeutic strategy for cartilage injury. In this study, we incorporated low-molecular-weight heparin (LMWH) into carboxymethyl chitosan-oxidized chondroitin sulfate (CMC-OCS) hydrogel for loading transforming growth factor-β3 (TGF-β3) as matrix of peripheral blood mesenchymal stem cells (PB-MSCs) to construct tissue-engineered cartilage. Meanwhile, three control hydrogels with or without LMWH and/or TGF-β3 were also prepared. The gelling time, microstructures, mechanical properties, degradation rate, cytotoxicity, and the release of TGF-β3 of different hydrogels were investigated. In vitro experiments evaluated the tri-lineage differentiation potential of PB-MSCs, combined with the proliferation, distribution, viability, morphology, and chondrogenic differentiation. Compared with non-LMWH-hydrogels, LMWH-hydrogels (LMWH-CMC-OCS-TGF-β3) have shorter gelling time, higher mechanical strength, slower degradation rate and more stable and lasting release of TGF-β3. After two weeks of culture in vitro, expression of cartilage-specific genes collagen type-2 (COL-2) and aggrecan (AGC), and secretion of glycosaminoglycan (GAG), and COL-2 proteins in LMWH-CMC-OCS-TGF-β3 group were significantly higher than those in other groups. COL-2 immunofluorescence staining showed that the proportion of COL-2 positive cells and immunofluorescence intensity in LMWH-CMC-OCS-TGF-β3 hydrogel were significantly higher than those in other groups. The LMWH-CMC-OCS-TGF-β3 hydrogel can slowly release TGF-β3 in a long term, and meanwhile the hydrogel can provide a biocompatible microenvironment for the growth and chondrogenic differentiation of PB-MSCs. Thus, LMWH functionalized CMC-OCS hydrogels proposed in this work will be beneficial for constructing functional scaffolds for tissue-engineered cartilage.

Articular cartilage (AC), tissue with the lowest volumetric cellular density, is not supplied with blood and nerve tissue resulting in limited ability for self-repair upon injury. Because there is no treatment capable of fully restoring damaged AC, tissue engineering is being investigated. The emphasis of this field is to engineer functional tissues in vitro in bioreactors capable of mimicking in vivo envi- ronments required for appropriate cellular growth and differentiation. In a step towards engineering AC, human adipose-derived stem cells were differentiated in a unique centrifugal bioreactor under oscillating hydrostatic pressure (OHP) and supply of transforming growth factor beta 3 (TGF-β3) that mimic in vivo environments. Static micromass and pellet cultures were used as controls. Since withstanding and absorbing loads are among the main functions of an AC, mechanical properties of the engineered AC tissues were assayed using atomic force microscopy (AFM) under a controlled indentation depth of 100 nm. Young's moduli of elasticity were quantified by modeling AFM force-indentation data using the Hertz model of contact mechanics. We found exposure to OHP causes cartilage constructs to have 45-fold higher Young's moduli compared to static cultures. Addition of TGF-β3 further increases Young's moduli in bioreactor samples by 1.9-fold bringing it within 70.6% of the values estimated for native cartilage. Our results imply that OHP and TGF-β3 act synergistically to improve the mechanics of engineered tissues.

Transforming growth factor (TGF) β is a pro-fibrotic cytokine. While three isoforms (TGF-β1, 2 and 3) are known, the functional differences between them are obscure. To investigate the roles of TGF-β isoforms during liver fibrogenesis, male Wistar rats were administrated carbon tetrachloride (CCl4) subcutaneously twice a week for two months. Livers were excised and sectioned for histochemical examinations. These livers were also used to quantitate the expression of genes associated with fibrogenesis, including TGF-β isoforms, as well as those associated with retinoid metabolism. Expression levels of Tgfb1 and Tgfb3 were up-regulated in CCl4-treated rat livers while that of Tgfb2 was not changed. The mRNAs for lecithin-retinol acyltransferase (Lrat) and retinoic acid hydroxylase, Cyp26a1, were also elevated. By immunohistochemical staining, TGF-β3 protein was found to be localized mainly in liver parenchymal cells (hepatocytes). These results indicate that retinoid mobilization likely takes place within the rat's liver following CCl4 treatment, and suggest the possibility that the expression of Tgfb mRNA is regulated by retinoic acid receptors. Reporter analyses of a region of the Tgfb3 gene were performed using the rat liver parenchymal cell line, RLC-16, and a positively responsive region was identified within its intron.

OBJECTIVE

This preliminary study aims to determine the differentially expressed proteins from chondrogenic differentiated multipotent stromal cells (cMSCs) in comparison to undifferentiated multipotent stromal cells (MSCs) and adult chondrocytes (ACs).

