Molecularly Tailored Interface for Long-Term Xenogeneic Cell Transplantation.

Encapsulation of therapeutic cells in a semipermeable device can mitigate the need for systemic immune suppression following cell transplantation by providing local immunoprotection while being permeable to nutrients, oxygen, and different cell-secreted biomolecules. However, fibrotic tissue deposition around the device has been shown to compromise the long-term function of the transplanted cells. Herein, a macroencapsulation device design that improves long-term survival and function of the transplanted cells is reported. The device is comprised of a semipermeable chitosan pouch with a tunable reservoir and molecularly engineered interface. The chitosan pouch interface decorated with 1,12-dodecanedioic acid (DDA), limits the cell adhesion and vigorous foreign body response while maintaining the barrier properties amenable to cell encapsulation. The device provides long-term protection to the encapsulated human primary hepatocytes in the subcutaneous space of immunocompetent mice. The device supports the encapsulated cells for up to 6 months as evident from cell viability and presence of human specific albumin in circulation. Solutions that integrate biomaterials and interfacial engineering such as the one described here may advance development of easy-to manufacture and retrievable devices for the transplantation of therapeutic cells in the absence of immunosuppression.

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.

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

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-ι-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 8 h 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 4 h 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 µg 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.

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 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.

Effective immobilization of BMP-2 mediated by polydopamine coating on biodegradable nanofibers for enhanced in vivo bone formation.

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(l-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(2), 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.