Dual Growth Factor-Loaded Core-Shell Polymer Microcapsules Can Promote Osteogenesis and Angiogenesis

Ramesh Subbiah,박귀덕,Ping Du,Mintai Peter Hwang,김인걸,반세영,노용관,박한수 한국고분자학회 2014 Macromolecular Research Vol.22 No.12

Growth factors (GFs) are very critical in stem cell differentiation and tissue regeneration. Therefore GFdelivery carriers have been a major subject in tissue engineering research. In this study, we prepare and optimizecore-shell microcapsules (C-S MCs) for dual GF delivery. The C-S MCs, composed of an alginate shell andpoly(lactic-co-glycolic) acid (PLGA) core, are fabricated using an electrodropping method via custom-made coaxialneedles. They are 198±38 μm in diameter with an average core size of 90±13 μm, and they are fabricated using analginate concentration of 1% (w/v), an electrical voltage of 11 kV, and an inner syringe flow rate of 50 μL/min. Usingthis platform, dual GFs, bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) areencapsulated in the alginate shell and PLGA core, respectively. In vitro release tests of dual GF-loaded C-S MCsreveal early release of BMP-2, followed by VEGF on a temporal release profile of 28 days. In vitro study of the dualGF-loaded MCs demonstrates their osteogenic activity with preosteoblasts; osteogenic markers (osteocalcin andtype I collagen) are upregulated and both calcium content and alkaline phosphatase (ALP) activity also increased. In addition, C-S MCs combined with collagen and preosteoblasts were subcutaneously transplanted to the dorsalregion of nude mice for 3 weeks. Analysis of the retrieved constructs exhibits that both osteogenesis and angiogenesiswere more active in the group containing dual GF-loaded MCs, along with deep penetration of blood vesselsinside the construct, compared to blank MCs or single GF (BMP-2)-loaded MCs. This study proposes a dual GFdelivery carrier using C-S MCs and demonstrates the feasibility of C-S MCs in the induction of osteogenesis andangiogenesis.

Osteogenic/Angiogenic Dual Growth Factor Delivery Microcapsules for Regeneration of Vascularized Bone Tissue.

Subbiah, Ramesh,Hwang, Mintai Peter,Van, Se Young,Do, Sun Hee,Park, Hansoo,Lee, Kangwon,Kim, Sang Heon,Yun, Kyusik,Park, Kwideok Wiley-VCH 2015 Advanced Healthcare Materials Vol.4 No.13

<P>Growth factors (GFs) are major biochemical cues for tissue regeneration. Herein, a novel dual GF delivery system is designed composed of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and alginate microcapsules (MCs) via an electrodropping method. While bone morphogenetic protein (BMP)-2 is encapsulated in the PLGA NPs, vascular endothelial growth factor (VEGF) is included in the alginate MCs, where BMP-2-loaded PLGA NPs are entrapped together in the fabrication process. The initial loading efficiencies of BMP-2 and VEGF are 78% 3.6% and 43% 1.7%, respectively. When our dual GF-loaded MCs are assessed for in vitro osteogenesis of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) on 2D and 3D environment, MCs contribute to much better UCB-MSCs osteogenesis as confirmed by von Kossa staining, immunofluorescence (osteocalcin, collagen 1), calcium content measurement, and osteogenic markers expression. In addition, when dual GF-encapsulated MCs are combined with collagen and then applied to 8 mm diameter rat calvarial defect model, the positive effects on vascularized bone regeneration are much more pronounced; micro computed tomography (CT) and histology analyses exhibit 82.3% bone healing coupled with 12.6% vessel occupied area. Put together, current study indicates a synergistic effect of BMP-2/VEGF and highlights the great potential of dual GF delivery modality (PLGA NPs-in-MC) for regeneration of vascularized bone.</P>

Investigation of the changes of biophysical/mechanical characteristics of differentiating preosteoblasts in vitro

Ramesh Subbiah,Muhammad Suhaeri,황민태,Woojun Kim,박귀덕 한국생체재료학회 2015 생체재료학회지 Vol.19 No.4

Background: Topography, stiffness, and composition of biomaterials play a crucial role in cell behaviors. In this study, we have investigated biochemical (gene markers), biophysical (roughness), and biomechanical (stiffness) changes during the osteogenic differentiation of preosteoblasts on gelatin matrices. Results: Our results demonstrate that gelatin matrices offer a favorable microenvironment for preosteoblasts as determined by focal adhesion and filopodia formation. The osteogenic differentiation potential of preosteoblasts on gelatin matrices is confirmed by qualitative (Alizarin red, von kossa staining, immunofluorescence, and gene expression) and quantitative analyses (alkaline phosphatase activity and calcium content). The biomechanical and biophysical properties of differentiating preosteoblasts are analyzed using atomic force microscopy (AFM) and micro indentation. The results show sequential and significant increases in preosteoblasts roughness and stiffness during osteogenic differentiation, both of which are directly proportional to the progress of osteogenesis. Cell proliferation, height, and spreading area seem to have no direct correlation with differentiation; however, they may be indirectly related to osteogenesis. Conclusions: The increased stiffness and roughness is attributed to the mineralized bone matrix and enhanced osteogenic extracellular matrix protein. This report indicates that biophysical and biomechanical aspects during in vitro cellular/extracellular changes can be used as biomarkers for the analysis of cell differentiation.

