Biomimetic natural biomaterials for tissue engineering and regenerative medicine:new biosynthesis methods,recent advances,and emerging applications

Biomimetic materials have emerged as attractive and competitive alternatives for tissue engineering(TE)and regenerative medicine.In contrast to conventional biomaterials or synthetic materials,biomimetic scaffolds based on natural biomaterial can offer cells a broad spectrum of biochemical and biophysical cues that mimic the in vivo extracellular matrix(ECM).Additionally,such materials have mechanical adaptability,micro-structure interconnectivity,and inherent bioactivity,making them ideal for the design of living implants for specific applications in TE and regenerative medicine.This paper provides an overview for recent progress of biomimetic natural biomaterials(BNBMs),including advances in their preparation,functionality,potential applications and future challenges.We highlight recent advances in the fabrication of BNBMs and outline general strategies for functionalizing and tailoring the BNBMs with various biological and physicochemical characteristics of native ECM.Moreover,we offer an overview of recent key advances in the functionalization and applications of versatile BNBMs for TE applications.Finally,we conclude by offering our perspective on open challenges and future developments in this rapidly-evolving field.

Promising use of metformin in treating neurological disorders:biomarker-guided therapies

Neurological disorders are a diverse group of conditions that affect the nervous system and include neurodegenerative diseases(Alzheimer’s disease,multiple sclerosis,Parkinson’s disease,Huntington’s disease),cerebrovascular conditions(stroke),and neurodevelopmental disorders(autism spectrum disorder).Although they affect millions of individuals around the world,only a limited number of effective treatment options are available today.Since most neurological disorders express mitochondria-related metabolic perturbations,metformin,a biguanide type II antidiabetic drug,has attracted a lot of attention to be repurposed to treat neurological disorders by correcting their perturbed energy metabolism.However,controversial research emerges regarding the beneficial/detrimental effects of metformin on these neurological disorders.Given that most neurological disorders have complex etiology in their pathophysiology and are influenced by various risk factors such as aging,lifestyle,genetics,and environment,it is important to identify perturbed molecular functions that can be targeted by metformin in these neurological disorders.These molecules can then be used as biomarkers to stratify subpopulations of patients who show distinct molecular/pathological properties and can respond to metformin treatment,ultimately developing targeted therapy.In this review,we will discuss mitochondria-related metabolic perturbations and impaired molecular pathways in these neurological disorders and how these can be used as biomarkers to guide metformin-responsive treatment for the targeted therapy to treat neurological disorders.

Biomarker-guided drug therapy: personalized medicine for treating Alzheimer’s disease

Alzheimer’s disease(AD)is a progressive neurodegenerative disorder associated with significant memory decline and cognitive impairment.AD is characterized by two classical neuropathological hal lmarks,namely the amyloid-beta(Aβ)plaques and neurofibril tangles.Currently,there are no disease-modifying treatments available for AD,except for a couple of the US Food and Drug Administration(FDA)-approved drugs to improve cognitive function by blocking N-methyl-D-aspartate receptors or cholinesterase activity(Panza et al.,2019).

Role of chondroitin sulfate proteoglycan signaling in regulating neuroinflammation following spinal cord injury

Spinal cord injury （SCI） elicits a robust inflammatory response that is a hallmark of the secondary injury mechanisms. Neuroinflammation is orchestrated initially by the response of resident astrocytes and microglia to injury, which subsequently facilitates the recruitment of peripheral immune cells into the SCI lesion （Orr and Gensel, 2018）. This inflammatory response contributes to cell death and tissue degeneration through the production of pro-inflammatory cytokines and chemokines, free radicals and proteolytic enzymes. However, neuroinflammatory cells also play beneficial regulatory role in repair mechanisms after SCI by adopting a reparative and wound healing phenotype （Orr and Gensel, 2018; Tran et al., 2018）. Hence, understanding the underlying mechanisms by which immune cells are reg- ulated within the microenvironment of injury would aid in harnessing the reparative potential of inflammation following SCI.

Nuclear accumulation of β-catenin and forkhead box O3a in colon cancer:Dangerous liaison

The WNT/-catenin and phosphoinositide 3-kinase(PI3K/AKT) signaling cascades both have been implicated in the formation and progression of colorectal cancer.Oncogenic PI3K/AKT signaling suppresses the activity of forkhead box O3a(FOXO3a) transcription factor through phosphorylation leading to its nuclear exclusion.Inhibition of the PI3K/AKT signaling by PI3K or AKT inhibitors results in the translocation of FOXO3a to the nucleus,and is considered to be a promising therapeutic strategy for many cancers including colon cancer.Now,however,a new study in Nature Medicine has revealed a nuclear interaction of-catenin with FOXO3a as a promoter of metastatic progression in colon cancer.The work has important implications for the treatment of colon cancers,suggests a companion biomarker strategy to enable a personalized medicine approach,and offers an alternative therapeutic strategy to overcome resistance to PI3K and AKT inhibitors.

