TGF-β₃ encapsulated PLCL scaffold by a supercritical CO₂-HFIP co-solvent system for cartilage tissue engineering

Kim, S.H.,Kim, S.H.,Jung, Y. Elsevier Science Publishers 2015 Journal of controlled release Vol.206 No.-

<P>Mimicking the native tissue microenvironment is critical for effective tissue regeneration. Mechanical cues and sustained biological cues are important factors, particularly in load-bearing tissues such as articular cartilage or bone. Carriers including hyclrogels and nanoparticles have been investigated to achieve sustained release of protein drugs. However, it is difficult to apply such carriers alone as scaffolds for cartilage regeneration because of their weak mechanical properties, and they must be combined with other biomaterials that have adequate mechanical strength. In this study, we developed the multifunctional scaffold which has similar mechanical properties to those of native cartilage and encapsulates TGF-beta 3 for chondrogenesis. In our previous work, we confirmed that poly(lacticle-co-caprolacton) (PLCL) did not foam when exposed to supercritical CO2 below 45 degrees C. Here, we used a supercritical carbon dioxide (scCO(2))-1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) co-solvent system to facilitate processing under mild conditions because high temperature causes protein denaturation and decreases bioactivity of the protein. This processing made it possible to fabricate a TGF-beta(3) encapsulated elastic porous PLCL scaffold at 37 C. We investigated the tissue regeneration efficiency of the TGF-beta(3) encapsulated PLCL scaffold using human adipose-derived stem cells (ADSCs) in vitro and in vivo (Groups; i. PLCL scaffold + Fibrin gel + TGF-beta(3), ii.TGF-133 encapsulated PLCL scaffold + Fibrin gel, iii.TGF-beta(3) encapsulated PLCL scaffold). We evaluated the chondrogenic abilities of the scaffolds at 4,8, and 12 weeks after subcutaneous implantation of the constructs in immune-deficient mice. Based on TGF-beta(3) release studies, we confirmed that TGF-beta(3) molecules were released by 8 weeks and remained in the PLCL matrix. Explants of TGF-beta(3)(encapsulated scaffolds by a co-solvent system exhibited distinct improvement in the compressive E-modulus and deposition of extracellular matrix. Furthermore, long-term delivery of TGF-beta(3) formed a hyaline cartilage-specific lacunae structure and prevented the hypertrophy of differentiated chondrocytes. TGF-beta(3) encapsulated PLCL scaffolds would be useful as functional scaffolds for cartilage tissue engineering. (C) 2015 Elsevier B.V. All rights reserved.</P>

TGF-β3 suppresses melanogenesis in human melanocytes cocultured with UV-irradiated neighboring cells and human skin

( Hye-rim Moon ),( Joon Min Jung ),( Su Yeon Kim ),( Mi Young Lee ),( Youngsup Song ),( Sung Eun Chang ) 대한피부과학회 2020 대한피부과학회 학술발표대회집 Vol.72 No.1

Background: Ultraviolet radiation (UVR) is the most well-known cause of skin pigmentation accompanied with photoaging. Transforming growth factor (TGF)-β1 was previously shown to have anti-melanogenic property; however, it can induce scarring in skin. Objectives: We investigated the effect of TGF-β3 on melanogenesis in human melanocytes cocultured with UV-irradiated skin constituent cells, and UV-irradiated human skin. Methods: UVB irradiation or treatment with stem cell factor (SCF) and endothelin-1 (ET-1) was applied to human melanocytes cocultured with keratinocytes and/or fibroblasts and ex vivo human skin. Mechanistic pathways were further explored after treatment with TGF-β3. Results: While UVB irradiation or SCF/ET-1 enhanced melanogenesis, TGF-β3 effectively inhibited melanin accumulation and tyrosinase activity via downregulation of the extracellular signal-regulated kinase (ERK)/microphthalmiaassociated transcription factor (MITF) pathway. TGF-β3 increased the expression of differentiation markers of keratinocytes. Conclusion: TGF-β3 effectively suppressed UVR-stimulated melanogenesis indicating that topical TGF-β3 may be a suitable candidate for the treatment of UV-associated hyperpigmentation disorders.

