RNAi functionalized scaffold for scarless skin regeneration

Abstract

Combination of a 3-D scaffold with the emerging RNA interference (RNAi) technique represents the latest paradigm of regenerative medicine. In our recent paper “RNAi functionalized collagen-chitosan/silicone membrane bilayer dermal equivalent for full-thickness skin regeneration with inhibited scarring” in the journal Biomaterials, we not only demonstrated a 3-D system for siRNA sustained delivery, but also presented a comprehensive in vivo study by targeting a vital problem in skin regeneration: scarring. It is expected that further development of this kind of RNAi functionalized scaffold can provide a better platform for directing cell fates by integrating the “down-regulating” biomolecular cues into the cellular microenvironment, leading to the complete functional regeneration of skin.

Keywords: :

• RNA interference
• scaffold
• skin
• scarring
• regenerative medicine

This article refers to:

Introduction

As an important aspect of regenerative medicine, tissue engineering has been brought ever closer to achieve its potential as life-saving and life-improving option for patients since Langer and Vacanti pioneered this strategy by culturing cells within a scaffold in the late 1980s.^1-3 As a general principle of tissue engineering, the scaffold, acting as an artificial extracellular matrix (ECM), should present well-designed multiple chemical, physical and biological cues to build a suitable cellular microenvironment to achieve proper tissue function and regeneration.^4,5 The spatial and temporal delivery of bioactive molecules (including low molecular weight drugs, peptides, growth factors, cytokines and mediators for gene therapy) mediated by the scaffold has been the subject of intensive researches.⁵ Compared with the instability and high cost of cell growth factors,⁶ the gene-activated matrix (GAM), which is prepared by loading of functional plasmid DNAs into the scaffolds, can locally transfect cells and produce the required cell growth factors at the wound site. It is exciting that another epoch-making gene therapy tool, i.e., RNA interference (RNAi) was discovered a decade ago, by which the expression of targeted genes can be downregulated through a potent endogenous pathway.⁷ Intuitively, the “down-regulating” RNAi functionalized scaffold has been considered to complement the “up-regulating” GAM since then. Although progress has been made toward developing GAM in regenerative medicine applications, the advent of RNAi functionalized scaffold is relatively recent.⁷

The acute skin injuries, burns as well as chronic wounds, present a worldwide growing health and economic burden.^8,9 Based on the principle of tissue engineering and regenerative medicine, bioengineered skin substitutes or dermal equivalents offer a fascinating therapeutic option for the treatment of skin loss.¹⁰ Although many significant milestones of bioengineered skins have been applied, challenges still remain to fulfill the criteria of “regenerated skin” with complete structural, aesthetic and functional properties of nature skin. The presence of fibrotic tissue in the repaired skin is a significant concern because the scarring indicates disfiguration and more importantly inferior functional quality (e.g., loss of sweat glands and hair follicles).¹¹ Therefore, anti-scarring technologies should be incorporated into the new-generation of bioengineered skin constructs.

Inspired by the mechanism of scar-free healing in embryonic wounds, the interruption of transforming growth factor (TGF)-β1 pathway may offer a solution to inhibiting scarring or even inducing scar-free regeneration of adult skin wounds.¹² With the advantage of high efficiency, specificity and accuracy over other “down-regulating” methods such as antibody neutralization and receptor blockage, RNAi is an attractive approach to inhibit TGF-β1.⁷ Therefore, the scarless regeneration of skin is an attractive target to verify the novel concept of regenerative medicine, i.e., RNA functionalized scaffold. Based on the importance of both RNAi functionalized scaffold and anti-scarring skin substitute, in our recent work published in the journal Biomaterials,¹³ we reported a collagen-chitosan/silicone membrane bilayer dermal equivalent (BDE) loaded with trimethylchitosan (TMC)/siRNA complexes targeting TGF-β1, aiming at interfering TGF-β1 signal pathway, directing cell fates and ultimately inhibiting scarring.

A 3-D Scaffolding System for Sustained siRNA Delivery

To achieve better protection and enhanced cellular uptake of siRNA, we took the advantage of using trimetylchitosan (TMC) as a siRNA vector to form nano-sized complexes. At an optimal N/P ratio (represents the molar ratio of nitrogen of TMC to phosphate of siRNA) 20, the TMC/siRNA complexes show suitable physicochemical properties for extracellular delivery. By 2-D culture of dermal fibroblasts, the applicability of the complexes to silence TGF-β1 is proved. We afterwards fabricated the RNAi-BDE by loading the TMC/siRNA complexes into porous collagen/chitosan scaffold and investigated its properties as reservoir for the sustained release of the incorporated complexes. We believe the initial burst release of siRNA can be attributed to the loosely physically adsorpted complexes and the remained complexes are retained dominantly due to tighter binding between TMC and scaffold component. These findings suggest that TMC plays an important role in both better protection and prolonged release of siRNA. This point is quite important because it embodies a prominent advantage of RNAi functionalized scaffolds, i.e., localized treatment and maintenance of effective amount of bioactive cues to match the possible long timescale of tissue regeneration.⁷ To access the biological properties of the RNAi-BDE at the in vitro level, a 3-D static culture model is applied. The RNAi-BDE demonstrates good cytocompatibility for fibroblast, which is the most predominant cell type involved in wound healing and produces much of TGF-β1. By comparing with the naked siRNA, the successful cellular internalization of TMC/siRNA complexes confirms again the vital role of TMC. We further evaluated the gene silencing efficiency of the RNAi-BDE by the same in vitro model. A sustained inhibition of TGF-β1 and Col I mRNA expression within 14 d was observed, which proves again that the BDE acts as a reservoir for the TMC/siRNA complexes. We assume that along with the cell infiltration and scaffold degradation the retained TMC/siRNA complexes gradually depart from the scaffold and enter the cells.