Re-evaluating the role of epithelial-mesenchymal-transition in cancer progression

Epithelial-mesenchymal transition（EMT） and mesenchymal-epithelial transition（MET） are essential for embryonic development and also important in cancer progression. In a conventional model, epithelial-like cancer cells transit to mesenchymal-like tumor cells with great motility via EMT transcription factors; these mesenchymallike cells migrate through the circulation system, relocate to a suitable site and then convert back to an epithelial-like phenotype to regenerate the tumor. However, recent findings challenge this conventional model and support the existence of a stable hybrid epithelial/mesenchymal（E/M） tumor population. Hybrid E/M tumor cells exhibit both epithelial and mesenchymal properties, possess great metastatic and tumorigenic capacity and are associated with poorer patient prognosis. The hybrid E/M model and associated regulatory networks represent a conceptual change regarding tumor metastasis and organ colonization. It may lead to the development of novel treatment strategies to ultimately stop cancer progression and improve disease-free survival.

Neuregulin-1:a novel regulator of glial response in spinal cord injury

Spinal cord injury （SCI） results in a dysregulated microenvi- ronment that is largely driven by the immediate and robust response of resident astrocytes and microglia （Filous and Silver, 2016）.

Fabricating 3-dimensional human brown adipose microtissues for transplantation studies

Transplanting cell cultured brown adipocytes(BAs)represents a promising approach to prevent and treat obesity(OB)and its associated metabolic disorders,including type 2 diabetes mellitus(T2DM).However,transplanted BAs have a very low survival rate in vivo.The enzymatic dissociation during the harvest of fully differentiated BAs also loses significant cells.There is a critical need for novel methods that can avoid cell death during cell preparation,transplantation,and in vivo.Here,we reported that preparing BAs as injectable microtissues could overcome the problem.We found that 3D culture promoted BA differentiation and UCP-1 expression,and the optimal initial cell aggregate size was 100μm.The microtissues could be produced at large scales via 3D suspension assisted with a PEG hydrogel and could be cryopreserved.Fabricated microtissues could survive in vivo for long term.They alleviated body weight and fat gain and improved glucose tolerance and insulin sensitivity in high-fat diet(HFD)-induced OB and T2DM mice.Transplanted microtissues impacted multiple organs,secreted protein factors,and influenced the secretion of endogenous adipokines.To our best knowledge,this is the first report on fabricating human BA microtissues and showing their safety and efficacy in T2DM mice.The proposal of transplanting fabricated BA microtissues,the microtissue fabrication method,and the demonstration of efficacy in T2DM mice are all new.Our results show that engineered 3D human BA microtissues have considerable advantages in product scalability,storage,purity,safety,dosage,survival,and efficacy.

3D printing of silk fibroin-based hybrid scaffold treated with platelet rich plasma for bone tissue engineering

3D printing/bioprinting are promising techniques to fabricate scaffolds with well controlled and patient-specific structures and architectures for bone tissue engineering.In this study,we developed a composite bioink consisting of silk fibroin(SF),gelatin(GEL),hyaluronic acid(HA),and tricalcium phosphate(TCP)and 3D bioprinted the silk fibroin-based hybrid scaffolds.The 3D bioprinted scaffolds with dual crosslinking were further treated with human platelet-rich plasma(PRP)to generate PRP coated scaffolds.Live/Dead and MTT assays demonstrated that PRP treatment could obviously promote the cell growth and proliferation of human adipose derived mesenchymal stem cells(HADMSC).In addition,the treatment of PRP did not significantly affect alkaline phosphatase(ALP)activity and expression,but significantly upregulated the gene expression levels of late osteogenic markers.This study demonstrated that the 3D printing of silk fibroin-based hybrid scaffolds,in combination with PRP post-treatment,might be a more efficient strategy to promote osteogenic differentiation of adult stem cells and has significant potential to be used for bone tissue engineering.

