Minh Khanh Nguyen

Sustained localized presentation of RNA interfering molecules from in situ forming hydrogels to guide stem cell osteogenic differentiation.

To date, RNA interfering molecules have been used to differentiate stem cells on two-dimensional (2D) substrates that do not mimic three-dimensional (3D) microenvironments in the body. Here, in situ forming poly(ethylene glycol) (PEG) hydrogels were engineered for controlled, localized and sustained delivery of RNA interfering molecules to differentiate stem cells encapsulated within the 3D polymer network. RNA interfering molecules were released from the hydrogels in a sustained and controlled manner over the course of 3-6 weeks, and exhibited high bioactivity. Importantly, it was demonstrated that the delivery of siRNA and/or miRNA from the hydrogel constructs enhanced the osteogenic differentiation of encapsulated stem cells. Prolonged delivery of siRNA and/or miRNA from this polymeric scaffold permitted extended regulation of cell behavior, unlike traditional siRNA experiments performed in vitro. This approach presents a powerful new methodology for controlling cell fate, and is promising for multiple applications in tissue engineering and regenerative medicine.

Photocleavable Hydrogels for Light-Triggered siRNA Release.

A photocleavable hydrogel system for on-demand delivery of genetic material is reported. The release of short interfering RNAs can be triggered by the application of UV light without any loss in bioactivity. This approach provides a promising external stimulus-based nucleic acid delivery platform for applications in disease therapeutics and tissue regeneration.

Spatial Control of Cell Gene Expression by siRNA Gradients in Biodegradable Hydrogels

The extracellular environment exposes cells to numerous biochemical and physical signals that regulate their behavior. Strategies for generating continuous gradients of signals in biomaterials may allow for spatial control and patterning of cell behavior, and ultimately aid in the engineering of complex tissues. Short interfering RNA (siRNA) can regulate gene expression by silencing specific mRNA molecules post-transcriptionally, which may be valuable when presented in a continuous gradient for regenerative or therapeutic applications. Here, a biodegradable hydrogel system containing a gradient of siRNA is presented, and its capacity to regulate protein expression of encapsulated cells in a spatially continuous manner is demonstrated. Photocross-linkable dextran hydrogels containing a gradient of siRNA have been successfully fabricated using a dual-programmable syringe pump system, and differential gene silencing in incorporated cells that is sustained over time has been shown using green fluorescent protein as a reporter. This platform technology may be applied in tissue engineering to spatially control biologically relevant cellular processes.

Endochondral Ossification in Critical‐Sized Bone Defects via Readily Implantable Scaffold‐Free Stem Cell Constructs

The growing socioeconomic burden of musculoskeletal injuries and limitations of current therapies have motivated tissue engineering approaches to generate functional tissues to aid in defect healing. A readily implantable scaffold‐free system comprised of human bone marrow‐derived mesenchymal stem cells embedded with bioactive microparticles capable of controlled delivery of transforming growth factor‐beta 1 (TGF‐β1) and bone morphogenetic protein‐2 (BMP‐2) was engineered to guide endochondral bone formation. The microparticles were formulated to release TGF‐β1 early to induce cartilage formation and BMP‐2 in a more sustained manner to promote remodeling into bone. Cell constructs containing microparticles, empty or loaded with one or both growth factors, were implanted into rat critical‐sized calvarial defects. Micro‐computed tomography and histological analyses after 4 weeks showed that microparticle‐incorporated constructs with or without growth factor promoted greater bone formation compared to sham controls, with the greatest degree of healing with bony bridging resulting from constructs loaded with BMP‐2 and TGF‐β1. Importantly, bone volume fraction increased significantly from 4 to 8 weeks in defects treated with both growth factors. Immunohistochemistry revealed the presence of types I, II, and X collagen, suggesting defect healing via endochondral ossification in all experimental groups. The presence of vascularized red bone marrow provided strong evidence for the ability of these constructs to stimulate angiogenesis. This system has great translational potential as a readily implantable combination therapy that can initiate and accelerate endochondral ossification in vivo. Importantly, construct implantation does not require prior lengthy in vitro culture for chondrogenic cell priming with growth factors that is necessary for current scaffold‐free combination therapies. Stem Cells Translational Medicine 2017;6:1644–1659