Structural and biological evaluation of a multifunctional SWCNT-AgNPs-DNA/PVA bio-nanofilm

Subbiah, Ramesh P.,Lee, Haisung,Veerapandian, Murugan,Sadhasivam, Sathya,Seo, Soo-won,Yun, Kyusik Springer-Verlag 2011 ANALYTICAL AND BIOANALYTICAL CHEMISTRY Vol.400 No.2

Evaluation of cytotoxicity, biophysics and biomechanics of cells treated with functionalized hybrid nanomaterials

Subbiah, Ramesh,Ramasundaram, Subramaniyan,Du, Ping,Hyojin, Kim,Sung, Dongkyung,Park, Kwideok,Lee, Nae-Eung,Yun, Kyusik,Choi, Kyoung Jin Royal Society 2013 Journal of the Royal Society, Interface Vol.10 No.88

<P>Hybrids consisting of carboxylated, single-walled carbon nanotube (c-SWNT)–silver nanoparticles (AgNPs)-DNA–poly vinyl alcohol (PVA) are synthesized via sequential functionalization to mimic the theragnostic (therapy and diagnosis) system. Carboxylation of SWNT has minimized the metal impurities with plenty of –COOH groups to produce hybrid (c-SWNT-AgNPs). The hybrid is further wrapped with DNA (hybrid-DNA) and encapsulated with PVA as hybrid composite (HC). Materials were tested against human alveolar epithelial cells (A549), mouse fibroblasts cells (NIH3T3) and human bone marrow stromal cells (HS-5). The composition-sensitive physico-chemical interactions, biophysics and biomechanics of materials-treated cells are evaluated. The cell viability was improved for HC, hybrid-PVA and c-SWNT when compared with SWNT and hybrid. SWNT and hybrid showed cell viability less than 60% at high dose (40 µg ml<SUP>−1</SUP>) and hybrid-PVA and HC retained 80% or more cell viability. The treatment of hybrid nanomaterials considerably changed cell morphology and intercellular interaction with respect to the composition of materials. Peculiarly, PVA-coated hybrid was found to minimize the growth of invadopodia of A549 cells, which is responsible for the proliferation of cancer cells. Surface roughness of cells increased after treatment with hybrid, where cytoplasmic regions specifically showed higher roughness. Nanoindentation results suggest that changes in biomechanics occurred owing to possible internalization of the hybrid. The changes in force spectra of treated cells indicated a possible greater interaction between the cells and hybrid with distinct stiffness and demonstrated the surface adherence and internalization of hybrid on or inside the cells.</P>

Investigation of cellular responses upon interaction with silver nanoparticles

Subbiah, Ramesh,Jeon, Seong Beom,Park, Kwideok,Ahn, Sang Jung,Yun, Kyusik Dove Medical Press 2015 INTERNATIONAL JOURNAL OF NANOMEDICINE Vol.10 No.specal

<P>In order for nanoparticles (NPs) to be applied in the biomedical field, a thorough investigation of their interactions with biological systems is required. Although this is a growing area of research, there is a paucity of comprehensive data in cell-based studies. To address this, we analyzed the physicomechanical responses of human alveolar epithelial cells (A549), mouse fibroblasts (NIH3T3), and human bone marrow stromal cells (HS-5), following their interaction with silver nanoparticles (AgNPs). When compared with kanamycin, AgNPs exhibited moderate antibacterial activity. Cell viability ranged from ≤80% at a high AgNPs dose (40 µg/mL) to >95% at a low dose (10 µg/mL). We also used atomic force microscopy-coupled force spectroscopy to evaluate the biophysical and biomechanical properties of cells. This revealed that AgNPs treatment increased the surface roughness (<I>P</I><0.001) and stiffness (<I>P</I><0.001) of cells. Certain cellular changes are likely due to interaction of the AgNPs with the cell surface. The degree to which cellular morphology was altered directly proportional to the level of AgNP-induced cytotoxicity. Together, these data suggest that atomic force microscopy can be used as a potential tool to develop a biomechanics-based biomarker for the evaluation of NP-dependent cytotoxicity and cytopathology.</P>

