Insulin‐like growth factor‐I improves chondrogenesis of predifferentiated human umbilical cord mesenchymal stromal cells

Human umbilical cord mesenchymal stromal cells (hUCMSCs) are an attractive cell source for tissue engineering with numerous advantages over other adult stem cell sources, such as great expansion ability in vitro and extensive availability. The objective of this 6‐week study was to test the hypothesis that switching from chondrogenic transforming growth factor‐beta3 (TGF‐β3) to anabolic insulin‐like growth factor‐I (IGF‐I) at the 3‐week time point would produce more cartilage‐like matrix than TGF‐β3 alone. hUCMSCs were seeded into polyglycolic acid (PGA) scaffolds and then cultured in chondrogenic medium containing TGF‐β3 for 3 weeks. The TGF‐β3‐treated hUCMSCs were then exposed for 3 more weeks to one of four different conditions: (1) continued in chondrogenic medium, (2) control medium (no TGF‐β3), (3) control medium with 10 ng/ml IGF‐I, or (4) control medium with 100 ng/ml IGF‐I. Compared to continuing with TGF‐β3, switching to IGF‐I increased collagen production, and furthermore increased both collagen type II gene expression and immunostaining. In conclusion, the shift from TGF‐β3 to IGF‐I at week 3 resulted in a significant increase of cartilage‐like extracellular matrix, confirming our hypothesis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1109–1115, 2009     

Mechanical Stimulation by Ultrasound Enhances Chondrogenic Differentiation of Mesenchymal Stem Cells in a Fibrin‐Hyaluronic Acid Hydrogel

Chondrogenic differentiation and cartilage tissue formation derived from stem cells are highly dependent on both biological and mechanical factors. This study investigated whether or not fibrin‐hyaluronic acid (HA) coupled with low‐intensity ultrasound (LIUS), a mechanical stimulation, produces an additive or synergistic effect on the chondrogenesis of rabbit mesenchymal stem cells (MSCs) derived from bone marrow. For the purpose of comparison, rabbit MSCs were first cultured in fibrin‐HA or alginate hydrogels, and then subjected to chondrogenic differentiation in chondrogenic‐defined medium for 4 weeks in the presence of either transforming growth factor‐beta3 (TGF‐β3) (10 ng/mL) or LIUS treatment (1.0 MHz and 200 mW/cm²). The resulting samples were evaluated at 1 and 4 weeks by histological observation, chemical assays, and mechanical analysis. The fibrin‐HA hydrogel was found to be more efficient than alginate in promoting chondrogenesis of the MSCs by producing a larger amount of sulfated glycosaminoglycans (GAGs) and collagen, and engineered constructs made with the hydrogel demonstrated higher mechanical strength. At 4 weeks of tissue culture, the chondrogenesis of the MSCs in fibrin‐HA were shown to be further enhanced by treatment with LIUS, as observed by analyses for the amounts of GAGs and collagen, and mechanical strength testing. In contrast, TGF‐β3, a well‐known chondrogenic inducer, showed a marginal additive effect in the amount of collagen only. These results revealed that LIUS further enhanced chondrogenesis of the MSCs cultured in fibrin‐HA, in vitro, and suggested that the combination of fibrin‐HA and LIUS is a useful tool in constructing high‐quality cartilage tissues from MSCs.     

Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells?