Photocrosslinkable, biodegradable hydrogels with controlled cell adhesivity for prolonged siRNA delivery to hMSCs to enhance their osteogenic differentiation

Photocrosslinked, biodegradable hydrogels have been extensively investigated for biomedical applications, including drug delivery and tissue engineering. Here, dextran (DEX) was chemically modified with mono(2-acryloyloxyethyl) succinate (MAES) via an esterification reaction, resulting in macromers that could be photocrosslinked to form hydrolytically degradable hydrogels. Hydrogel swelling ratio and degradation rate were controlled by varying the degree of MAES modification. Thiolated cell adhesion peptides (GRGDSPC) were conjugated to acrylated dextran via thiol-acrylate reaction to regulate the interactions of human mesenchymal stem cells (hMSCs) with the photocrosslinkable hydrogels. The hydrogels permitted sustained release of short interfering RNA (siRNA) over 7 weeks and were cytocompatible with hMSCs. Sustained presentation of siRNA from these photocrosslinked DEX hydrogels enhanced the osteogenic differentiation of encapsulated hMSCs. These DEX hydrogels with tunable siRNA delivery and cell adhesive properties may provide an excellent platform for bioactive molecule delivery and tissue regeneration applications.

RNA interfering molecule delivery from in situ forming biodegradable hydrogels for enhancement of bone formation in rat calvarial bone defects

RNA interference (RNAi) may be an effective and valuable tool for promoting the growth of functional tissue, as short interfering RNA (siRNA) and microRNA (miRNA) can block the expression of genes that have negative effects on tissue regeneration. Our group has recently reported that the localized and sustained presentation of siRNA against noggin (siNoggin) and miRNA-20a from in situ forming poly(ethylene glycol) (PEG) hydrogels enhanced osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hMSCs). Here, the capacity of the hydrogel system to accelerate bone formation in a rat calvarial bone defect model is presented. After 12 weeks post-implantation, the hydrogels containing encapsulated hMSCs and miRNA-20a resulted in more bone formation in the defects than the hydrogels containing hMSCs without siRNA or with negative control siRNA. This localized and sustained RNA interfering molecule delivery system may provide an excellent platform for healing bony defects and other tissues. STATEMENT OF SIGNIFICANCE Delivery of RNAi molecules may be a valuable strategy to guide cell behavior for tissue engineering applications, but to date there have been no reports of a biomaterial system capable of both encapsulation of cells and controlled delivery of incorporated RNA. Here, we present PEG hydrogels that form in situ via Michael type reaction, and that permit encapsulation of hMSCs and the concomitant controlled delivery of siNoggin and/or miRNA-20a. These RNAs were chosen to suppress noggin, a BMP-2 antagonist, and/or PPAR-γ, a negative regulator of BMP-2-mediated osteogenesis, and therefore promote osteogenic differentiation of hMSCs and subsequent bone repair in critical-sized rat calvarial defects. Simultaneous delivery of hMSCs and miRNA-20a enhanced repair of these defects compared to hydrogels containing hMSCs without siRNA or with negative control siRNA. This in situ forming PEG hydrogel system offers an exciting platform for healing critical-sized bone defects by localized, controlled delivery of RNAi molecules to encapsulated hMSCs and surrounding cells.

Corrigendum to “Sustained localized presentation of RNA interfering molecules from in situ forming hydrogels to guide stem cell osteogenic differentiation” [Biomaterials 35/24 (2014) 6278–6286]

Corrigendum to “Sustained localized presentation of RNA interfering molecules from in situ forming hydrogels to guide stem cell osteogenic differentiation” [Biomaterials 35/24 (2014) 6278e6286] Minh K. Nguyen , Oju Jeon , Melissa D. Krebs , Daniel Schapira , Eben Alsberg a, b, c, * a Department of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44016, USA b Department of Orthopaedic Surgery, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44016, USA c National Center for Regenerative Medicine, Division of General Medical Sciences, Case Western Reserve University, Cleveland, OH, 44106, USA