Crystalline Structure and Ferroelectric Properties of Poly(vinelidene fluoride)-Chemically Converted Graphene Nanocomposites

RAMASUNDARAM,RAMESH SUBBIAH,안유진,SHAMIM-ARA PERVIN,최경진,김갑진 한국고분자학회 2012 한국고분자학회 학술대회 연구논문 초록집 Vol.2012 No.4

Development of Contrast Agents for The Selective Labeling for Cancerous Lesions using A Hydrogel-based Nanoemulsions

정윤,Ramesh Subbiah,박귀덕,이강원 한국고분자학회 2016 한국고분자학회 학술대회 연구논문 초록집 Vol.41 No.1

Novel Platform of Cardiomyocyte Culture and Coculture via Fibroblast-Derived Matrix-Coupled Aligned Electrospun Nanofiber

Suhaeri, Muhammad,Subbiah, Ramesh,Kim, Su-Hyun,Kim, Chong-Hyun,Oh, Seung Ja,Kim, Sang-Heon,Park, Kwideok American Chemical Society 2017 ACS APPLIED MATERIALS & INTERFACES Vol.9 No.1

<P>For cardiac tissue engineering, much attention has been given to the artificial cardiac microenvironment in winch anisotropic design of scaffold and extracellular matrix (ECM) are the major cues. Here we propose poly(L-lactide-co-caprolactone) and fibroblast-derived ECM (PLCL/FDM), a hybrid scaffold that combines aligned electrospun PLCL fibers and FDM. Fibroblasts were grown on the PLCL fibers for 5 7 days and subsequently decellularized to produce PLCL/FDM. Various analyses confirmed aligned, FDM-deposited PLCL fibers. Compared to fibronectin (FN)-coated electrospun PLCL fibers (control), H9c2 cardiomyoblast differentiation was significantly effective, and neonatal rat cardiomyocyte (CM) phenotype and maturation was improved on PLCL/FDM. Moreover, a coculture platform was created using multilayer PLCL/FDM in which two different cells make indirect or direct cell cell contacts. Such coculture platforms demonstrate their feasibility in terms of higher cell viability, efficiency of target cell harvest (>95% in noncontact; 85% in contact mode), and molecular diffusion through the PLCL/FDM layer. Coculture of primary CMs and fibroblasts exhibited much better CM phenotype and improvement of CM maturity upon either direct or indirect interactions, compared to the conventional coculture systems (transwell insert and tissue culture plate (TCP)). Taken together, our platform should be very useful and have significant contributions in investigating some scientific or practical issues of crosstalks between multiple cell types.</P>

Cardiomyoblast (H9c2) Differentiation on Tunable Extracellular Matrix Microenvironment

Suhaeri, Muhammad,Subbiah, Ramesh,Van, Se Young,Du, Ping,Kim, In Gul,Lee, Kangwon,Park, Kwideok Mary Ann Liebert 2015 Tissue engineering. Part A Vol.21 No.11

<P>Extracellular matrices (ECM) obtained from in vitro-cultured cells have been given much attention, but its application in cardiac tissue engineering is still limited. This study investigates cardiomyogenic potential of fibroblast-derived matrix (FDM) as a novel ECM platform over gelatin or fibronectin, in generating cardiac cell lineages derived from H9c2 cardiomyoblasts. As characterized through SEM and AFM, FDM exhibits unique surface texture and biomechanical property. Immunofluorescence also found fibronectin, collagen, and laminin in the FDM. Cells on FDM showed a more circular shape and slightly less proliferation in a growth medium. After being cultured in a differentiation medium for 7 days, H9c2 cells on FDM differentiated into cardiomyocytes, as identified by stronger positive markers, such as α-actinin and cTnT, along with more elevated gene expression of Myl2 and Tnnt compared to the cells on gelatin and fibronectin. The gap junction protein connexin 43 was also significantly upregulated for the cells differentiated on FDM. A successive work enabled matrix stiffness tunable; FDM crosslinked by 2wt% genipin increased the stiffness up to 8.5?kPa, 100 times harder than that of natural FDM. The gene expression of integrin subunit α5 was significantly more upregulated on FDM than on crosslinked FDM (X-FDM), whereas no difference was observed for β1 expression. Interestingly, X-FDM showed a much greater effect on the cardiomyoblast differentiation into cardiomyocytes over natural one. This study strongly indicates that FDM can be a favorable ECM microenvironment for cardiomyogenesis of H9c2 and that tunable mechanical compliance induced by crosslinking further provides a valuable insight into the role of matrix stiffness on cardiomyogenesis.</P>