METHODS

ACs and bone marrow-derived MSCs were harvested from New Zealand White rabbits (n = 3). ACs and cMSCs were embedded in alginate and were cultured using a defined chondrogenic medium containing transforming growth factor-beta 3 (TGF-β3). Chondrogenic expression was determined using type-II collagen, Safranin-O staining and glycosaminoglycan analyses. Two-dimensional gel electrophoresis (2-DE) was used to isolate proteins from MSCs, cMSCs and ACs before being identified using liquid chromatography-mass spectrometry (LC-MS). The differentially expressed proteins were then analyzed using image analysis software.

RESULTS

Both cMSCs and ACs were positively stained with type-II collagen and safranin-O. The expression of glycosaminoglycan in cMSCs was comparable to AC at which the highest level was observed at day-21 (p>0.05). Six protein spots were found to be most differentially expressed between MSCs, cMSCs and ACs. The protein spots cofilin-1 (CFL1) and glycealdehyde-3-phosphate dehydrogenase (GAPD) from cMSCs had expression levels similar to that of ACs whereas the others (ie. MYL6B, ALDOA, TAGLN2, EF1-alpha), did not match the expression level of ACs.

CONCLUSIONS

Despite having similar phenotypic expressions to ACs, cMSCs expressed proteins which were not typically expected. This may explain the reason for the unexplained lack of improvement in cartilage repair outcomes reported in previous studies.

The purpose of study was to investigate the maturation of mesenchymal stem cells (MSC) laden in HA constructs with various combinations of chemically defined medium (CM) components and determine the impact of dexamethasone and serum on construct properties. Constructs were cultured in CM with the addition or withdrawal of media components or were transferred to serum containing media that partially represents an in vivo-like condition where pro-inflammatory signals are present. Constructs cultured in CM+ (CM with TGF-β3) and DEX- (CM+ without dexamethasone) conditions produced robust matrix, while those in ITS/BSA/LA- (CM+ without ITS/BSA/LA) and Serum+ (10% FBS with TGF-β3) produced little matrix. While construct properties in DEX- were greater than those in CM+ at 4 weeks, properties in CM+ and DEX- reversed by 8 weeks. While construct properties in DEX- were greater than those in CM+ at 4 weeks, the continued absence or removal of dexamethasone resulted in marked GAG loss by 8 weeks. Conversely, the continued presence or new addition of dexamethasone at 4 weeks further improved or maintained construct properties through 8 weeks. Finally, when constructs were converted to Serum (in the continued presence of TGF-β3 with or without dexamethasone) after pre-culture in CM+ for 4 weeks, GAG loss was attenuated with addition of dexamethasone. Interestingly, however, collagen content and type was not impacted. In conclusion, dexamethasone influences the functional maturation of MSC-laden HA constructs, and may help to maintain properties during long-term culture or with in vivo translation by repressing pro-inflammatory signals. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1717-1727, 2018.

Intervertebral disc (IVD) herniation can be caused by both degeneration and traumatic injury, ultimately resulting in back pain or sciatica due to disc protrusion. Replacement of the nucleus pulposus (NP) tissue during surgical intervention post herniation could improve the long-term stability of the functional spinal unit. Tissue engineering strategies may potentially restore both biological and mechanical function of the NP. Recently, photocrosslinked carboxymethylcellulose (CMC) hydrogels were shown to support chondrogenic, NP-like extracellular matrix (ECM) elaboration by human mesenchymal stromal cells (hMSCs) when supplemented with TGF-β3. However, long-term preconditioning with soluble growth factors in vitro or the use of sustained growth factor delivery vehicles in vivo can be expensive and difficult to control. Transient supplementation with growth factors has been shown to maintain or improve maturation of tissue-engineered constructs. The objective of this study was to evaluate the influence of TGF-β3 exposure time on hydrogel bulk properties and NP-like matrix elaboration in hMSC-laden CMC hydrogels. Constructs were exposed to TGF-β3 for 2 weeks (Transient), 8 weeks (Continuous) or 0 weeks (controls). After 8 weeks of culture, both the Transient and Continuous groups exhibited increased ECM accumulation compared to 2 weeks and controls. The Transient group displayed significantly greater accumulation of collagens I and II, while GAG content was significantly higher in the Continuous group by 8 weeks. Distribution of ECM was more homogeneous in the Continuous group, while the Transient group exhibited more concentrated accumulation in the periphery of the hydrogel by 8 weeks. Mechanical properties improved over time in both groups, however, Continuous constructs demonstrated significantly more robust mechanical properties (equilibrium modulus and peak stress) compared to Transient gels at 8 weeks. Although the functional properties of Transient constructs did not surpass those achieved by Continuous scaffolds, they increased and were maintained upon growth factor removal at 2 weeks, and were greater than controls. Additionally, Transient construct mechanical properties (equilibrium modulus, % relaxation) were similar to those of native NP tissue. The differences seen in ECM distribution and subsequent construct functional maturation are likely due to the time available for diffusion of growth factors through the construct. Overall, these findings support the use of short-term TGF-β3 treatment to promote sufficient long-term tissue maturation in vitro in this hMSC-laden CMC hydrogel system.

