Graded functionalization of biomaterial surfaces using mussel-inspired adhesive coating of polydopamine

Perikamana, Sajeesh Kumar Madhurakkat,Shin, Young Min,Lee, Jin Kyu,Lee, Yu Bin,Heo, Yunhoe,Ahmad, Taufiq,Park, So Yeon,Shin, Jisoo,Park, Kyung Min,Jung, Hyun Suk,Cho, Seung-Woo,Shin, Heungsoo Elsevier 2017 Colloids and Surfaces B Vol.159 No.-

<P><B>Abstract</B></P> <P>Biomaterials with graded functionality have various applications in cell and tissue engineering. In this study, by controlling oxidative polymerization of dopamine, we demonstrated universal techniques for generating chemical gradients on various materials with adaptability for secondary molecule immobilization. Diffusion-controlled oxygen supply was successfully exploited for coating of polydopamine (PD) in a gradient manner on different materials, regardless of their surface chemistry, which resulted in gradient in hydrophilicity and surface roughness. The PD gradient controlled graded adhesion and spreading of human mesenchymal stem cells (hMSCs) and endothelial cells. Furthermore, the PD gradient on these surfaces served as a template to allow for graded immobilization of different secondary biomolecules such as cell adhesive arginine-glycine-aspartate (RGD) peptides and siRNA lipidoid nanoparticles (sLNP) complex, for site-specific adhesion of human mesenchymal stem cells, and silencing of green fluorescent protein (GFP) expression on GFP-HeLa cells, respectively. In addition, the same approach was adapted for generation of nanofibers with surface in graded biomineralization under simulated body fluid (SBF). Collectively, oxygen-dependent generation of PD gradient on biomaterial substrates can serve as a simple and versatile platform that can be used for various applications realizing <I>in vivo</I> tissue regeneration and in vitro high-throughput screening of biomaterials.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A method for generating polydopamine (PD) gradient on biomaterial has been reported. </LI> <LI> PD gradient substrates showed gradient in surface chemistry and hydrophilicity. </LI> <LI> PD gradient substrates were able to modulate the spatial distribution of biomolecules. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Harnessing biochemical and structural cues for tenogenic differentiation of adipose derived stem cells (ADSCs) and development of an <i>in vitro</i> tissue interface mimicking tendon-bone insertion graft

Madhurakkat Perikamana, Sajeesh Kumar,Lee, Jinkyu,Ahmad, Taufiq,Kim, Eun Mi,Byun, Hayeon,Lee, Sangmin,Shin, Heungsoo Elsevier 2018 Biomaterials Vol.165 No.-

<P><B>Abstract</B></P> <P>Tendon-bone interface tissue is extremely challenging to engineer because it exhibits complex gradients of structure, composition, biologics, and cellular phenotypes. As a step toward engineering these transitional zones, we initially analyzed how different (topographical or biological) cues affect tenogenic differentiation of adipose-derived stem cells (ADSCs). We immobilized platelet-derived growth factor - BB (PDGF-BB) using polydopamine (PD) chemistry on random and aligned nanofibers and investigated ADSC proliferation and tenogenic differentiation. Immobilized PDGF greatly enhanced the proliferation and tenogenic differentiation of ADSCs; however, nanofiber alignment had no effect. Interestingly, the PDGF immobilized aligned nanofiber group showed a synergistic effect with maximum expression of tenogenic markers for 14 days. We also generated a nanofiber surface with spatially controlled presentation of immobilized PDGF on an aligned architecture, mimicking native tendon tissue. A gradient of immobilized PDGF was able to control the phenotypic differentiation of ADSCs into tenocytes in a spatially controlled manner, as confirmed by analysis of the expression of tenogenic markers and immunofluorescence staining. We further explored the gradient formation strategy by generation of a symmetrical gradient on the nanofiber surface for the generation of a structure mimicking bone-patellar-tendon-bone with provision for gradient immobilization of PDGF and controlled mineralization. Our study reveals that, together with biochemical cues, favorable topographical cues are important for tenogenic differentiation of ADSCs, and gradient presentation of PDGF can be used as a tool for engineering stem cell–based bone-tendon interface tissues.</P>

Effects of Immobilized BMP-2 and Nanofiber Morphology on In Vitro Osteogenic Differentiation of hMSCs and In Vivo Collagen Assembly of Regenerated Bone

