Taufiq Ahmad

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

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(L-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 Youngs 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.

Delivery of a Cell Patch of Cocultured Endothelial Cells and Smooth Muscle Cells Using Thermoresponsive Hydrogels for Enhanced Angiogenesis.

Cell-based therapy has been studied as an attractive strategy for therapeutic angiogenesis. However, obtaining a stable vascular structure remains a challenge due to the poor interaction of transplanted cells with native tissue and the difficulty in selecting the optimal cell source. In this study, we developed a cell patch of cocultured human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) using thermosensitive hydrogels for regeneration of mature vasculatures. In vitro characterization of HUVECs in the cocultured group revealed the formation of a mesh-like morphology over 5 days of culture. Vascular endothelial growth factor expression was also upregulated in the cocultured group compared with HUVECs only. The cell patch seeded with HUVECs, SMCs, or both cell type was prepared on the synthetic thermosensitive and cell interactive hydrogels, and readily detached from the hydrogel within 10 min by expansion of the hydrogel when the temperature was decreased to 4°C. We then investigated the therapeutic effect of the cell patch using a hind limb ischemic model of an athymic mouse. Overall, the group that received a cell patch of cocultured HUVECs and SMCs had a significantly retarded rate of necrosis with a significant increase in the number of arterioles and capillaries for 4 weeks compared with the groups transplanted with only HUVECs or SMCs. Dual staining of smooth muscle alpha actin and human nuclear antigen showed that the implanted cell patch was partially involved in vessel formation. In summary, the simple transplantation of a cocultured cell patch using a hydrogel system could enhance therapeutic angiogenesis through the regeneration of matured vascular structures.

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

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 in vivo tissue regeneration and in vitro high-throughput screening of biomaterials.

Spatially Assembled Bilayer Cell Sheets of Stem Cells and Endothelial Cells Using Thermosensitive Hydrogels for Therapeutic Angiogenesis

Although the coculture of multiple cell types has been widely employed in regenerative medicine, in vivo transplantation of cocultured cells while maintaining the hierarchical structure remains challenging. Here, a spatially assembled bilayer cell sheet of human mesenchymal stem cells and human umbilical vein endothelial cells on a thermally expandable hydrogel containing fibronectin is prepared and its effect on in vitro proangiogenic functions and in vivo ischemic injury is investigated. The expansion of hydrogels in response to a temperature change from 37 to 4 °C allows rapid harvest and delivery of the bilayer cell sheet to two different targets (an in vitro model glass surface and in vivo tissue). The in vitro study confirms that the bilayer sheet significantly increases proangiogenic functions such as the release of nitric oxide and expression of vascular endothelial cell genes. In addition, transplantation of the cell sheet from the hydrogels into a hindlimb ischemia mice model demonstrates significant retardation of necrosis particularly in the group transplated with the bilayer sheet. Collectively, the bilayer cell sheet is readily transferrable from the thermally expandable hydrogel and represents an alternative approach for recovery from ischemic injury, potentially via improved cell-cell communication.

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

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 α2, α5, and β1 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. STATEMENT OF SIGNIFICANCE 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 in vitro assay.

One-step delivery of a functional multi-layered cell sheet using a thermally expandable hydrogel with controlled presentation of cell adhesive proteins

In this study, we developed a new system enabling rapid delivery of a multi-layered cell sheet by combining layer-by-layer (LBL) coating of a cell membrane and surface engineered thermally expandable hydrogel. Human dermal fibroblasts were LBL-coated with fibronectin (FN) and gelatin to form a multi-layered cell sheet in a single seeding step via spontaneous 3D cell-cell interactions. FN was covalently immobilized onto the surface of a Tetronic®-based hydrogel at two different concentrations (1 and 5 μg ml-1) for stable adhesion of the multi-layered cell sheet, followed by polydopamine coating. In both conditions, a multi-layered cell sheet was stably formed. Then, the cell sheet on the hydrogel modified with 1 μg ml-1 FN rapidly detached (>90% efficiency) in response to the expansion of the hydrogel when temperature changed from 37 °C to 4 °C, while the other group had a reduced detachment due to excessive cell-hydrogel interaction. The multi-layered cell sheet was evident in cell-extracellular matrix and cell-cell junction formation, and bFGF was continuously secreted over 7 days of in vitro culture. The multi-layered transplanted to the mouse subcutaneous tissue also exhibited evidence of vascular ingrowth, which collectively suggest that the delivery system maintaining cellular functions is applicable for regenerative medicine.

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

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.

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

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 NaHCO3 (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. In vitro 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 in vitro bone models, in vivo cell carriers, and as building blocks for bioprinting 3D bone tissue. STATEMENT OF SIGNIFICANCE 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 in vitro and mineralized in the growth media. These spheroids were fused to form in vitro 3D mineralized tissue with larger size.

Oxygen-dependent generation of a graded polydopamine coating on nanofibrous materials for controlling stem cell functions

Substrates modified with gradient surface chemistry are of fundamental importance for designing a new bio-interface in biomaterial research and tissue engineering. However, current gradient fabrication strategies are not easily accessible to most laboratories due to complex, expensive, and expertise-requiring procedures. In this study, we generated a gradient of polydopamine (PD) coating on a PLLA nanofiber surface using a spatially restricted supply of oxygen in the reaction solution. Analysis of the oxygen distribution revealed that oxygen availability varied along different reaction solution depths during dopamine polymerization. We then extensively investigated the effects of different parameters, such as tilting angle, reaction time, pH of the reaction solution, and concentration of dopamine, on PD gradient formation, which should be appropriately modulated for PD gradient on nanofibers. Further, culturing of human mesenchymal stem cells (hMSCs) on the PD gradient nanofiber resulted in a gradient of adhesion and spreading from high to low PD coating. However, the proliferation rate was not affected by the PD gradient, with an approximately 3-fold change after 5 days of culture. Maintenance of the stem cell density gradient on the PD gradient nanofiber resulted in controlled osteogenic differentiation, which was greater in the higher PD-coated area. Interestingly, stemness analysis showed a reverse trend relative to osteogenic differentiation of hMSCs. In summary, the spatially controlled polymerization of dopamine can be a versatile tool to generate substrates with gradient surface chemistry, which holds promise to direct stem cell behavior.