Prediction of recurrence in patients with unprovoked venous thromboembolism (VTE) remains a challenge. Studies of atherosclerosis suggest a protective role of transforming growth factor (TGF)-β. However, the role of TGF-β has not been studied in VTE. The aim of this study was to investigate TGF-β as a predictive marker of recurrent VTE in patients with a first episode of unprovoked VTE. Patients in the Malmö Thrombophilia Study (MATS) were followed after the discontinuation of anticoagulant treatment until the diagnosis of recurrent VTE or the end of the study in December 2008 (mean ± SD 38.5 months ± 27). Among patients with a first episode of unprovoked VTE, we identified 42 patients with recurrent VTE during the follow-up period. Two age- and sex-matched control subjects without recurrent VTE were selected for each patient (n = 84). Plasma levels of the three isoforms of TGF-β (TGF-β1, TGF-β2 and TGF-β3) were quantified simultaneously by TGF-β 3-plex immunoassay. Compared to controls, plasma levels of TGF-β1 and TGF-β2 were significantly lower in patients with recurrent VTE (p < 0.05), whereas no difference was found for TGF-β3. In a multivariate Cox regression analyses, adjusted for inherited thrombophilia, age, sex and BMI, low levels of TGF-β1 [hazard ratio (HR) = 2.2, 95% confidence interval (CI) 1.1-4.3; p = 0.02] and TGF-β2 (HR = 2.4, 95% CI 1.2-4.7; p = 0.01) were independently associated with a higher risk of recurrent VTE. We propose TGF-β1 and TGF-β2 as potential predictive markers for recurrence in patients with unprovoked VTE.

Growth and feeding traits such as BW, BW gain (BWG), feed intake (FI), and feed conversion ratio (FCR) are of economic importance in poultry production. In this study, 8 SNP of the transforming growth factor β3 (TGF-β3) gene, which are located in the proximity of quantitative trait loci affecting BW and FCR, were selected to be genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in 2 yellow meat-type chicken populations with 724 birds in total. Body weights at 49 (BW49) and 70 (BW70) d of age and FI in the interval were recorded, and respective BWG and FCR were calculated for each bird. Two SNP with a very low minor allele frequency (<1%) were discarded from further analysis. The results showed that both rs13586818 and rs14535174 had significant effects (P < 0.05) on BWG and FCR, and that rs14535177 was significantly (P < 0.05) associated with BW49, BW70, and FI. Furthermore, birds with the GA genotype of rs13586818 showed strongly higher FCR and lower BWG (P < 0.05) compared with AA individuals. The TT birds had significantly larger BWG (P < 0.05) than GT birds in rs14535174, whereas FCR was the opposite. Individuals with the GC genotype of rs14535177 had significantly higher BW49, BW70, FCR, and FI (P < 0.05) than those with the GG genotype. Additionally, haplotypes based on 3 SNP of rs13586818, rs14535174, and rs14535177 were significantly associated with FCR (P < 0.05). The SNP and analyzed haplotypes identified in this study might be used as potential genetic markers in meat-type chicken breeding.

Members of the TGF-β superfamily are involved in numerous cell functions; however, except for myostatin, their roles in the regulation of muscle growth in fish are completely unknown. We measured tgf-β1, tgf-β2, tgf-β3, inhibin βA (inh) and follistatin (fst) gene expression during muscle growth recovery following a fasting period. We observed that tgf-β1a and tgf-β2 expression were quickly down-regulated after refeeding and that tgf-β3 reached its highest level of expression 7days post-refeeding, mirroring myogenin expression. Inh βA1 mRNA levels decreased sharply after refeeding, in contrast to fst b2 expression, which peaked at day 2. No significant modification of expression was observed for tgf-β1a, tgf-β1b, tgf-β1c and tgf-β6 during refeeding. In vitro, tgf-β2 and inh βA1 expression decreased during the differentiation of satellite cells, whereas tgf-β3 expression increased following the same pattern as myogenin. Surprisingly, fst b1 and fst b2 expression decreased during differentiation, whereas no variation was observed in fst a1 and fst a2 expression levels. In vitro analyses also indicated that IGF1 treatment up-regulated tgf-β3, inh βA1 and myogenin expression, and that MSTN treatment increased fst b1 and fst b2 expression. In conclusion, we showed that the expression of tgf-β2, tgf-β3 and inh βA1 is dynamically regulated during muscle growth resumption and satellite cell differentiation, strongly suggesting that these genes have a role in the regulation of muscle growth.