Madhurakkat Perikamana, Sajeesh Kumar,Lee, Jinkyu,Ahmad, Taufiq,Jeong, Yonghoon,Kim, Do-Gyoon,Kim, Kyobum,Shin, Heungsoo American Chemical Society 2015 ACS APPLIED MATERIALS & INTERFACES Vol.7 No.16

<P>Engineering bone tissue is particularly challenging because of the distinctive structural features of bone within a complex biochemical environment. In the present study, we fabricated poly(<SMALL>l</SMALL>-lactic acid) (PLLA) electrospun nanofibers with random and aligned morphology immobilized with bone morphogenic protein-2 (BMP-2) and investigated how these signals modulate (1) in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs) and (2) in vivo bone growth rate, mechanical properties, and collagen assembly of newly formed bone. The orientation of adherent cells followed the underlying nanofiber morphology; however, nanofiber alignment did not show any difference in alkaline phosphate activity or in calcium mineralization of hMSCs after 14 days of in vitro culture in osteogenic differentiation media. In vivo bone regeneration was significantly higher in the nanofiber implanted groups (approximately 65–79%) as compared to the defect-only group (11.8 ± 0.2%), while no significant difference in bone regeneration was observed between random and aligned groups. However, nanoindentation studies of regenerated bone revealed Young’s modulus and contact hardness with anisotropic feature for aligned group as compared to random group. More importantly, structural analysis of collagen at de novo bone showed the ability of nanofiber morphology to guide collagen deposition. SEM and TEM images revealed regular, highly ordered collagen assemblies on aligned nanofibers as compared to random fibers, which showed irregular, randomly organized collagen deposition. Taken together, we conclude that nanofibers in the presence of osteoinductive signals are a potent tool for bone regeneration, and nanofiber alignment can be used for engineering bone tissues with structurally assembled collagen fibers with defined direction.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/aamick/2015/aamick.2015.7.issue-16/acsami.5b01340/production/images/medium/am-2015-01340t_0011.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/am5b01340'>ACS Electronic Supporting Info</A></P>

Materials from Mussel-Inspired Chemistry for Cell and Tissue Engineering Applications

Madhurakkat Perikamana, Sajeesh Kumar,Lee, Jinkyu,Lee, Yu Bin,Shin, Young Min,Lee, Esther J.,Mikos, Antonios G.,Shin, Heungsoo American Chemical Society 2015 Biomacromolecules Vol.16 No.9

<P>Current advances in biomaterial fabrication techniques have broadened their application in different realms of biomedical engineering, spanning from drug delivery to tissue engineering. The success of biomaterials depends highly on the ability to modulate cell and tissue responses, including cell adhesion, as well as induction of repair and immune processes. Thus, most recent approaches in the field have concentrated on functionalizing biomaterials with different biomolecules intended to evoke cell- and tissue-specific reactions. Marine mussels produce mussel adhesive proteins (MAPs), which help them strongly attach to different surfaces, even under wet conditions in the ocean. Inspired by mussel adhesiveness, scientists discovered that dopamine undergoes self-polymerization at alkaline conditions. This reaction provides a universal coating for metals, polymers, and ceramics, regardless of their chemical and physical properties. Furthermore, this polymerized layer is enriched with catechol groups that enable immobilization of primary amine or thiol-based biomolecules via a simple dipping process. Herein, this review explores the versatile surface modification techniques that have recently been exploited in tissue engineering and summarizes polydopamine polymerization mechanisms, coating process parameters, and effects on substrate properties. A brief discussion of polydopamine-based reactions in the context of engineering various tissue types, including bone, blood vessels, cartilage, nerves, and muscle, is also provided.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bomaf6/2015/bomaf6.2015.16.issue-9/acs.biomac.5b00852/production/images/medium/bm-2015-00852b_0006.gif'></P>

Controlled Retention of BMP-2-Derived Peptide on Nanofibers Based on Mussel-Inspired Adhesion for Bone Formation

Lee, Jinkyu,Perikamana, Sajeesh Kumar Madhurakkat,Ahmad, Taufiq,Lee, Min Suk,Yang, Hee Seok,Kim, Do-Gyoon,Kim, Kyobum,Kwon, Bosun,Shin, Heungsoo Mary Ann Liebert 2017 Tissue engineering. Part A Vol.23 No.7