OBJECTIVE: Adipose tissue-derived mesenchymal stem cells (ATMSCs) have been shown to differentiate into bone, cartilage, fat or muscle. However, it is not certain that ATMSCs are equal to bone marrow-derived mesenchymal stem cells (BMMSC) for their bone and cartilage forming potential. The purpose of this study was to answer the question. METHODS: BMMSCs were obtained from the medullary canal of femur and ATMSCs were isolated from the fat harvested during liposuction procedures. After cell expansion in culture media and two passages, the immunofluorescent studies for STRO-1 and CD34 were performed to characterize the BMMSCs and ATMSCs. Osteogenesis was induced on a monolayer culture with osteogenic medium containing dexamethasone, beta-glycerophosphate and ascorbate. After 2-3 weeks, alkaline phosphatase (AP) and Von Kossa staining were done. To test for chondrogenesis, mesenchymal stem cells (MSCs) were cultured in a pellet culture and in a fibrin scaffold with a chondrogenic medium (CM) containing transforming growth factor-beta(2) and insulin-like growth factor-I. After 4 weeks, Safranin-O staining and immunohistochemical staining for type II collagen were done to evaluate the chondrogenic differentiation and the matrix production. A histological scale was used to semiquantitatively assess the degree of chondrogenesis. RESULTS: Both BMMSCs and ATMSCs were STRO-1 positive and CD34 negative. On the test of osteogenesis, the osteoblastic differentiation of ATMSCs as demonstrated by AP staining was much less than that of the BMMSCs (P=0.002). The amount of matrix mineralization shown by Von Kossa staining also showed statistical differences between the two MSCs (P=0.011). On the test for chondrogenesis by the pellet culture ATMSCs showed much weaker presentation as chondrogenic cells in both cell morphology and the matrix production. The histological score was 6.5 (SD1.3) for the BMMSCs, and 4.3 (SD1.6) for the ATMSCs cultured in CM, which was statistically significant (P=0.023). The results from fibrin gel paralleled those from the pellet culture in general. CONCLUSION: The results of our study suggest that the ATMSCs may have an inferior potential for both osteogenesis and chondrogenesis compared with the BMMSCs, and these cast doubts on the value of adipose tissue as a source of MSCs.     

Growth factor regulation of intracellular pH homeostasis under hypoxic conditions in isolated equine articular chondrocytes

Hypoxia and acidosis are recognized features of inflammatory arthroses. This study describes the effects of IGF‐1 and TGF‐β₁ on pH regulatory mechanisms in articular cartilage under hypoxic conditions. Acid efflux, reactive oxygen species (ROS), and mitochondrial membrane potential were measured in equine articular chondrocytes isolated in the presence of serum (10% fetal calf serum), IGF‐1 (1, 10, 50, 100 ng/ml) or TGF‐β₁ (0.1, 1, 10 ng/ml) and then exposed to a short‐term (3 h) hypoxic insult (1% O₂). Serum and 100 ng/ml IGF‐1 but not TGF‐β₁ attenuated hypoxic regulation of pH homeostasis. IGF‐1 appeared to act through mitochondrial membrane potential stabilization and maintenance of intracellular ROS levels in very low levels of oxygen. Using protein phosphorylation inhibitors PD98059 (25 µM) and wortmannin (200 nM) and Western blotting, ERK1/2 and PI‐3 kinase pathways are important for the effect of IGF‐1 downstream to ROS generation in normoxia but only PI‐3 kinase is implicated in hypoxia. These results show that oxygen and growth factors interact to regulate pH recovery in articular chondrocytes by modulating intracellular oxygen metabolites. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 197–203, 2013     

In vitro chondrogenic differentiation of human articular cartilage derived chondroprogenitors using pulsed electromagnetic field

BackgroundThe ability to grow new cartilage remains the standard goal of any treatment strategy directed at cartilage repair. Chondroprogenitors have garnered interest due to their applicability in cell therapy. Pulsed electromagnetic field (PEMF) favors chondrogenesis by possible upregulation of genes belonging to TGFβ superfamily. Since TGFβ is implicated in chondrogenic signalling, the aim of the study was to evaluate the ability of PEMF to induce chondrogenesis via endogenous TGFβ production in chondroprogenitors vs differentiation using chondrogenic medium inclusive of TGFβ.MethodsChondroprogenitors were harvested from three non-diseased human knee joints via fibronectin assay. Passage 3 pellets were subjected to four different culture conditions: a) negative control contained chondrogenic medium without TGFβ2, b) positive control contained medium with TGFβ2, c) PEMF 1 contained medium of negative control plus single exposure to PEMF and d) PEMF 2 contained medium of negative control plus multiple exposures to PEMF. Following differentiation (day 21), pellets were assessed for gene expression of ACAN, SOX9, COL2A1, TGFβ1, TGFβ2, and TGFβ3. Alcian blue staining to detect glycosaminoglycan deposition was also performed. Medium supernatant was used to detect endogenous latent TGF-β1 levels using ELISA.ResultsAll study arms exhibited comparable gene expression without any significant difference. Although positive control and PEMF study arms demonstrated notably better staining than negative control, the level of latent TGF-β1 was seen to be significantly high in supernatant from positive control (P < 0.05) when compared to other groups.ConclusionOur results indicate that PEMF induced chondrogenesis might involve other signalling molecules, which require further evaluation.     

