Decellularized extracellular matrix scaffolds: Recent trends and emerging strategies in tissue engineering

Advancing application of mesenchymal stem cell-based bone tissue regeneration

Functional Biomaterials for Bone Regeneration: A Lesson in Complex Biology

Globally, more than 1.5 million patients undergo bone graft surgeries annually, and the development of biomaterial scaffolds that mimic natural bone for bone grafting remains a tremendous challenge. In recent decades, due to the improved understanding of the mechanisms of bone remodeling and the rapid development of gene therapy, RNA (including messenger RNA (mRNA), microRNA (miRNA), and short interfering RNA (siRNA)) has attracted increased attention as a new tool for bone tissue engineering due to its unique nature and great potential to cure bone defects. Different types of RNA play roles via a variety of mechanisms in bone-related cells in vivo as well as after synthesis in vitro. In addition, RNAs are delivered to injured sites by loading into scaffolds or systemic administration after combination with vectors for bone tissue engineering. However, the challenge of effectively and stably delivering RNA into local tissue remains to be solved. This review describes the mechanisms of the three types of RNAs and the application of the relevant types of RNA delivery vectors and scaffolds in bone regeneration. The improvements in their development are also discussed.

RNA-based scaffolds for bone regeneration: application and mechanisms of mRNA, miRNA and siRNA.

Increasing evidence has indicated that bone morphogenetic protein 2 (BMP2) coordinates with microRNAs (miRNAs) to form intracellular networks regulating mesenchymal stem cells (MSCs) osteogenesis. This study aimed to identify specific miRNAs in rat adipose-derived mesenchymal stem cells (ADSCs) during BMP2-induced osteogenesis, we selected the most significantly down-regulated miRNA, miR-146a, to systematically investigate its role in regulating osteogenesis and bone regeneration. Overexpressing miR-146a notably repressed ADSC osteogenesis, whereas knocking down miR-146a greatly promoted this process. Drosophila mothers against decapentaplegic protein 4 (SMAD4), an important co-activator in the BMP signaling pathway, was miR-146a's direct target and miR-146a exerted its repressive effect on SMAD4 through interacting with 3'-untranslated region (3'-UTR) of SMAD4 mRNA. Furthermore, knocking down SMAD4 attenuated the ability of miR-146a inhibitor to promote ADSC osteogenesis. Next, transduced ADSCs were incorporated with poly(sebacoyl diglyceride) (PSeD) porous scaffolds for repairing critical-sized cranial defect, the treatment of miR-146a inhibitor greatly enhanced ADSC-mediated bone regeneration with higher expression levels of SMAD4, Runt-related transcription factor 2 (Runx2) and Osterix in newly formed bone. In summary, our study showed that miR-146a negatively regulates the osteogenesis and bone regeneration from ADSCs both in vitro and in vivo.

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Effects of miR-146a on the osteogenesis of adipose-derived mesenchymal stem cells and bone regeneration

Tissue engineering technology that adopts mesenchymal stem cells combined with scaffolds presents a promising strategy for tissue regeneration. Human adipose-derived stem cells (hADSCs) have attracted considerable attention in bone engineering for their osteogenic potential. The extracellular matrix (ECM) is critical for the stem cell niche as a physical support and is known to be able to maintain stem cell properties. In this study, the ECM derived from ADSCs was produced and termed the ADM. The ADM was demonstrated to markedly promote proliferation of bone marrow derived stem cells (BMSCs) and exhibited strongly osteogenic simulative effects in vitro. The results showed that alkaline phosphatase (ALP) activity, Alizarin red S (ARS) staining, osteogenic gene markers and proteins were significantly up-regulated. Next, we developed a poly(sebacoyl diglyceride) (PSeD) mesh scaffold coated with the ADM and evaluated its capacity to create an osteogenic microenvironment. BMSCs were cultured on the composite scaffolds and subjected to osteogenic differentiation in vitro. The results showed that the composite scaffolds facilitated the osteogenesis more than a simple PSeD scaffold. Then the PSeD/ADM scaffold seeded with BMSCs was used to repair critical-sized calvarial defects in rats, which significantly enhanced the reparative effects as confirmed via micro-CT, sequential fluorescent labeling and histological observation. In conclusion, we demonstrated that the ADM could promote both proliferation and osteogenesis of BMSCs, and the combination of ADM and PSeD synergistically stimulated bone formation, which may provide a novel scheme for bone regeneration.

In vitro osteogenic induction of bone marrow mesenchymal stem cells with a decellularized matrix derived from human adipose stem cells and in vivo implantation for bone regeneration.

