Enhanced effects of slowly co-released TGF-ß3 and BMP-2 from biomimetic calcium phosphate-coated silk fibroin scaffolds in the repair of osteochondral defects.

Bioactive agents have demonstrated regenerative potential for cell-free bone tissue engineering. Nevertheless, certain challenges persist, including ineffective delivery methods and confined therapeutic potency. Here, we demonstrated that the biomimetic calcium phosphate coating system (BioCaP) could effectively uptake and slowly release the incorporated bioactive agents compared to the surface absorption system via osteoclast-mediated degradation of BioCaP coatings. The release kinetics were determined as a function of time. The release rate was stable without remarkable burst release during the first 1 day, followed by a sustained release from day 7 to day 19. Then, we developed the bi-functional BioCaP-coated silk fibroin scaffolds enabling the effective co-delivery of TGF-ß3 and BMP-2 (SFI-T/SFI-B) and the corresponding slow release of TGF-ß3 and BMP-2 exhibited superior potential in promoting chondrogenesis and osteogenesis without impairing cell vitality in vitro. The SFI-T/SFI-B scaffolds could improve cartilage and bone regeneration in 5 × 4 mm rabbit osteochondral (OC) defect. These findings indicate that the biomimetic calcium-phosphate coated silk fibroin scaffolds with slowly co-released TGF-ß3 and BMP-2 effectively promote the repair of OC defects, hence facilitating the future clinical translation of controlled drug delivery in tissue engineering.

Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel.

OBJECTIVES: We genetically modified dedifferentiated chondrocytes (DCs) using lentiviral vectors and adenoviral vectors encoding TGF-ß3 (referred to as transgenic groups below) and encapsulated these DCs in the microcavitary hydrogel and investigated the combinational effect on redifferentiation of the genetically manipulated DCs. RESULTS: The Cell Counting Kit-8 data indicated that both transgenic groups exhibited significantly higher cell viability in the first week but inferior cell viability in the subsequent timepoints compared with those of the control group. Real-time polymerase chain reaction and western blot analysis results demonstrated that both transgenic groups had a better effect on redifferentiation to some extent, as evidenced by higher expression levels of chondrogenic genes, suggesting the validity of combination with transgenic DCs and the microcavitary hydrogel on redifferentiation. Although transgenic DCs with adenoviral vectors presented a superior extent of redifferentiation, they also expressed greater levels of the hypertrophic gene type X collagen. It is still worth further exploring how to deliver TGF-ß3 more efficiently and optimizing the appropriate parameters, including concentration and duration. CONCLUSIONS: The results demonstrated the better redifferentiation effect of DCs with the combinational use of transgenic TGF-ß3 and a microcavitary alginate hydrogel and implied that DCs would be alternative seed cells for cartilage tissue engineering due to their easily achieved sufficient cell amounts through multiple passages and great potential to redifferentiate to produce cartilaginous extracellular matrix.

Role of TGF-ß3 in modulating inflammatory responses and wound healing processes in ischemic ulcers in atherosclerotic patients.

Ischemic ulcers pose a multifaceted clinical dilemma for patients with atherosclerosis, frequently compounded by suboptimal wound healing mechanisms. The dual function of Transforming Growth Factor Beta 3 (TGF-ß3) in ischemic ulcer healing is not fully comprehended, despite its involvement in modulating inflammatory responses and tissue regeneration. The main aim of this investigation was to clarify the functions and mechanisms by which TGF-ß3 regulates inflammatory responses and promotes wound healing in patients with ischemic ulcers who have atherosclerosis. Between August 2022 and November 2023, this cross-sectional investigation was conducted on 428 patients diagnosed with atherosclerotic ischemic ulcers in Haikou, China. The expression and function of TGF-ß3 were examined throughout the different stages of wound healing, including inflammation, proliferation and remodelling. In addition to documenting patient demographics and ulcer characteristics, an analysis was conducted on biopsy samples to determine the expression of TGF-ß3, pro-inflammatory and anti-inflammatory markers. A subset of patients were administered topical TGF-ß3 in order to evaluate its therapeutic effects. The expression pattern of TGF-ß3 was found to be stage-dependent and significant, exhibiting increased levels during the phase of inflammation and reduced activity in subsequent phases. TGF-ß3 levels were found to be greater in ulcers that were larger and deeper, especially in inflammatory phase. TGF-ß3 applied topically induced discernible enhancement in ulcer healing parameters, such as reduction in ulcer depth and size. The therapeutic significance of TGF-ß3 was emphasised due to its twofold function of regulating the inflammatory environment and facilitating the regeneration of damaged tissues. Ischemic ulcer lesion healing is significantly influenced by TGF-ß3, which functions as an anti-inflammatory and pro-inflammatory mediator. Its correlation with ulcer characteristics and stages of healing suggests that it may have utility as a targeted therapeutic agent.