Enhancement of the chondrogenic differentiation of mesenchymal stem cells and cartilage repair by ghrelin

Transforming growth factor beta (TGF‐β) is commonly utilized in chondrogenic differentiation protocols, but this often results in incomplete maturation of the derived chondrocytes. Gene expression analysis, quantitation of sulfated glycosaminoglycan and collagen, and histological staining were performed to assess the effects of ghrelin. The signaling pathways involved were investigated with inhibitors or targeted by shRNAs. Joint cavity delivery of TGF‐β with or without ghrelin, within a rat cartilage defect model was performed to evaluate the in vivo effects of ghrelin. Ghrelin dramatically enhanced gene expression levels of SOX9, ACAN, and COL II and resulted in increased synthesis of sulfated glycosaminoglycan (sGAG) and collagen in vitro. Combined treatment with TGF‐β and ghrelin synergistically enhanced the phosphorylation of ERK1/2 and DMNT3A, which accounted for increased expression of chondrogenic genes. Delivery of ghrelin in combination with TGF‐β after MSC implantation within a rat osteochondral defect model significantly enhanced de novo cartilage regeneration, as compared to delivery with TGF‐β alone. In conclusion, ghrelin could significantly enhance MSC chondrogenic differentiation in vitro and can also enhance cartilage regeneration in vivo when used in combination with TGF‐β. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1387–1397, 2019.     

Release of growth factors from a reinforced collagen GAG matrix supplemented with platelet rich plasma: Influence on cultured human meniscal cells

Damage to meniscal cartilage has been strongly linked to accelerated articular wear and consequently to osteoarthritis. Damage might be ameliorated by delivery of growth factors from platelet rich plasma (PRP) via a fiber reinforced collagen matrix designed for meniscal repair. PRP composition, release of growth factors, and influence on meniscal cell growth and gene expression were investigated. PRP was prepared using Harvest Smartprep (HS‐PRP), Cascade Fibrinet (CF‐PRP), and a simple centrifuge protocol (DC‐PRP) from four donors each. CF‐PRP had the highest ratio of platelets, with very few other blood cell types. HS‐PRP had the highest total number of platelets but also contained high levels of red and white blood cells. Absorbed to collagen matrices HS‐PRP released the highest levels of TGF‐β1 and PDGF‐AB with DC‐PRP the most IGF‐1. Cumulative release from collagen matrix was 48 ng/cm³ IGF‐1, 96 ng/cm³ TGF‐β1, and 9.6 ng/cm³ PDGF‐AB. Collagen matrix with PRP was able to increase meniscal cell number above peripheral whole blood and up‐regulated gene expression of Aggrecan, Collagen type I (α1), and Elastin (3.3 ± 0.8‐fold, 2.9 ± 0.6‐fold, 4.0 ± 1.4‐fold, respectively). Demonstrating that PRP combined with fiber reinforced collagen matrix could influence meniscal cells and might be of use for treating meniscal defects. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:273–278, 2014.     

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

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

Fluocinolone Acetonide Is a Potent Synergistic Factor of TGF‐β3–Associated Chondrogenesis of Bone Marrow–Derived Mesenchymal Stem Cells for Articular Surface Regeneration

Articular cartilage repair remains a challenging problem. Based on a high‐throughput screening and functional analysis, we found that fluocinolone acetonide (FA) in combination with transforming growth factor beta 3 (TGF‐β3) strongly potentiated chondrogenic differentiation of human bone marrow–derived mesenchymal stem cells (hBMSCs). In an in vivo cartilage defect model in knee joints of immunocompromised mice, transplantation of FA/TGF‐β3–treated hBMSCs could completely repair the articular surface. Analysis of the intracellular pathways revealed that FA enhanced TGF‐β3–induced phosphorylation of Smad2 and Smad3. Additionally, we performed a pathway array and found that FA activates the mTORC1/AKT pathway. Chemical inhibition of mTORC1 with rapamycin substantially suppressed FA effect, and inhibition of AKT completely repressed chondrogenesis of hBMSCs. Inhibition of glucocorticoid receptor with mifepristone also suppressed FA effect, suggesting that FA involves binding to the glucocorticoid receptor. Comparative analysis with other glucocorticoids (triamcinolone acetonide [TA] and dexamethasone [DEX]) revealed the unique ability of FA to repair articular cartilage surgical defects. Analysis of intracellular pathways showed that the mTORC1/AKT pathway and the glucocorticoid receptor was highly activated with FA and TA, but to a lesser extent with DEX. Collectively, these results show a unique ability of FA to enhance TGF‐β3–associated chondrogenesis, and suggest that the FA/TGF‐β3 combination may be used as major inducer of chondrogenesis in vitro. Additionally, FA/TGF‐β3 could be potentially applied in a clinical setting to increase the efficiency of regenerative approaches based on chondrogenic differentiation of stem cells. © 2015 American Society for Bone and Mineral Research.     