Bone tissue engineering has emerged as an effective alternative treatment to the problem of bone defect. To repair a bone defect, antibiosis and osteogenesis are two essential aspects of the repair process. By searching the literature and performing exploratory experiments, we found that β defensin 2 (BD2), with bifunctional properties of antibiosis and osteogenesis, was a feasible alternative for traditional growth factors. The antimicrobial ability of BD2 against Staphylococcus aureus and Escherichia coli was studied by the spread plate and live/dead staining methods (low effective concentration of 20 ng/mL). BD2 was also demonstrated to enhance osteogenesis, with higher messenger RNA (mRNA) and protein expression of the osteogenic markers collagen I (Col1), runt-related transcription factor 2 (Runx2), osteopontin (Opn), and osteocalcin (Ocn) in vitro (1.5-2.5-fold increase compared with the control group in the most effective concentration group), which was consistent with the alkaline phosphatase (ALP) and alizarin red S (ARS) staining results. We implanted poly(sebacoyl diglyceride) (PSeD) combined with BD2 and rat bone tissue-derived mesenchymal stem cells (rBMSCs) under the back skin of rats and found that the inflammatory response was significantly lower with this combination than with the PSeD/rBMSCs scaffold without BD2 and the pure PSeD group and was similar to the control group. Importantly, when assessed in a critical-sized in vivo rat 8 m diameter calvaria defect model, a scaffold we developed combining bifunctional BD2 with porous organic polymer displayed an osteogenic effect that was 160-200% greater than the control group. The in vivo study results revealed a significant osteogenic response and antimicrobial effect and were consistent with the in vitro results. In summary, BD2 displayed a great potential of simultaneously promoting bone regeneration and preventing infection and could provide a viable alternative to traditional growth factors applied in bone defect repair.

https://pubs.acs.org/doi/pdf/10.1021/acsomega.9b04249

Effect of Bifunctional β Defensin 2-Modified Scaffold on Bone Defect Reconstruction.

Bone regeneration, a complex physiological process, remains a challenge due to the susceptibility to the environment and absence of osteogenic growth factors around the defect region. Although autologous bone grafting is regarded as the gold standard for bone defect treatment, guided bone regeneration membranes in combination with multiple functional growth factors show a striking regeneration effect. Here, a biomimetic nanofibrous hybrid hydrogel composed of bacterial cellulose membranes and alginate/CaCl2 for sustained growth factor delivery was developed. The antibacterial peptide beta-defensin 2 served as an antibacterial, osteogenic, and angiogenic growth factor and was loaded into the aforementioned hydrogel. The mechanical and physical properties of the biomimetic nanofibrous hybrid hydrogel were investigated. Then, the in vitro osteogenic and angiogenic differentiation was confirmed by alkaline phosphatase (ALP) activity, Alizarin Red S staining, qPCR, western blot analysis and tube formation assays. After implantation into a rat calvarial defect model for 12 weeks, nanofibrous hybrid hydrogel membranes could adhere to the defect surface and promote new bone and vessel regeneration.

Biomimetic nanofibrous hybrid hydrogel membranes with sustained growth factor release for guided bone regeneration.

Abstract Objective To explore the therapeutic potential and the underlying mechanism of metformin, an adenosine monophosphate‐activated kinase (AMPK) activator, in ocular melanoma. Methods CCK8, transwell, and colony formation assays were performed to detect the proliferation and migration ability of ocular melanoma cells. A mouse orthotopic xenograft model was built to detect ocular tumor growth in vivo. Western blot, immunofluorescence, and electron microscopy were adopted to evaluate the autophagy levels of ocular melanoma cells, and high‐throughput proteomics and CUT & Tag assays were performed to analyze the candidate for autophagy alteration. Results Here, we revealed for the first time that a relatively low dose of metformin induced significant tumorspecific inhibition of the proliferation and migration of ocular melanoma cells both in vitro and in vivo. Intriguingly, we found that metformin significantly attenuated autophagic influx in ocular melanoma cells. Through high‐throughput proteomics analysis, we revealed that optineurin (OPTN), which is a key candidate for autophagosome formation and maturation, was significantly downregulated after metformin treatment. Moreover, excessive OPTN expression was associated with an unfavorable prognosis of patients. Most importantly, we found that a histone deacetylase, SIRT1, was significantly upregulated after AMPK activation, resulting in histone deacetylation in the OPTN promoter. Conclusions Overall, we revealed for the first time that metformin significantly inhibited the progression of ocular melanoma, and verified that metformin acted as an autophagy inhibitor through histone deacetylation of OPTN. This study provides novel insights into metformin ‐ guided suppression of ocular melanoma and the potential mechanism underlying the dual role of metformin in autophagy regulation.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ctm2.660

Ai Zhuang

Papers

Effects of miR-146a on the osteogenesis of adipose-derived mesenchymal stem cells and bone regeneration

In vitro osteogenic induction of bone marrow mesenchymal stem cells with a decellularized matrix derived from human adipose stem cells and in vivo implantation for bone regeneration.

Effect of Bifunctional β Defensin 2-Modified Scaffold on Bone Defect Reconstruction.

Biomimetic nanofibrous hybrid hydrogel membranes with sustained growth factor release for guided bone regeneration.

Metformin promotes histone deacetylation of optineurin and suppresses tumour growth through autophagy inhibition in ocular melanoma