Heterogeneous response to TGF-ß1/3 isoforms in fibroblasts of different origins: implications for wound healing and tumorigenesis.

Identification of therapeutic targets for treating fibrotic diseases and cancer remains challenging. Our study aimed to investigate the effects of TGF-ß1 and TGF-ß3 on myofibroblast differentiation and extracellular matrix deposition in different types of fibroblasts, including normal/dermal, cancer-associated, and scar-derived fibroblasts. When comparing the phenotype and signaling pathways activation we observed extreme heterogeneity of studied markers across different fibroblast populations, even within those isolated from the same tissue. Specifically, the presence of myofibroblast and deposition of extracellular matrix were dependent on the origin of the fibroblasts and the type of treatment they received (TGF-ß1 vs. TGF-ß3). In parallel, we detected activation of canonical signaling (pSMAD2/3) across all studied fibroblasts, albeit to various extents. Treatment with TGF-ß1 and TGF-ß3 resulted in the activation of canonical and several non-canonical pathways, including AKT, ERK, and ROCK. Among studied cells, cancer-associated fibroblasts displayed the most heterogenic response to TGF-ß1/3 treatments. In general, TGF-ß1 demonstrated a more potent activation of signaling pathways compared to TGF-ß3, whereas TGF-ß3 exhibited rather an inhibitory effect in keloid- and hypertrophic scar-derived fibroblasts suggesting its clinical potential for scar treatment. In summary, our study has implications for comprehending the role of TGF-ß signaling in fibroblast biology, fibrotic diseases, and cancer. Future research should focus on unraveling the mechanisms beyond differential fibroblast responses to TGF-ß isomers considering inherent fibroblast heterogeneity.

TGF-ß3 Protects Neurons Against Intermittent Hypoxia-Induced Oxidative Stress and Apoptosis Through Activation of the Nrf-2/KEAP1/HO-1 Pathway via Binding to TGF-ßRI.

Intermittent hypoxia (IH) is the primary pathological manifestation of obstructive sleep apnea (OSA) and the main cause of OSA-induced cognitive impairment. Hippocampal neurons are considered to be critical cells affected by IH. Transforming growth factor-ß3 (TGF-ß3) is a cytokine with a neuroprotective effect, which plays a crucial role in resisting hypoxic brain injury, while its role in IH-induced neuronal injury is still unclear. Here, we aimed to clarify the mechanism of TGF-ß3 protecting IH-exposed neurons by regulating oxidative stress and secondary apoptosis. Morris water maze results revealed that IH exposure was unable to affect the vision and motor ability of rats, but significantly affected their spatial cognition. Second-generation sequencing (RNA-seq) and subsequent experiments supported that IH decreased TGF-ß3 expression and stimulated reactive oxygen species (ROS)-induced oxidative stress and apoptosis in rat hippocampus. In vitro, IH exposure significantly activated oxidative stress within HT-22 cells. Exogenous administration of Recombinant Human Transforming Growth Factor-ß3 (rhTGF-ß3) prevented ROS surge and secondary apoptosis in HT-22 cells caused by IH, while TGF-ß type receptor I (TGF-ßRI) inhibitor SB431542 blocked the neuroprotective effect of rhTGF-ß3. Nuclear factor erythroid 2-related factor 2 (Nrf-2) is a transcription factor preserving intracellular redox homeostasis. rhTGF-ß3 improved the nuclear translocation of Nrf-2 and activated downstream pathway. However, Nrf-2 inhibitor ML385 suppressed the activation of the Nrf-2 mechanism by rhTGF-3 and restored the effects of oxidative stress damage. These results indicate that TGF-ß3 binding to TGF-ßRI activates the intracellular Nrf-2/KEAP1/HO-1 pathway, reduces ROS creation, and attenuates oxidative stress and apoptosis in IH-exposed HT-22 cells.