Wnt-5a is involved in TGF-β3-stimulated chondrogenic differentiation of chick wing bud mesenchymal cells

Jin, Eun-Jung,Park, Jae-Han,Lee, Sun-Young,Chun, Jang-Soo,Bang, Ok-Sun,Kang, Shin-Sung Elsevier 2006 The international journal of biochemistry & cell b Vol.38 No.2

<P><B>Abstract</B></P><P>In this study we investigated whether signalling by TGF-β3 and Wnt-5a cross-talk during chondrogenic differentiation of chick wing mesenchyme. Using differential display polymerase chain reaction screening, we found the expression of Wnt-5a to be significantly increased during transforming growth factor-β3 (TGF-β3)-induced precartilage condensation in mesenchyme micromass cultures. Transfection of cells with a Wnt-5a expression construct promoted precartilage condensation and chondrogenesis in micromass cultures, similar to that observed when chondrogenic-competent cells were exposed to TGF-β3. Overexpression of Wnt-5a or treatment with TGF-β3 stimulated the activation of protein kinase C-α (PKC-α) and p38 mitogen-activated protein kinase (MAPK), both positive regulators of chondrogenic differentiation. Inactivation of PKC-α and p38 MAPK by specific inhibitors abrogated chondrogenesis stimulated by both TGF-β3 and Wnt-5a. Similarly, partial reduction in TGF-β3-induced Wnt-5a expression by small interfering RNA resulted in decreased activities of PKC-α and p38 MAPK, and abolished the chondro-stimulatory effect of TGF-β3. Collectively, these findings indicate that Wnt-5a, a non-canonical Wnt, can mediate the chondro-stimulatory effect of TGF-β3 through upregulation of PKC-α and p38MAPK signaling.</P>

TGF-β3 Inhibits Chondrogenesis by Suppressing Precartilage Condensation through Stimulation of N-cadherin Shedding and Reduction of cRREB-1 Expression

Eun-Jung Jin,Kwang Sook Park,김동균,Young-Sup Lee,손종경,Jae Chang Jung,Ok-Sun Bang,Shin-Sung Kang 한국분자세포생물학회 2010 Molecules and cells Vol.29 No.4

Transforming growth factor-β (TGF-β) plays crucial roles in controlling cell differentiation and maintaining tissue integrity. Previously we reported that TGF-β3 treatment decreased the mRNA expression of the gap junction protein,connexin 43 as well as cell number, which lead to the inhibition of chondrogenic condensation in cultured chick leg bud mesenchymal cells. The present study demonstrates that TGF-β3 can induce cleavage in the ectodomain of neuronal cadherin (N-cadherin) at the initiation stage of chondrogenesis and reduce cell numbers, cellular adhesion and the expression level of connexin 43. Differential displayed PCR (DD-PCR) comparison of adherent- and non-adherent chick leg chondrogenic progenitor cells showed increased expression of the chick ras-responsive element binding transcription factor, cRREB-1, in adherent cells. In chick leg bud mesenchymal cells, cRREB-1 transcription was inhibited by TGF-β3 at the early stage of chondrogenesis. Small interfering RNA (siRNA)-mediated knockdown of cRREB-1 reduced cell numbers, cellular adhesion, and the expression level of connexin 43 resulting in the inhibition of precartilage condensation. Taken together, these findings indicate that TGF-β3 mediates the inhibitory signal necessary for precartilage condensation by stimulating N-cadherin shedding and reducing cRREB-1 expression levels.