Carbon nanomaterials for cardiovascular theranostics:Promises and challenges

Cardiovascular diseases(CVDs)are the leading cause of death worldwide.Heart attack and stroke cause irreversible tissue damage.The currently available treatment options are limited to“damage-control”rather than tissue repair.The recent advances in nanomaterials have offered novel approaches to restore tissue function after injury.In particular,carbon nanomaterials(CNMs)have shown significant promise to bridge the gap in clinical translation of biomaterial based therapies.This family of carbon allotropes(including graphenes,carbon nanotubes and fullerenes)have unique physiochemical properties,including exceptional mechanical strength,electrical conductivity,chemical behaviour,thermal stability and optical properties.These intrinsic properties make CNMs ideal materials for use in cardiovascular theranostics.This review is focused on recent efforts in the diagnosis and treatment of heart diseases using graphenes and carbon nanotubes.The first section introduces currently available derivatives of graphenes and carbon nanotubes and discusses some of the key characteristics of these materials.The second section covers their application in drug delivery,biosensors,tissue engineering and immunomodulation with a focus on cardiovascular applications.The final section discusses current shortcomings and limitations of CNMs in cardiovascular applications and reviews ongoing efforts to address these concerns and to bring CNMs from bench to bedside.

3D printing of multilayered scaffolds for rotator cuff tendon regeneration

Repairing massive rotator cuff tendon defects remains a challenge due to the high retear rate after surgical intervention.3D printing has emerged as a promising technique that enables the fabrication of engineered tissues with heterogeneous structures and mechanical properties,as well as controllable microenvironments for tendon regeneration.In this study,we developed a new strategy for rotator cuff tendon repair by combining a 3D printed scaffold of polylactic-co-glycolic acid(PLGA)with cell-laden collagen-fibrin hydrogels.We designed and fabricated two types of scaffolds:one featuring a separate layer-by-layer structure and another with a tri-layered structure as a whole.Uniaxial tensile tests showed that both types of scaffolds had improved mechanical properties compared to single-layered PLGA scaffolds.The printed scaffold with collagen-fibrin hydrogels effectively supported the growth,proliferation,and tenogenic differentiation of human adipose-derived mesenchymal stem cells.Subcutaneous implantation of the multilayered scaffolds demonstrated their excellent in vivo biocompatibility.This study demonstrates the feasibility of 3D printing multilayered scaffolds for application in rotator cuff tendon regeneration.

Exosomes derived from differentiated human ADMSC with the Schwann cell phenotype modulate peripheral nerve-related cellular functions

Peripheral nerve regeneration remains a significant clinical challenge due to the unsatisfactory functional recovery and public health burden.Exosomes,especially those derived from mesenchymal stem cells(MSCs),are promising as potential cell-free therapeutics and gene therapy vehicles for promoting neural regeneration.In this study,we reported the differentiation of human adipose derived MSCs(hADMSCs)towards the Schwann cell(SC)phenotype(hADMSC-SCs)and then isolated exosomes from hADMSCs with and without differentiation(i.e.,dExo vs uExo).We assessed and compared the effects of uExo and dExo on antioxidative,angiogenic,anti-inflammatory,and axon growth promoting properties by using various peripheral nerve-related cells.Our results demonstrated that hADMSC-SCs secreted more neurotrophic factors and other growth factors,compared to hADMSCs without differentiation.The dExo isolated from hADMSC-SCs protected rat SCs from oxidative stress and enhanced HUVEC migration and angiogenesis.Compared to uExo,dExo also had improved performances in downregulating pro-inflammatory gene expressions and cytokine secretions and promoting axonal growth of sensory neurons differentiated from human induced pluripotent stem cells.Furthermore,microRNA(miRNA)sequencing analysis revealed that exosomes and their parent cells shared some similarities in their miRNA profiles and exosomes displayed a distinct miRNA signature.Many more miRNAs were identified in dExo than in uExo.Several upregulated miRNAs,like miRNA-132-3p and miRNA-199b-5p,were highly related to neuroprotection,anti-inflammation,and angiogenesis.The dExo can effectively modulate various peripheral nerve-related cellular functions and is promising for cell-free biological therapeutics to enhance neural regeneration.