Influence of Transforming Growth Factors beta 1 and beta 3 in the Scar Formation Process.

BACKGROUND: Transforming growth factor-beta (TGF-ß) plays an instrumental role in forming scars and keloids. TGF-ß isoforms exhibit differential expression, indicating distinct wound healing and scar formation functions. However, the role of TGF-ß1 and TGF-ß3 in wound healing and scar formation remains unclear. This study aimed to compare the specific roles of TGF-ß1 and TGF-ß3 in wound healing and scar formation by biomolecular analysis. MATERIALS AND METHODS: The study was conducted by cell isolation and culture cells from a total of 20 human samples. Normal human fibroblasts (NHF) were isolated from normal human samples and myofibroblasts from the different scar types, namely hypertrophic (HT) and keloid (K) scars. NHF and cells from the HT, and K scar, each of which were divided into 3 sample groups: the untreated control, TGF-ß1 (10 µg/mL)-treated group, and TGF-ß3 (10 µg/mL)-treated group. The results of confocal microscopy and fluorescence-activated cell sorting experiments were compared. RESULTS: Both the HT and K groups had higher α-smooth muscle actin (α-SMA) expression than the NHF group in the untreated control group. In comparison with the untreated group, NHFs showed a significant increase in α-SMA expression in the TGF-ß1-treated group. HT showed a high α-SMA level, which was statistically significant compared with the normal fibroblasts. In the TGF-ß3-treated group, α-SMA expression was slightly increased in NHF as compared with the untreated group. TGF-ß3 treated HT exhibited a greater reduction in α-SMA expression than in the TGF-ß1 treated HT. K, on the other hand, had only a minimal effect on the treatment of TGF-ß1 and TGF-ß3. CONCLUSIONS: The findings suggest that TGF-ß3 may play a regulatory role in the wound repair process, which could be useful in the development of scar-reducing therapies for patients with scar-related cosmetic concerns.

TGF-ß3 enhances cell-to-cell communication in chondrocytes via the ALK5/p-Smad3 axis.

Gap junctional intercellular communication (GJIC) is indispensable for the maintenance of physiological balance in articular cartilage. Transforming growth factor-ß3 (TGF-ß3), an important growth factor of TGF-ß superfamily, is well recognized to play a unique regulatory role in cartilage development and diseases. However, the role of TGF-ß3 in GJIC in adult chondrocytes remains elusive. This work aims to investigate the effect of TGF-ß3 on gap-junction mediated intercellular communication in chondrocytes. We first showed that TGF-ß3 could enhance the synaptic connections between chondrocytes by scanning electron microscopy (SEM) and promote the cell-to-cell communication in living chondrocytes by scrape loading/dye transfer assay. We then confirmed that TGF-ß3 enhanced cell-to-cell communication via up-regulation of connexin 43 (Cx43). We next found that TGF-ß3-enhanced GJIC required the participation of TGF-beta type I receptor ALK5 and depended on the activation of p-Smad3 signalling. Finally, through inhibitor experiments of SB525334 and SIS3, we demonstrated that TGF-ß3-induced functional GJIC in chondrocytes via the axis of ALK5/p-Smad3 signalling. Taking together, these results demonstrate a strong correlation between TGF-ß3 and GJIC in chondrocytes, which provides a new perspective on the importance of TGF-ß3 on cartilage physiology and pathobiology.