<P>Although bone morphogenetic protein-2 (BMP-2) has been frequently used to stimulate bone formation, it has several side effects to be addressed, including the difficulty in optimization of clinically relevant doses and unwanted induction of cancerous signaling processes. In this study, an osteogenic peptide (OP) derived from BMP-2 was investigated as a substitute for BMP-2. In vitro studies showed that OP was able to enhance the osteogenic differentiation and mineralization of human mesenchymal stem cells (hMSCs). The peptides were then conjugated onto biocompatible poly--lactide electrospun nanofibers through polydopamine chemistry. Surface chemical analysis proved that more than 80% of the peptides were stably retained on the nanofiber surface after 8h of polydopamine coating during at least 28 days, and the amount of peptides that was retained increased depending on the polydopamine coating time. For instance, about 65% of the peptides were retained on nanofibers after 4h of polydopamine coating. Also, a relatively small dose of peptides could effectively induce bone formation in in vivo critical-sized defects on the calvarial bones of mice. More than 50.4%+/- 16.9% of newly formed bone was filled within the defect after treatment with only 10.5 +/- 0.6 mu g of peptides. Moreover, these groups had similar elastic moduli and contact hardnesses with host bone. Taken together, our results suggest that polydopamine-mediated OP immobilized on nanofibers can modulate the retention of relatively short lengths of peptides, which might make this an effective therapeutic remedy to guide bone regeneration using a relatively small amount of peptides.</P>

Effective Immobilization of BMP-2 Mediated by Polydopamine Coating on Biodegradable Nanofibers for Enhanced in Vivo Bone Formation

Cho, Hyeong-jin,Madhurakkat Perikamana, Sajeesh Kumar,Lee, Ji-hye,Lee, Jinkyu,Lee, Kyung-Mi,Shin, Choongsoo S.,Shin, Heungsoo American Chemical Society 2014 ACS APPLIED MATERIALS & INTERFACES Vol.6 No.14

<P>Although bone morphogenic proteins (BMPs) have been widely used for bone regeneration, the ideal delivery system with optimized dose and minimized side effects is still active area of research. In this study, we developed bone morphogenetic protein-2(BMP-2) immobilized poly(<SMALL>l</SMALL>-lactide) (PLLA) nanofibers inspired by polydopamine, which could be ultimately used as membranes for guided bone regeneration, and investigated their effect on guidance of in vitro cell behavior and in vivo bone formation. Surface chemical analysis of the nanofibers confirmed successful immobilization of BMP-2 mediated by polydopamine, and about 90% of BMP-2 was stably retained on the nanofiber surface for at least 28 days. The alkaline phosphatase activity and calcium mineralization of human mesenchymal stem cells (hMSCs) after 14 days of in vitro culture was significantly enhanced on nanofibers immobilized with BMP-2. More importantly, BMP-2 at a relatively small dose was highly active following implantation to the critical-sized defect in the cranium of mice; radiographic analysis demonstrated that 77.8 ± 11.7% of newly formed bone was filled within the defect for a BMP-2-immobilized groups at the concentration of 124 ± 9 ng/cm<SUP>2</SUP>, as compared to 5.9 ± 1.0 and 34.1 ± 5.5% recovery, for a defect-only and a polydopamine-only group, respectively. Scanning and transmission electron microscopy of samples from the BMP-2 immobilized group showed fibroblasts and osteoblasts with nanofiber strands in the middle of regenerated bone tissue, revealing the importance of interaction between implanted nanofibers and the neighboring extracellular environment. Taken together, our data support that the presentation of BMP-2 on the surface of nanofibers as immobilized by utilizing polydopamine chemistry may be an effective method to direct bone growth at relatively low local concentration.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/aamick/2014/aamick.2014.6.issue-14/am501391z/production/images/medium/am-2014-01391z_0011.gif'></P>

Stem cell spheroids incorporating fibers coated with adenosine and polydopamine as a modular building blocks for bone tissue engineering

Ahmad, Taufiq,Byun, Hayeon,Lee, Jinkyu,Madhurakat Perikamana, Sajeesh Kumar,Shin, Young Min,Kim, Eun Mi,Shin, Heungsoo Elsevier 2020 Biomaterials Vol.230 No.-