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

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

Simulated microgravity affects chondrogenesis and hypertrophy of human mesenchymal stem cells

Purpose

During in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) hypertrophy is an inadvertent event associated with cell differentiation toward the osteogenic lineage. Up to now, there is no stringent experimental control mechanism to prevent hypertrophy of MSCs. Microgravity is known to have an impact on osteogenesis. In this study, the influence of simulated microgravity (SMG) on both chondrogenesis and hypertrophy of hMSCs was evaluated.

Methods

A bioreactor using a rotating wall vessel was constructed to simulate microgravity. Pellet cultures formed from hMSCs (P5) were supplemented with human transforming growth factor-β₃ (TGF-β₃). The hMSC pellet cultures treated with TGF-β₃ were either kept in SMG or in a control system. After three weeks of culture, the chondrogenic differentiation status and level of hypertrophy were examined by safranin-O staining, immunohistochemistry and quantitative real-time PCR.

Results

SMG reduced the staining for safranin-O and collagen type II. The expression of collagen type X α1 chain (COL10A1) and collagen type II α₁ chain (COL2A1) were both significantly reduced. There was a higher decrease in COL2A1 than in COL10A1 expression, resulting in a low COL2A1/COL10A1 ratio.

Conclusions

SMG reduced hypertrophy of hMSCs during chondrogenic differentiation. However, the expression of COL2A1 was likewise reduced. Even more, the COL2A1/COL10A1 ratio decreased under SMG conditions. We therefore assume that SMG has a significant impact on the chondrogenic differentiation of hMSCs. However, due to the high COL2A1 suppression under SMG, this culture system does not yet seem to be suitable for a potential application in cartilage repair.     

Osteogenic potential of reamer irrigator aspirator (RIA) aspirate collected from patients undergoing hip arthroplasty

Intramedullary nailing preceded by canal reaming is the current standard of treatment for long‐bone fractures requiring stabilization. However, conventional reaming methods can elevate intramedullary temperature and pressure, potentially resulting in necrotic bone, systemic embolism, and pulmonary complications. To address this problem, a reamer irrigator aspirator (RIA) has been developed that combines irrigation and suction for reduced‐pressure reaming with temperature modulation. Osseous particles aspirated by the RIA can be recovered by filtration for use as an autograft, but the flow‐through is typically discarded. The purpose of this study was to assess whether this discarded filtrate has osteogenic properties that could be used to enhance the total repair potential of aspirate. RIA aspirate was collected from five patients (ages 71–78) undergoing hip hemiarthroplasty. Osseous particles were removed using an open‐pore filter, and the resulting filtrate (230 ± 200 mL) was processed by Ficoll‐gradient centrifugation to isolate mononuclear cells (6.2 ± 5.2 × 10⁶ cells/mL). The aqueous supernatant contained FGF‐2, IGF‐I, and latent TGF‐β1, but BMP‐2 was below the limit of detection. The cell fraction included culture plastic‐adherent, fibroblastic cells that displayed a surface marker profile indicative of mesenchymal stem cells and that could be induced along the osteogenic, adipogenic, and chondrogenic lineages in vitro. When compared to outgrowth cells from the culture of osseous particles, filtrate cells were more sensitive to seeding density during osteogenic culture but had similar capacity for chondrogenesis. These results suggest using RIA aspirate to develop improved, clinically expeditious, cost‐effective technologies for accelerating the healing of bone and other musculoskeletal tissues. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:42–49, 2009     

Development of arthrogenic joint contracture as a result of pathological changes in remobilized rat knees