TGF-ß isoforms inhibit hepatitis C virus propagation in transforming growth factor beta/SMAD protein signalling pathway dependent and independent manners.

Transforming growth factor beta (TGF-ß) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-ß isoforms, including TGF-ß1, TGF-ß2 and TGF-ß3, remain unclear. Here, we demonstrated that all of the three TGF-ß isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-ß isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-ß isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-ß/SMAD signalling pathway-dependent and TGF-ß/SMAD signalling pathway-independent manners. TGF-ß isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-ß1 and TGF-ß2, not TGF-ß3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-ß isoforms in the HCV-related liver disease progression.

Synergistic short-term and long-term effects of TGF-ß1 and 3 on collagen production in differentiating myoblasts.

Skeletal muscle fibrosis and regeneration are modulated by transforming growth factor ß (TGF-ß) superfamily. Amongst them, TGF-ß1 is a highly potent pro-fibrotic factor, while TGF-ß3 has been implicated to reduce scar formation and collagen production in skin and vocal mucosa. However, little is known about the individual and combined short- and long-term effects of TGF-ß1 and TGF-ß3 on collagen expression in myoblasts and myotubes. Here we show that in C2C12 myoblasts TGF-ß1 and/or TGF-ß3 increased mRNA expression of Ctgf and Fgf-2 persistently after 3 h and of Col1A1 after 24 h, while TGF-ß1+TGF-ß3 mitigated these effects after 48 h incubation. Gene expression of Tgf-ß1 was enhanced by TGF-ß1 and/or TGF-ß3 after 24 h and 48 h. However, Tgfbr1 mRNA expression was reduced at 48 h. After 48 h incubation with TGF-ß1 and/or TGF-ß3, Col3A1 and Col4A1 mRNA expression levels were decreased. Myoblasts produced collagen after three days incubation with TGF-ß1 and/or TGF-ß3 in a dose independent manner. Collagen deposition was doubled when myoblasts differentiated into myotubes and TGF-ß1 and/or TGF-ß3 did not stimulate collagen production any further. TGF-ß type I receptor (TGFBR1) inhibitor, LY364947, suppressed TGF-ßs-induced collagen production. Collagen I expression was higher in myotubes than in myoblasts. TGF-ß1 and/or TGF-ß3 inhibited myotube differentiation which was antagonized by LY364947. These results indicate that both C2C12 myoblasts and myotubes produce collagen. Whereas TGF-ß1 and TGF-ß3 individually and simultaneously stimulate collagen production in C2C12 differentiating myoblasts, in myotubes these effects are less prominent. In muscle cells, TGF-ß3 is ineffective to antagonize TGF-ß1-induced collagen production.

Conventional immunomarkers stain a fraction of astrocytes in vitro: A comparison of rat cortical and spinal cord astrocytes in naïve and stimulated cultures.

Astrocytes are responsible for a wide variety of essential functions throughout the central nervous system. The protein markers glial fibrillary acidic protein (GFAP), glutamate aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), glutamine synthetase (GS), 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), and the transcription factor SOX9 are routinely used to label astrocytes in primary rodent cultures. However, GLAST, GLT-1, GS, and SOX9 are also produced by microglia and oligodendrocytes and GFAP, GLAST, GLT-1, and GS production levels are affected by astrocyte phenotypic changes associated with reactive astrogliosis. No group has performed a comprehensive immunocytochemical evaluation to quantify the percentage of cells labeled by these markers in vitro, nor compared changes in staining between cortex- and spinal cord-derived cells in naïve and stimulated cultures. Here, we quantified the percentage of cells positively stained for these six markers in astrocyte, microglia, and oligodendrocyte cultures isolated from neonatal rat cortices and spinal cords. Additionally, we incubated the astrocytes with transforming growth factor (TGF)-ß1 or TGF-ß3 to determine if the labeling of these markers is altered by these stimuli. We found that only SOX9 in cortical cultures and ALDH1L1 in spinal cord cultures labeled more than 75% of the cells in naïve and stimulated astrocyte cultures and stained less than 5% of the cells in microglia and oligodendrocyte cultures. Furthermore, significantly more cortical than spinal cord astrocytes stained for GFAP, GLAST, and ALDH1L1 in naïve cultures, whereas significantly more spinal cord than cortical astrocytes stained for GLAST and GS in TGF-ß1-treated cultures. These findings are important as variability in marker staining may lead to misinterpretation of the astrocyte response in cocultures, migration assays, or engineered disease models.