<P><B>Abstract</B></P> <P>Although stem cell spheroids offer great potential as functional building blocks for bottom-up bone tissue engineering, delivery of bioactive signals remain challenging. Here, we engineered adenosine-ligand-modified fiber fragments to create a 3D cell-instructive microenvironment for bone. Briefly, the Poly(ι-lactic acid) (PLLA) nanofiber sheet was partially degraded into fragmented fibers (FFs) through aminolysis and adenosine was stably incorporated via one-step polydopamine coating. The SEM and XPS analysis demonstrated that polydopamine assisted adenosine coating efficiency was significantly increased, which led to high coating efficiency of adenosine and its significant retention. The engineered fibers were then assembled into stable spheroids with human-adipose-derived stem cells (hADSCs). The adenosine in the spheroids effectively stimulated A2bR (1.768 ± 0.08) signaling, which further significantly induced the expression of osteogenic markers such as Runx2 (3.216 ± 0.25), OPN (4.136 ± 0.14), OCN (10.16 ± 0.34), and OSX (2.27 ± 0.11) with improved mineral deposition (1.375 ± 0.05 μg per spheroid). In contrast, the adipogenic differentiation of hADSCs was significantly suppressed within the engineered spheroids. Transplantation of engineered spheroids strongly induced osteogenic differentiation of hADSCs in ectopic subcutaneous tissue. Finally, the bone regeneration was significantly enhanced by implanting AP-FF group (59.97 ± 18.33%) as compared to P-FF (27.96 ± 11.14) and defect only (7.97 ± 3.76%). We propose that stem cell spheroids impregnated with engineered fibers enabling adenosine delivery could be promising building blocks for a bottom-up approach to create large tissues for regeneration of damaged bone.</P>

Agglomeration of human dermal fibroblasts with ECM mimicking nano-fragments and their effects on proliferation and cell/ECM interactions

Taufiq Ahmad,신영민,이진규,신혁준,Sajeesh Kumar Madhurakart Perikamana,신흥수 한국공업화학회 2018 Journal of Industrial and Engineering Chemistry Vol.67 No.-

Here, we engineered spheroids by using ECM mimicking nano-fragments (NFs) with fibroblasts and investigated their effect on proliferation and cell/ECM interactions. NF incorporation resulted in formation of a stable spheroid, which improved proliferation and viability of cells by assisting oxygen transport confirmed by LOX-1 staining. In addition, hypoxic and apoptotic genes were significantly downregulated in spheroids with PD-NFs. Furthermore, ECM and cell junction proteins were highly expressed. Overall, our findings suggest that incorporation of NFs within spheroids for assembly with various cell types can be an alternative approach for 3D cell culture in many applications.

Fabrication of <i>in vitro</i> 3D mineralized tissue by fusion of composite spheroids incorporating biomineral-coated nanofibers and human adipose-derived stem cells

Ahmad, Taufiq,Shin, Hyeok Jun,Lee, Jinkyu,Shin, Young Min,Perikamana, Sajeesh Kumar Madhurakat,Park, So Yeon,Jung, Hyun Suk,Shin, Heungsoo Elsevier 2018 ACTA BIOMATERIALIA Vol.74 No.-

<P><B>Abstract</B></P> <P>Development of a bone-like 3D microenvironment with stem cells has always been intriguing in bone tissue engineering. In this study, we fabricated composite spheroids by combining functionalized fibers and human adipose-derived stem cells (hADSCs), which were fused to form a 3D mineralized tissue construct. We prepared fragmented poly (ι-lactic acid) (PLLA) fibers approximately 100 μm long by partial aminolysis of electrospun fibrous mesh. PLLA fibers were then biomineralized with various concentrations of NaHCO<SUB>3</SUB> (0.005, 0.01, and 0.04 M) to form mineralized fragmented fibers (mFF1, mFF2, and mFF3, respectively). SEM analysis showed that the minerals in mFF2 and mFF3 completely covered the fiber surface, and surface chemistry analysis confirmed the presence of hydroxyapatite peaks. Additionally, mFFs formed composite spheroids with hADSCs, demonstrating that the cells were strongly attached to mFFs and homogeneously distributed throughout the spheroid. <I>In vitro</I> culture of spheroids in the media without osteogenic supplements showed significantly enhanced expression of osteogenic genes including Runx2 (20.83 ± 2.83 and 22.36 ± 2.18 fold increase), OPN (14.24 ± 1.71 and 15.076 ± 1.38 fold increase), and OCN (4.36 ± 0.41 and 5.63 ± 0.51 fold increase) in mFF2 and mFF3, respectively, compared to the no mineral fiber group. In addition, mineral contents were significantly increased at day 7. Blocking the biomineral-mediated signaling by PSB 603 significantly down regulated the expression of these genes in mFF3 at day 7. Finally, we fused composite spheroids to form a mineralized 3D tissue construct, which maintained the viability of cells and showed pervasively distributed minerals within the structure. Our composite spheroids could be used as an alternative platform for the development of <I>in vitro</I> bone models, <I>in vivo</I> cell carriers, and as building blocks for bioprinting 3D bone tissue.</P> <P><B>Statement of Significance</B></P> <P>This manuscript described our recent work for the preparation of biomimeral-coated fibers that can be assembled with mesenchymal stem cells and provide bone-like environment for directed control over osteogenic differentiation. Biomineral coating onto synthetic, biodegradable single fibers was successfully carried out using multiple steps, combination of template protein coating inspired from mussel adhesion and charge-charge interactions between template proteins and mineral ions. The biomineral-coated single micro-scale fibers (1–2.5 μm in diameter) were then assembled with human adipose tissue derived stem cells (hADSCs). The assembled structure exhibited spheroidal architecture with few hundred micrometers. hADSCs within the spheroids were differentiated into osteogenic lineage <I>in vitro</I> and mineralized in the growth media. These spheroids were fused to form <I>in vitro</I> 3D mineralized tissue with larger size.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Hybrid-spheroids incorporating ECM like engineered fragmented fibers potentiate stem cell function by improved cell/cell and cell/ECM interactions