A TGF-β-induced gene, βig-h3, is crucial for the apoptotic disappearance of the medial edge epithelium in palate fusion

Choi, Kang-Young,Kim, Hyun-Jung,Cho, Byung-Chae,Kim, In-San,Kim, Hyun-Jung,Ryoo, Hyun-Mo Wiley Subscription Services, Inc., A Wiley Company 2009 Journal of cellular biochemistry Vol.107 No.4

<P>TGF-β3, TβR-I, and TGF-β-activated Smad2 has been suggested to be a series of signaling molecules for secondary palate fusion. In this article, we show that a gene induced by TGF-β, βig-h3, is coincidentally expressed with TGF-β3 in medial edge epithelial (MEE) cells undergoing apoptosis during normal palatal fusion. βig-h3 was also highly expressed in the areas of post-weaning mammary gland cells and developing phalangeal joints in which TGF-β3 or BMP-4-induced apoptosis occurs, respectively. Blocking of βig-h3 expression in E12.5 embryos with antisense oligodeoxynucleotides (ODN) resulted in cleft of the secondary palate in 84% of the treated mice that were born. Moreover, the antisense ODN treatment resulted in a failure of apoptosis in the MEE between palatal shelves in physical contact in organ culture. We conclude that βig-h3 expression in the MEE is stimulated by TGF-β3, causes cell death, and consequently results in complete fusion of the apposed palatal shelves. J. Cell. Biochem. 107: 818–825, 2009. © 2009 Wiley-Liss, Inc.</P>

Chondrogenic differentiation of mesenchymal stem cells embedded in a scaffold by long-term release of TGF-β3 complexed with chondroitin sulfate

Park, Ji Sun,Yang, Hyun Jung,Woo, Dae Gyun,Yang, Han Na,Na, Kun,Park, Keun-Hong Wiley Subscription Services, Inc., A Wiley Company 2010 Journal of biomedical materials research. Part A Vol.a92 No.2

<P>In this study, mesenchymal stem cells (MSCs) embedded in biodegradable and water-swollen, elastic block copolymer scaffolds were assessed for MSC chondrogenesis. To determine the optimal conditions for chondrogenesis of the embedded rMSCs, transforming growth factor-β3 (TGF-β3) was physically conjugated with chondroitin sulfate (CS) and mixed into scaffolds, which were subsequently evaluated for the differentiation of transplanted rMSCs. In determination of CS-bound growth factors for chondrogenesis, scaffold mixed with rMSCs and TGF-β3 was then tested by growth factor release profiles, confocal laser microscopy, RT-PCR analysis, real time-QPCR, and histology. The results of several different analyses of the transplanted rMSCs embedded in the scaffolds showed that rMSCs coupled with a CS-bound TGF-β3 encapsulated scaffold evidenced superior cartilage tissue formation as measured by an assay of specific gene and protein expression. Moreover, the scaffold exhibited more rapid and more distinct morphology of differentiated rMSCs than was observed with other scaffolds, as determined by histology and immunochemical histology analysis. These results indicate that the elastic block copolymer scaffolds combined with a CS-bound TGF-β3 should prove very suitable matrix for cell-based cartilage tissue engineering. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010</P>

PKCη Regulates the TGFβ3-induced Chondrogenic Differentiation of Human Mesenchymal Stem Cell

구보미,이은신,하영술,JAE YONG PARK,Joo Yeon Jeong,이동훈,Gyeong Jae Cho,Wan Sung Choi,Young Phil Yune,강상수 한국발생생물학회 2013 발생과 생식 Vol.17 No.4

Transforming growth factor (TGF) family is well known to induce the chondrogenic differentiation of mesenchymal stem cells (MSC). However, the precise signal transduction pathways and underlying factors are not well known. Thus the present study aims to evaluate the possible role of C2 domain in the chondrogenic differentiation of human mesenchymal stem cells. To this end, 145 C2 domains in the adenovirus were individually transfected to hMSC, and morphological changes were examined. Among 145 C2 domains, C2 domain of protein kinase C eta (PKCη) was selected as a possible chondrogenic differentiation factor for hMSC. To confirm this possibility, we treated TGFβ3, a well known chondrogenic differentiation factor of hMSC, and examined the increased-expression of glycosaminoglycan (GAG), collagen type II (COL II) as well as PKCη using PT-PCR, immunocytochemistry and Western blot analysis. To further evaluation of C2 domain of PKCη, we examined morphological changes, expressions of GAG and COL II after transfection of PKCη-C2 domain in hMSC. Overexpression of PKCη-C2 domain induced morphological change and increased GAG and COL II expressions. The present results demonstrate that PKCη involves in the TGF-β3-induced chondrogenic differentiation of hMSC, and C2 domain of PKCη has important role in this process.