Dual Delivery of TGF-ß3 and Ghrelin in Microsphere/Hydrogel Systems for Cartilage Regeneration.

Articular cartilage (AC) damage is quite common, but due to AC's poor self-healing ability, the damage can easily develop into osteoarthritis (OA). To solve this problem, we developed a microsphere/hydrogel system that provides two growth factors that promote cartilage repair: transforming growth factor-ß3 (TGF-ß3) to enhance cartilage tissue formation and ghrelin synergy TGF-ß to significantly enhance the chondrogenic differentiation. The hydrogel and microspheres were characterized in vitro, and the biocompatibility of the system was verified. Double emulsion solvent extraction technology (w/o/w) is used to encapsulate TGF-ß3 and ghrelin into microspheres, and these microspheres are encapsulated in a hydrogel to continuously release TGF-ß3 and ghrelin. According to the chondrogenic differentiation ability of mesenchymal stem cells (MSCs) in vitro, the concentrations of the two growth factors were optimized to promote cartilage regeneration.

TGF-ß3/Smad3 Contributes to Isoflurane Postconditioning Against Cerebral Ischemia-Reperfusion Injury by Upregulating MEF2C.

Isoflurane postconditioning alleviates cerebral ischemic-reperfusion injury (CIRI), but the underlying mechanism has not been fully clarified. We previously demonstrated that the transforming growth factor beta-1 (TGF-ß1)/Smads signaling pathway is involved in the neuroprotective effect of isoflurane postconditioning. TGF-ß3 has a highly homologous sequence relative to that of TGF-ß1. In this study, we explored the roles of the TGF-ß3/Smad3 signaling pathway and myocyte enhancer factor 2C (MEF2C) in neuroprotection induced by isoflurane postconditioning. A CIRI rat model was established by middle cerebral artery occlusion for 1.5 h, followed by 24 h of reperfusion. Isoflurane postconditioning led to lower infarct volumes and neurologic deficit scores, more surviving neurons, and less damaged and apoptotic neurons as compared with those of CIRI rats. Moreover, isoflurane postconditioning upregulated the expressions of TGF-ß3, p-Smad3, and MEF2C. However, the neuroprotective effect was reversed by pirfenidone, a TGF-ß3/Smad3 signaling pathway inhibitor. Also, pirfenidone treatment downregulated the expression of MEF2C. These results indicate that the TGF-ß3/Smad3 signaling pathway contributes to the neuroprotection of isoflurane postconditioning after CIRI and is possibly related to MEF2C.

Crosslinker concentration controls TGFß-3 release and annulus fibrosus cell apoptosis in genipin-crosslinked fibrin hydrogels.