Ahmad, Taufiq,Lee, Jinkyu,Shin, Young Min,Shin, Hyeok Jun,Madhurakat Perikamana, Sajeesh Kumar,Park, Sun Hwa,Kim, Sung Won,Shin, Heungsoo Elsevier 2017 ACTA BIOMATERIALIA Vol.64 No.-

<P><B>Abstract</B></P> <P>Extracellular matrix (ECM) microenvironment is critical for the viability, stemness, and differentiation of stem cells. In this study, we developed hybrid-spheroids of human turbinate mesenchymal stem cells (hTMSCs) by using extracellular matrix (ECM) mimicking fragmented fibers (FFs) for improvement of the viability and functions of hTMSCs. We prepared FFs with average size of 68.26 µm by partial aminolysis of poly L-lactide (PLLA) fibrous sheet (FS), which was coated with polydopamine for improved cell adhesion. The proliferation of hTMSCs within the hybrid-spheroids mixed with fragmented fibers was significantly increased as compared to that from the cell-only group. Cells and fragmented fibers were homogenously distributed with the presence of pore like empty spaces in the structure. LOX-1 staining revealed that the hybrid-spheroids improved the cell viability, which was potentially due to enhanced transport of oxygen through void space generated by engineered ECM. Transmission electron microscopy (TEM) analysis confirmed that cells within the hybrid-spheroid formed strong cell junctions and contacts with fragmented fibers. The expression of cell junction proteins including connexin 43 and E-cadherin was significantly upregulated in hybrid-spheroids by 16.53 ± 0.04 and 28.26 ± 0.11-fold greater than that from cell-only group. Similarly, expression of integrin α<SUB>2,</SUB> α<SUB>5</SUB>, and β<SUB>1</SUB> was significantly enhanced at the same group by 25.72 ± 0.13, 27.48 ± 0.49, and 592.78 ± 0.06-fold, respectively. In addition, stemness markers including Oct-4, Nanog, and Sox2 were significantly upregulated in hybrid-spheroids by 96.56 ± 0.06, 158.95 ± 0.06, and 115.46 ± 0.47-fold, respectively, relative to the cell-only group. Additionally, hTMSCs within the hybrid-spheroids showed significantly greater osteogenic differentiation under osteogenic media conditions. Taken together, our hybrid-spheroids can be an ideal approach for stem cell expansion and serve as a potential carrier for bone regeneration.</P> <P><B>Statement of Significance</B></P> <P>Cells are spatially arranged within extracellular matrix (ECM) and cell/ECM interactions are crucial for cellular functions. Here, we developed a hybrid-spheroid system incorporating engineered ECM prepared from fragmented electrospun fibers to tune stem cell functions. Conventionally prepared cell spheroids with large diameters (>200 µm) is often prone to hypoxia. In contrast, the hybrid-spheroids significantly enhanced viability and proliferation of human turbinate mesenchymal stem cells (hTMSCs) as compared to spheroid prepared from cell only. Under these conditions, the presence of fragmented fibers also improved maintenance of stemness of hTMSCs for longer time cultured in growth media and demonstrated significantly greater osteogenic differentiation under osteogenic media conditions. Thus, the hybrid-spheroids can be used as a delivery carrier for stem cell based therapy or a 3D culture model for <I>in vitro</I> assay.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>