Transforming Growth Factor-β3 Gene SfaN1 Polymorphism in Korean Nonsyndromic Cleft Lip and Palate Patients

( Myung Hee Kim ),( Hyo Jin Kim ),( Je Yong Choi ),( Dong Seok Nahm ) 생화학분자생물학회 2003 BMB Reports Vol.36 No.6

The nonsyndromic cleft lip and palate (NSCL/P) is a congenital deformity of multifactorial origin with a relatively high incidence in the oriental population. Various etiologic candidate genes have been reported with conflicting results, according to race and analysis methods. Recently, the ablation of the TGF-β3 gene function induced cleft palates in experimental animals. Also, polymorphisms in the TGF-β3 gene have been studied in different races; however, they have not been studied in Koreans. A novel A →G single nucleotide polymorphism (defined by the endonuclease SfaN1) was identified in intron 5 of TGF-β3 (IVS5+104A)G). It resulted in different genotypes, AA,AG, and GG. The objective of this study was to investigate the relationship between the SfaN1 polymorphism in TGF-β3 and the risk of NSCL/P in the Korean population. The population of this study consisted of 28 NSCL/P patients and 41 healthy controls. The distribution of the SfaN1 genotypes was different between the cases and controls. The frequency of the G allele was significantly associated with the increased risk of NSCL/P [oddsratio (OR)=15.92, 95% confidence interval (CI)=6.3-41.0]. The risk for the disease increased as the G allele numbers increased (GA genotype: OR =2.11, 95% CI= 0.38-11.68; GG genotype: OR = 110.2, 95% CI = 10.67 2783.29) in NSCL/P. A stratified study in patients revealed that the SfaN1 site IVS5+104A>G substitution was strongly associated with an increased risk of NSCL/P in males (p<0.001), but not in females. In conclusion, the polymorphism of the SfaN1 site in TGF-β3 was significantly different between the NSCL/P patients and the control. This may be a good screening marker for NSCL/P patients among Koreans.

Chondrogenesis of human mesenchymal stem cells encapsulated in a hydrogel construct: Neocartilage formation in animal models as both mice and rabbits

Park, Ji Sun,Woo, Dae Gyun,Yang, Han Na,Lim, Hye Jin,Park, Kyong Mi,Na, Kun,Park, Keun-Hong Wiley Subscription Services, Inc., A Wiley Company 2010 Journal of biomedical materials research. Part A Vol.a92 No.3

<P>In this study, in vivo studies, both nude mouse and rabbit cartilage defect, were tested for chondrogenesis using stem cells (SCs) using growth factor. Specifically, human mesenchymal stem cells (hMSCs) were embedded in a hydrogel scaffold, which was coencapsulated with transforming growth factor-β3 (TGF-β3). The specific extracellular matrices (ECMs) released from hMSCs transplanted into the animal were assessed via glycosaminoglycan (GAG)/DNA content, RT-PCR, real time-QPCR, immunohistochemical (IHC), and Safranin-O staining and were observed up to 7 weeks after injection. By detection of ECMs the GAG content per cell remained constant for all formulations, indicating that the dramatic increase in cell number for samples with TGF-β3 was accompanied by the maintenance of the cell phenotypes. The histological and IHC staining of the newly repaired tissues observed after treatment with TGF-β3 mixed with hMSCs evidenced hyaline cartilage-like characteristics. Moreover, the results observed with the animal model (rabbit) treated with hMSCs embedded in the growth factor-containing hydrogel indicate that the implantation of mixed cells with TGF-β3 may constitute a clinically efficient method for the regeneration of hyaline articular cartilage. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010</P>