Back pain is a leading cause of global disability associated with intervertebral disc (IVD) pathologies. Discectomy alleviates disabling pain caused by IVD herniation without repairing annulus fibrosus (AF) defects, which can cause accelerated degeneration and recurrent pain. Biological therapies show promise for IVD repair but developing high-modulus biomaterials capable of providing biomechanical stabilisation and delivering biologics remains an unmet challenge. The present study identified critical factors and developed an optimal formulation to enhance the delivery of AF cells and transforming growth beta-3 (TGFß-3) in genipin-crosslinked fibrin (FibGen) hydrogels. Part 1 showed that AF cells encapsulated in TGFß-3-supplemented high-modulus FibGen synthesised little extracellular matrix (ECM) but could release TGFß-3 at physiologically relevant levels. Part 2 showed that AF cells underwent apoptosis when encapsulated in FibGen, even after reducing fibrin concentration from 70 to 5 mg/mL. Mechanistic experiments, modifying genipin concentration and integrin binding site presence demonstrated that genipin crosslinking caused AF cell apoptosis by inhibiting cell-biomaterial binding. Adding integrin binding sites with fibronectin partially rescued apoptosis, indicating genipin also caused acute cytotoxicity. Part 3 showed that FibGen formulations with 1 mg/mL genipin had enhanced ECM synthesis when supplemented with fibronectin and TGFß-3. In conclusion, FibGen could be used for delivering biologically active compounds and AF cells, provided that formulations supplied additional sites for cell-biomaterial binding and genipin concentrations were low. Results also highlighted a need for developing strategies that protect cells against acute crosslinker cytotoxicity to overcome challenges of engineering high-modulus cell carriers for musculoskeletal tissues that experience high mechanical demands.

Intervertebral Disc Regeneration Using Stem Cell/Growth Factor-Loaded Porous Particles with a Leaf-Stacked Structure.

Although biological therapies based on growth factors and transplanted cells have demonstrated some positive outcomes for intervertebral disc (IVD) regeneration, repeated injection of growth factors and cell leakage from the injection site remain considerable challenges for human therapeutic use. Herein, we prepare human bone marrow-derived mesenchymal stem cells (hBMSCs) and transforming growth factor-ß3 (TGF-ß3)-loaded porous particles with a unique leaf-stack structural morphology (LSS particles) as a combination bioactive delivery matrix for degenerated IVD. The LSS particles are fabricated with clinically acceptable biomaterials (polycaprolactone and tetraglycol) and procedures (simple heating and cooling). The LSS particles allow sustained release of TGF-ß3 for 18 days and stable cell adhesiveness without additional modifications of the particles. On the basis of in vitro and in vivo studies, it was observed that the hBMSCs/TGF-ß3-loaded LSS particles can provide a suitable milieu for chondrogenic differentiation of hBMSCs and effectively induce IVD regeneration in a beagle dog model. Thus, therapeutically loaded LSS particles offer the promise of an effective bioactive delivery system for regeneration of various tissues including IVD.

Mechanisms of TGFß3 Action as a Therapeutic Agent for Promoting the Synthesis of Extracellular Matrix Proteins in Hyaline Cartilage.

Hyaline cartilage is a nonvascular connective tissue covering the joint surface. It consists mostly of the extracellular matrix proteins and a small number of highly differentiated chondrocytes. At present, various techniques for repairing joint surfaces damage, for example, the use of modified cell cultures and biodegradable scaffolds, are under investigation. Molecular mechanisms of cartilage tissue proliferation have been also actively studied in recent years. TGFß3, which plays a critical role in the proliferation of normal cartilage tissue, is one of the most important protein among cytokines and growth factors affecting chondrogenesis. By interacting directly with receptors on the cell membrane surface, TGFß3 triggers a cascade of molecular interactions involving transcription factor Sox9. In this review, we describe the effects of TGFß3 on the receptor complex activation and subsequent intracellular trafficking of Smad proteins and analyze the relation between these processes and upregulation of expression of major extracellular matrix genes, such as col2a1 and acan.

Repair of Rotator Cuff Tendon Defects in Aged Rats Using a Growth Factor Injectable Gel Scaffold.