This study aimed to elucidate how rats recover from immobilization‐induced knee joint contracture. Rats’ right knees were immobilized by an external fixator at a flexion of 140° for 3 weeks. After removal of the fixator, the joints were allowed to move freely (remobilization) for 0, 1, 3, 7, or 14 days (n = 5 each). To distinguish myogenic and arthrogenic contractures, the passive extension range of motion was measured before and after myotomy of the knee flexors. Knee joints were histologically analyzed and the expression of genes encoding inflammatory or fibrosis‐related mediators, interleukin‐1β (1L‐1β ), fibrosis‐related transforming growth factor‐β1 (TGF‐β1 ), and collagen type I (COL1A1 ) and III (COL3A1 ), were examined in the knee joint posterior capsules using real‐time PCR. Both myogenic and arthrogenic contractures were established within 3 weeks of immobilization. During remobilization, the myogenic contracture decreased over time. In contrast, the arthrogenic contracture developed further during the remobilization period. On day 1 of remobilization, inflammatory changes characterized by edema, inflammatory cell infiltration, and upregulation of IL‐1β gene started in the knee joint posterior capsule. In addition, collagen deposition accompanied by fibroblast proliferation, with upregulation of TGF‐β1 , COL1A1 , and COL3A1 genes, appeared in the joint capsule between days 7 and 14. These results suggest the progression of arthrogenic contracture following remobilization, which is characterized by fibrosis development, is possibly triggered by inflammation in the joint capsule. It is therefore necessary to focus on developing new treatment strategies for immobilization‐induced joint contracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1414–1423, 2017.

     

Three‐dimensional high‐density co‐culture with primary tenocytes induces tenogenic differentiation in mesenchymal stem cells

Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and tissue engineering and may represent an attractive option for tendon repair and regeneration. Thus far the ability of MSCs to differentiate into tenocytes in vitro has not been investigated. Experiments were performed with and without growth factors (IGF‐1, TGF‐β1, IGF‐1/TGF‐β1, PDGF‐BB, and BMP‐12), in co‐cultures of tenocytes and MSCs mixed in different ratios and by culturing MSCs with spent media obtained from primary tenocytes. Tenogenesis was induced in MSCs through a combination of treatment with IGF‐1 and TGF‐β1, in high‐density co‐cultures and through cultivation with the spent media from primary tenocytes. Electron microscopy and immunoblotting were used to demonstrate up‐regulation of collagen I/III, decorin, tenomodulin, β1‐Integrin, MAPKinase pathway (Shc, Erk1/2), and scleraxis in the co‐cultures and provide simultaneous evidence for the inhibition of apoptosis. In monolayer co‐cultures extensive intercellular contacts between MSCs and tenocytes were observed. Cells actively exchanged vesicles, which were labeled by using immunofluorescence and immunogold techniques, suggesting the uptake and interchange of soluble factors produced by the MSCs and/or tenocytes. We conclude that MSCs possess tenogenic differentiation potential when provided with relevant stimuli and a suitable microenvironment. This approach may prove to be of practical benefit in future tissue engineering and tendon regenerative medicine research. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1351–1360, 2011     

Osteoblast‐Derived TGF‐β1 Stimulates IL‐8 Release Through AP‐1 and NF‐κB in Human Cancer Cells

Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)‐8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone‐derived growth factors on the IL‐8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast‐derived TGF‐β1 is associated with osteolytic bone diseases. Materials and Methods: IL‐8 mRNA levels were measured using RT‐PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF‐β1, BMP‐2, and IGF‐1. DNA affinity protein‐binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c‐fos, c‐jun, p65, and p50 to the IL‐8 promoter. A transient transfection protocol was used to examine IL‐8, NF‐κB, and activator protein (AP)‐1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL‐8, AP‐1, and NF‐κB promoter in human cancer cells. Osteoblasts were transfected with TGF‐β1, BMP‐2, or IGF‐1 small interfering RNA, and the medium was collected after 48 h. TGF‐β1 but not BMP‐2 or IGF‐1 siRNA inhibited OBCM‐induced IL‐8 release in human cancer cells. In addition, TGF‐β1 also directly induced IL‐8 release in human cancer cells. Activation of AP‐1 and NF‐κB DNA‐protein binding and MAPKs after TGF‐β1 treatment was shown, and TGF‐β1–induced IL‐8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF‐β1, BMP‐2, and IGF‐1. TGF‐β1 is the major contributor to the activation of extracellular signal‐related kinase (ERK), p38, and c‐Jun N‐terminal kinase (JNK), leading to the activation of AP‐1 and NF‐κB on the IL‐8 promoter and initiation of IL‐8 mRNA and protein release, thereby promoting osteoclastogenesis.     