A Dermal Equivalent for Scarless Skin Regeneration

Since the RNAi-BDE performs well in vitro, we further applied it to full-thickness skin defects on the back of pigs, and performed in vivo research at two levels. The first level studies the expression of TGF-β1 and related factors. The encouraging results from both qualitative and quantitative evaluations within the timescale of 30 d confirm the in vivo gene inhibition effect of the scaffold-based siRNA delivery system, i.e., RNAi-BDE. This phenomenon may be caused by two reasons: (1) the reservoir function of the BDE for the TMC/siRNA complexes and (2) the interruption of “auto-induction” effect of TGF-β1. Based on these findings, we assume that the implanted RNAi-BDE starts to work in the early stage of wound healing due to the “burst-released” siRNA from the scaffold. When fibroblasts and other types of cells further invade into and degrade the scaffold, more TMC/siRNA complexes are exposed to and internalized by cells, resulting in further inhibition of ECM over-accumulation and myofibroblast differentiation. The second level investigates the scar inhibition as a result of TGF-β1 alteration. We performed ultra-thin skin grafting on the wounds at day 14 post-implantation and assessed the wounds treated by RNAi-BDE for 73 d. Excitingly, the regenerated skin shows a structure extremely similar to that of normal skin with significant scar inhibition, where collagen bundles distribute in multiple planes with a complex architecture of 3-D woven reticular array.

Conclusion Remarks and Perspectives

The importance and novel feature of this study lies in the following two aspects: (1) It reflects the latest approach of tissue engineering and regenerative medicine to integrate the powerful and promising tool of RNAi. Because RNAi has been studied only in the past years, the number of reports on 3D-scaffold-mediated RNAi is very small. The molecular properties and action mechanism of siRNA differ from those of plasmid DNA, so it is quite possible that the designs for RNAi functionalized scaffold and GAM have different developmental concerns. Although the “up-regulating” GAM has been extensively studied,^14-16 more researches on the “down-regulated” RNAi functionalized scaffold are needed. For sure these advances in matrices and scaffolds with genetic cues will have important implications for a new generation of biomaterials for tissue regeneration and regenerative medicine. (2) It reports the use and analysis of RNAi-BDE in a pig model, providing a potential solution to achieving scarless skin regeneration mediated by a scaffold. With a systematic and comprehensive analysis from in vitro to in vivo level, it provides a solution to the vital problem of bioengineered skin. It may also inspire a new category of RNAi functionalized biomaterials for other tissues and organs.

Indeed this article expands our previous work and reflects our years of research on biomaterials for skin regeneration.^13,17-28 In the context of scaffold-mediated gene therapy and GAM, we have also developed gene-activated BDE with a plasmid encoding vascular endothelial growth factor (VEGF) to enhance angiogenesis in both full-thicknesses incisional wounds and burns.^19,26 Delayed or poor angiogenesis of reconstructed skin will fail to nourish the overlaying epidermal layer.²⁹ This would result in the loss of graft and hamper the timely healing of damaged skin and improve the risk of death.²⁹ Therefore, acceleration of the angiogenesis rate to achieve rapid formation of new blood capillaries is urgently required and “life-saving.” Scarring is also quite important and is a “life-improving” issue related to bioengineered skin. The scar-free regeneration should have features including complete restoration of skin structure and function, which reflect a focus of interest for the emerging fields of regenerative medicine. The key to the materials-based skin regeneration is to build a suitable environment, where cells are exposed to a complex pattern of biosignal molecules that direct cell behaviors and guide tissue regeneration. We believe that this currently discussed paper represents such a paradigm. Further investigations are encouraged to achieve “complete-regenerated” skin with normal appendages (hair follicles, sweat glands and sebaceous glands) and the functions of touch, temperature sensation, excretion, perspiration and thermoregulation.

With the ability to differentiate into varied somatic cells under appropriate micro-environmental cues, the emerging stem cell technology has greatly promoted the development of regenerative medicine.³⁰ By both pre-loading stem cells or recruiting them in situ, the biomimic scaffold (or preferably self-adaptive biomaterials) with desired compositions, microstructures and bio-signals can be well-defined to induce the differentiation of stem cells and may finally regenerate a neo-skin with complete structure and functions. It is expected that RNAi functionalized scaffold will play a crucial role in modulating stem cell fate, leading to significant progress in bioengineered skin for complete regeneration, and will also provide a tremendous potential for both basic research and clinical therapies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.