The ethnic difference of the prevalence of SfaN polymorphism in the nonsyndromic cleft palate

Kim, Myung-Hee,Nahm, Dong-Seok,Horatiu Rotaru,Lucia Hurubeanu,Choi, Je-Yong,Chae, Chang-Hoon,Kim, Seong-Gon 대한치과교정학회 2004 대한치과교정학회지 Vol.34 No.3

비증후군성 구순구개열은 가장 빈도가 높은 선천성 기형 중의 하나로 특히 한국이나 일본과 같은 극동 지방에서 높은 발생율을 보이고 동유럽에서는 드물게 보고되고 있다. 이러한 인종에 따른 차이는 이 질환에 유전적인 배경이 있음을 의미한다. 본 연구의 목적은 한국인의 비증후군성 구개열과 연관이 있다고 알려진 SfaN1 단일 염기 다형성증의 발현빈도가 한국인 구개열 가족과 루마니아 구순구개열 가족 사이에 통계적으로 유의할만한 차이가 있는지를 알아보기 위하여 시행하였다. 한국인 26가족과 루마니아 18가족을 대상으로 하였다. 전체 인원수는 한국인의 경우 78명이었고 루마니아의 경우 41명이었다. 유전자 서열분석에 사용된 샘플은 각 참여자의 혈액이나 타액을 채취하여 분석하였다. SfaN1 단일 염기 다형 성증은 TCF-β3 유전자의 5번 인트론에서 관찰된다 (A18141G). 결과를 보면 한국인과 루마니아인의 비증후군성 구개열 가족 사이에는 통계적으로 유의할만한 차이가 인지되었다. 전체 샘플 중에서 AA allele는 한국인에서는 18명 (23.1%)이었으나 루마니아는 27명 (65.9%)이었다. AS allele는 한국인에서는 27명 (34.6%)이었으나 루마니아에서는 13명 (31.7%)이었다. GG allele는 한국인에서는 33명 (42.3%)이었으나 루마니아에서는 1명 (2.4%)이었다. 두 집단 사이의 차이는 통계적으로 유의하였다 (p<0.001). 결론적으로 한국인과 루마니아인의 비증후군성 구개열 가족 사이에 SfaN1 단일 염기 다형성증의 발현빈도는 한국인에서 통계적으로 유의할만하게 높게 나타났으며, 이는 한국에서 루마니아보다 비증후군성 구개열의 빈도가 높게 나타나는 현상을 부분적으로 설명하여 주는 것으로 사료된다. Nonsyndromic cleft lip and/or palate (NSCLP) is one of the most common congenital deformities and its prevalence in Far East Asia, such as within Korean and Japanese populations, is relatively high. However, in the eastern part of Europe, clefts are relatively rare situations. These ethnic differences infer a genetic background of the disease. The objective of this study was to compare the frequency of single nucleotide polymorphism (SNP) in TGF-β3 between Korean and Romanian cleft families. Korean cleft families samples were collected from twenty-six families (n=78) and Romanian cleft families samples were collected from eighteen families (n=41). For sequencing, the blood or saliva of the subjects was sampled. A single nucleotide plolymorphism was observed in the intron 5 of TGF-β3 (A18141G). The frequency of each allele was significantly different between the Korean and Romanian samples. The AA allele was present in 18 out of 78 Korean samples (23.1%) and in 27 out of 41 Romanian samples (65.9%). The AG was present in 27 (34.6%) out of 78 Koreans and in 13 (31.7%) out of 41 Romanians. The GG was found in 33 (42.3%) Koreans and in 1 (2.4%) Romanian. The difference between the groups was significant (p<0.001). In conclusion, the frequency of observed SNP was significantly different between the two countries. SNP in TGF-β3 in the Korean population seemed to have a higher possibility of occurrence for nonsyndromic cleft palate than the Romanian population.