Simvastatin inhibits transforming growth factor‐β1‐induced expression of type I collagen,CTGF, and α‐SMA in keloid fibroblasts

Simvastatin, a 3‐hydroxy‐3‐methylglutaryl coenzyme‐A reductase inhibitor, is used to reduce cholesterol levels. Accumulating evidence has revealed the immunomodulatory and anti‐inflammatory effects of simvastatin that prevent cardiovascular diseases. In addition, the beneficial effects of statins on fibrosis of various organs have been reported. However, the functional effect of statins on dermal fibrosis of keloids has not yet been explored. The objective of this study was to determine whether simvastatin could affect dermal fibrosis associated with keloids. We examined the effect of simvastatin on transforming growth factor (TGF)‐β1‐induced production of type I collagen, connective tissue growth factor (CTGF or CCN2), and α‐smooth muscle actin (α‐SMA). Keloid fibroblasts were cultured and exposed to different concentrations of simvastatin in the presence of TGF‐β1, and the effects of simvastatin on TGF‐β1‐induced collagen and CTGF production in keloid fibroblasts were determined. The type I collagen, CTGF, and α‐SMA expression levels and the Smad2 and Smad3 phosphorylation levels were assessed by Western blotting. The effect of simvastatin on cell viability was evaluated by assessing the colorimetric conversion of 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide. Simvastatin suppressed TGF‐β1‐induced type I collagen, CTGF, and α‐SMA production in a concentration‐dependent manner. The TGF‐β1‐induced Smad2 and Smad3 phosphorylation levels were abrogated by simvastatin pretreatment. The inhibition of type I collagen, CTGF, and α‐SMA expression by simvastatin was reversed by geranylgeranyl pyrophosphate, suggesting that the simvastatin‐induced cellular responses were due to inhibition of small GTPase Rho involvement. A RhoA activation assay showed that preincubation with simvastatin significantly blocked TGF‐β1‐induced RhoA activation. The Rho‐associated coiled kinase inhibitor Y27632 abrogated TGF‐β1‐induced production of type I collagen, CTGF, and α‐SMA. However, Y27632 had no significant effect on TGF‐β1‐induced phosphorylation of Smad2 and Smad3. In conclusion, the present study suggests that simvastatin is an effective inhibitor of TGF‐β1‐induced type I collagen, CTGF, and α‐SMA production in keloid fibroblasts.     

Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy

Objective

This study investigated a novel approach to induce chondrogenic differentiation of human mesenchymal stem cells (hMSC). We hypothesized that a structured three-dimensional co-culture using hMSC and chondrocytes would provide chondroinductive cues to hMSC without inducing hypertrophy.

Method

In an effort to promote optimal chondrogenic differentiation of hMSC, we created bilaminar cell pellets (BCPs), which consist of a spherical population of hMSC encased within a layer of juvenile chondrocytes (JC). In addition to histologic analyses, we examined proteoglycan content and expression of chondrogenic and hypertrophic genes in BCPs, JC pellets, and hMSC pellets grown in the presence or absence of transforming growth factor-β (TGFβ) following 21 days of culture in either growth or chondrogenic media.

Results

In either growth or chondrogenic media, we observed that BCPs and JC pellets produced more proteoglycan than hMSC pellets treated with TGFβ. BCPs and JC pellets also exhibited higher expression of the chondrogenic genes Sox9, aggrecan, and collagen 2A1, and lower expression of the hypertrophic genes matrix metalloproteinase-13, Runx2, collagen 1A1, and collagen 10A1 than hMSC pellets. Histologic analyses suggest that JC promote chondrogenic differentiation of cells in BCPs without hypertrophy. Furthermore, when cultured in hypoxic and inflammatory conditions intended to mimic the injured joint microenvironment, BCPs produced significantly more proteoglycan than either JC pellets or hMSC pellets.

Conclusion

The BCP co-culture promotes a chondrogenic phenotype without hypertrophy and, relative to pellet cultures of hMSCs or JCs alone, is more resistant to the adverse conditions anticipated at the site of articular cartilage repair.