OBJECTIVE

The aim of this study was to improve a method that induce cartilage differentiation of human embryoid stem cells (hESCs) in vitro, and test the effect of in vivo environments on the further maturation of hESCs derived cells.

METHODS

Embryoid bodies (EBs) formed from hESCs, with serum-free KSR-based medium and mesodermal specification related factors, CHIR, and Noggin for first 8days. Then cells were digested and cultured as micropellets in serum-free KSR-based chondrogenic medium that was supplemented with PDGF-BB, TGF β3, BMP4 in sequence for 24days. The morphology, FACS, histological staining as well as the expression of chondrogenic specific genes were detected in each stage, and further in vivo experiments, cell injections and tissue transplantations, further verified the formation of chondrocytes.

RESULTS

We were able to obtain chondrocyte/cartilage from hESCs using serum-free KSR-based conditioned medium. qPCR analysis showed that expression of the chondroprogenitor genes and the chondrocyte/cartilage matrix genes. Morphology analysis demonstrated we got PG+COL2+COL1-particles. It indicated we obtained hyaline cartilage-like particles. 32-Day differential cells were injected subcutaneous. Staining results showed grafts developed further mature in vivo. But when transplanted in subrenal capsule, their effect was not good as in subcutaneous. Microenvironment might affect the cartilage formation.

CONCLUSIONS

The results of this study provide an absolute serum-free and efficient approach for generation of hESC-derived chondrocytes, and cells will become further maturation in vivo. It provides evidence and technology for the hypothesis that hESCs may be a promising therapy for the treatment of cartilage disease.

Exposure to electromagnetic pulses in certain doses may lead to increase in the permeability of the blood testes barrier (BTB) in mice, which in turn affects spermatogenesis, penetration and spermiation. TGF-β3 is a key molecule involved in BTB permeability via regulation of tight junction proteins, and it participates in regulating spermatogenesis, synthesis of steroids and production of the extracellular matrix in testicular tissue. Therefore, it is hypothesized that TGF-β3 plays important roles in electromagnetic pulse (EMP)-induced changes in BTB permeability. In the present study, we carried out whole-body irradiation on mice using EMP of different intensities. No obvious pathological changes or significant increase in apoptosis was detected in testicular tissues after exposure to 100 and 200 pulses of intensity 200kV/m; however, with 400 pulses we observed the degeneration and shrinkage of testicular tissues along with a significant increase in apoptotic rate. Moreover, in the 100- and 200-EMP groups, a non-significant increase in TGF-β3 mRNA and protein expression was observed, whereas in the 400-EMP group a significant increase was observed (P<0.05). These results indicate that increase in the apoptotic rate of testicular tissues and increase in TGF-β3 expression may be one of the mechanisms for EMP-induced increase in BTB permeability in mice.

Osteoarthritis is the leading cause of disability in the US. Consequently, there is a pressing need for restoring the structural and functional properties of diseased articular cartilage. Yet the search for the right combination of proper target cells and growth factors for cartilage regeneration remains challenging. In this study, we first tested the intrinsic chondrogenic differentiation ability of human perivascular stem cells (hPSCs), a novel source of mesenchymal stem cells (MSCs) isolated by fluorescence-activated cell sorting (FACS) from human adipose tissue. A putative prochondrogenic growth factor, NEL-like molecule-1 (NELL-1), was added to the hPSC pellets to upregulate gene expression of chondrogenic markers, including AGGRECAN, COLLAGEN II, and COMP. Furthermore, the addition of NELL-1 to a transforming growth factor beta 3 (TGF-β3) + bone morphogenetic protein-6 (BMP-6) "cocktail" resulted in the best combinatorial stimulation in accelerating the chondrogenic differentiation of hPSCs, as evidenced by increased gene and protein expression of chondrogenic markers in a shortened induction time without elevating expression of hypertrophic, fibrotic, and osteogenic markers. Mechanistically, this acceleration rendered by NELL-1 may be partially attributed to NELL-1's upregulation of BMP receptors and TGF-β receptor type I in hPSCs for increased responsiveness to BMPs + TGF-βs. In conclusion, lipoaspirate-derived hPSCs present a novel and abundant cell source of MSCs for cartilage regeneration, and the combinatorial application of NELL-1, TGF-β3, and BMP-6 with hPSCs may remarkably enhance and accelerate cartilage repair.