PURPOSE: To determine if the tendon-specific crosslinking gelatin (Col-Tgel) impregnated with growth factors promotes tendon healing at the bone interface and in a tendon window model. METHODS: Two different Col-Tgel formulations were first tested in vitro by evaluating cell morphology and tendogenic differentiation. After the optimum formulation was determined, the gel was mixed with either transforming growth factor-ß3 (TGF-ß3) or growth differentiation factor-7 (GDF-7) growth factor and prepared for injections. Window defects were induced in 12 animals, which were randomized into the following treatments: (1) sham, (2) empty Col-Tgel, (3) Col-Tgel containing TGF-ß3, or (4) Col-Tgel containing GDF-7. Based on these results, the sham, empty Col-Tgel, and Col-Tgel containing TGF-ß3 were applied to the supraspinatus repair interface. Tendons were analyzed biomechanically and histologically using hematoxylin and eosin and Masson's trichrome staining. RESULTS: In the window defect model, histologic scores were the best in rats treated with TGF-ß3 containing Col-Tgel, followed by the empty Col-Tgel scaffold, and finally the sham control. The GDF-7 Col-Tgel was not further tested because occasional ectopic cartilage and bone formation was found in the prior window defect model. In the supraspinatus repair model, there was no statistical difference (P > .05) in the biomechanical strength among the 3 treatment groups, but load-to-failure ratio improved when TGF-ß3 was added to the scaffold, suggesting improved tendon healing. CONCLUSIONS: This pilot study evaluated the performance of an injectable gel tendon graft in a population of retired breeder rats. The results suggest that Col-Tgel containing TGF-ß3 may be a useful adjunctive treatment for surgical repair of full-thickness rotator cuff tears. Histologic and biomechanical scores suggest that Col-Tgel containing TGF-ß3 promotes tendon healing. CLINICAL RELEVANCE: The results of this study suggest that shoulders injected with Col-Tgel may be a useful adjunctive treatment for repair of rotator cuff tears.

Co-Culture of Adipose-Derived Stem Cells and Chondrocytes With Transforming Growth Factor-Beta 3 Promotes Chondrogenic Differentiation.

Tissue engineering cartilage is a promising strategy to reconstruct the craniofacial cartilaginous defects. It demands plenty of chondrocytes to generate human-sized craniofacial frameworks. Partly replacement of chondrocytes by adipose-derived stem cells (ADSCs) can be an alternative strategy.The study aimed at evaluating the chondrogenic outcome of ADSCs and chondrocytes in direct co-culture with transforming growth factor-beta (TGF-ß3). Porcine ADSCs and chondrocytes were obtained from abdominal wall and external ears. Four groups: ADSCs or chondrocytes monocultured in medium added with TGF-ß3; ADSCs and ACs co-cultured with or without TGF-ß3. Cell growth rate was performed to evaluate the cell proliferation. Morphological, histologic and real-time polymerase chain reaction analysis were performed to characterize the chondrogenic outcome of pellets. ADSCs had favorable multi-lineage differentiation potential. Further, when ADSCs were co-cultured with chondrocytes in medium added with TGF-ß3, the cell proliferation was promoted and the chondrogenic differentiation of ADSCs was enhanced. We demonstrate that pellet co-culture of ADSCs and chondrocyte with TGF-ß3 could construct high quantity cartilages. It suggests that this strategy might be useful in future cartilage repair.

Synergistic effects of recombinant Lentiviral-mediated BMP2 and TGF-beta3 on the osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are considered good candidates for seed cells in bone engineering. The study aim to investigate the synergistic effects of human bone morphogenetic protein 2 (hBMP2) and transforming growth factor beta3 (hTGF-beta3) modified BMSCs on inducing osteogenic differentiation in vitro. METHODS: Lentivirus (LV) carrying hBMP2 and/or hTGF-beta3 genes were constructed and used to transduce rat BMSCs. The expression of osteogenic molecules was detected by qRT-PCR and western blotting. RESULTS: Targeted genes were PCR-amplified and confirmed by DNA sequencing and BLAST analysis. BMSCs infected by vectors effectively resulted in the overexpressions of hBMP2 and hTGF-beta3 and higher levels of hBMP2 and hTGF-beta3 in the culture supernatant. The co-transduction of hBMP2 and hTGF-beta3 induced BMSCs osteogenic differentiation more effectively than the transduction of hBMP2 or hTGF-beta3 individually. The expression levels of osteopontin (OPN), osteocalcin (OCN), and osteoprotegerin (OPG) in LV-hBMP2 + LV-hTGF-beta3 group (BMSCs transfected by vectors respectively carrying hBMP-2 gene and hTGF-beta3 gene) and LV-hBMP2-hTGF-beta3 group (BMSCs transfected by vector carrying hBMP2 and hTGF-beta3 fusion gene) were significantly higher than in LV-BMP2 (BMSCs transfected by vector carrying hBMP2 gene) and LV-TGF-beta3 (BMSCs transfected by vector carrying hTGF-beta3 gene) groups (P < 0.05). The hBMP2 and/or hTGF-beta3 overexpression upregulated alkaline phosphatase (ALP) activity. CONCLUSION: The present study showed that hBMP2 and/or hTGF-beta3 genes can be successfully overexpressed in BMSCs. Our study proved that the two cytokines (hBMP2 and hTGF-beta3) could induce bone differentiation synergistically, which foresees the use of the combination of these two cytokines as a therapeutic strategy in the future.

TGF-ß1 Suppresses IL-33-Induced Mast Cell Function.

TGF-ß1 is involved in many pathological conditions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGF-ß1 can suppress IgE-mediated mast cell activation of human and mouse mast cells. IL-33 is a member of the IL-1 family capable of inducing mast cell responses and enhancing IgE-mediated activation. In this study, we investigated the effects of TGF-ß on IL-33-mediated mast cell activation. Bone marrow-derived mast cells cultured in TGF-ß1, ß2, or ß3 showed reduced IL-33-mediated production of TNF, IL-6, IL-13, and MCP-1 in a concentration-dependent manner. TGF-ß1 inhibited IL-33-mediated Akt and ERK phosphorylation as well as NF-κB- and AP-1-mediated transcription. These effects were functionally important, as TGF-ß1 injection suppressed IL-33-induced systemic cytokines in vivo and inhibited IL-33-mediated cytokine release from human mast cells. TGF-ß1 also suppressed the combined effects of IL-33 and IgE-mediated activation on mouse and human mast cells. The role of IL-33 in the pathogenesis of allergic diseases is incompletely understood. These findings, consistent with our previously reported effects of TGF-ß1 on IgE-mediated activation, demonstrate that TGF-ß1 can provide broad inhibitory signals to activated mast cells.

Effects of host fatty acid-binding protein 4 on Eimeria tenella sporozoites invasion of cells.

In our previous study, proteomics analyses of host cells infected with Eimeria tenella sporozoites coupled with isobaric tags for relative and absolute quantitation, identified several host proteins related to Eimeria invasion. In this study, A 458-bp Gallus gallus fatty acid-binding protein 4 (FABP4) gene was cloned and subcloned to pET-28c(+) vector to construct the prokaryotic recombinant expression plasmid pET-28c(+)-FABP4. The 18.5 kDa recombinant FABP4 protein (rFABP4) was expressed and identified by western blotting. Expression of FABP4 in E. tenella sporozoite-infected DF-1 cells was downregulated significantly than in non-infected cells detected by western blotting and immunohistochemistry. The antibody inhibition assay showed that antibodies against FABP4 at 50, 100, 200, 300, and 400 µg/mL had no significant effect on sporozoite invasion. BMS-309403 and transforming growth factor-ß3 (TGF-ß3) was used to inhibit and improve the expression of FABP4 in DF-1 cells, respectively, and their effect on the sporozoite invasion of cells was detected by flow cytometry. Sporozoite invasion rate in the BMS-309403-treated group was not significantly affected; however, the invasion rate in the TGF-ß3-treated group declined significantly. These results show that host FABP4 plays a negative role in Eimeria invasion. However, further studies are needed to elucidate the exact mechanism of how FABP4 negatively regulates Eimeria invasion.