Novel fibrin-fibronectin matrix accelerates mice skin wound healing

Plasma fibrinogen(F1)and fibronectin(pFN)polymerize to form a fibrin clot that is both a hemostatic and provisional matrix for wound healing.About 90%of plasma F1 has a homodimeric pair ofγchains(γγF1),and 10%has a heterodimeric pair ofγand more acidicγ′chains(γγ′F1).We have synthesized a novel fibrin matrix exclusively from a 1:1(molar ratio)complex ofγγ′F1 and pFN in the presence of highly active thrombin and recombinant Factor XIII(rFXIIIa).In this matrix,the fibrin nanofibers were decorated with pFN nanoclusters(termedγγ′F1:pFN fibrin).In contrast,fibrin made from 1:1 mixture ofγγF1 and pFN formed a sporadic distribution of“pFN droplets”(termedγγF1+pFN fibrin).Theγγ′F1:pFN fibrin enhanced the adhesion of primary human umbilical vein endothelium cells(HUVECs)relative to theγγF1+FN fibrin.Three dimensional(3D)culturing showed that theγγ′F1:pFN complex fibrin matrix enhanced the proliferation of both HUVECs and primary human fibroblasts.HUVECs in the 3Dγγ′F1:pFN fibrin exhibited a starkly enhanced vascular morphogenesis while an apoptotic growth profile was observed in theγγF1+pFN fibrin.Relative toγγF1+pFN fibrin,mouse dermal wounds that were sealed byγγ′F1:pFN fibrin exhibited accelerated and enhanced healing.This study suggests that a 3D pFN presentation on a fibrin matrix promotes wound healing.

Anisotropic scaffolds for peripheral nerve and spinal cord regeneration

The treatment of long-gap(>10 mm)peripheral nerve injury(PNI)and spinal cord injury(SCI)remains a continuous challenge due to limited native tissue regeneration capabilities.The current clinical strategy of using autografts for PNI suffers from a source shortage,while the pharmacological treatment for SCI presents dissatisfactory results.Tissue engineering,as an alternative,is a promising approach for regenerating peripheral nerves and spinal cords.Through providing a beneficial environment,a scaffold is the primary element in tissue engineering.In particular,scaffolds with anisotropic structures resembling the native extracellular matrix(ECM)can effectively guide neural outgrowth and reconnection.In this review,the anatomy of peripheral nerves and spinal cords,as well as current clinical treatments for PNI and SCI,is first summarized.An overview of the critical components in peripheral nerve and spinal cord tissue engineering and the current status of regeneration approaches are also discussed.Recent advances in the fabrication of anisotropic surface patterns,aligned fibrous substrates,and 3D hydrogel scaffolds,as well as their in vitro and in vivo effects are highlighted.Finally,we summarize potential mechanisms underlying the anisotropic architectures in orienting axonal and glial cell growth,along with their challenges and prospects.

Bioengineering strategies for the treatment of peripheral arterial disease

Peripheral arterial disease(PAD)is a progressive atherosclerotic disorder characterized by narrowing and occlusion of arteries supplying the lower extremities.Approximately 200 million people worldwide are affected by PAD.The current standard of operative care is open or endovascular revascularization in which blood flow restoration is the goal.However,many patients are not appropriate candidates for these treatments and are subject to continuous ischemia of their lower limbs.Current research in the therapy of PAD involves developing modalities that induce angiogenesis,but the results of simple cell transplantation or growth factor delivery have been found to be relatively poor mainly due to difficulties in stem cell retention and survival and rapid diffusion and enzymolysis of growth factors following injection of these agents in the affected tissues.Biomaterials,including hydrogels,have the capability to protect stem cells during injection and to support cell survival.Hydrogels can also provide a sustained release of growth factors at the injection site.This review will focus on biomaterial systems currently being investigated as carriers for cell and growth factor delivery,and will also discuss biomaterials as a potential stand-alone method for the treatment of PAD.Finally,the challenges of development and use of biomaterials systems for PAD treatment will be reviewed.

Microfabricated platforms to investigate cell mechanical properties

Mechanical stimulation has been imposed on living cells using several approaches.Most early investigations were conducted on groups of cells,utilizing techniques such as substrate deformation and flow-induced shear.To investigate the properties of cells individually,many conventional techniques were utilized,such as AFM,optical traps/optical tweezers,magnetic beads,and micropipette aspiration.In specific mechanical interrogations,microelectro-mechanical systems(MEMS)have been designed to probe single cells in different interrogation modes.To exert loads on the cells,these devices often comprise piezo-electric driven actuators that attach directly to the cell or move a structure on which cells are attached.Uniaxial and biaxial pullers,micropillars,and cantilever beams are examples of MEMS devices.In this review,the methodologies to analyze single cell activity under external loads using microfabricated devices will be examined.We will focus on the mechanical interrogation in three different regimes:compression,traction,and tension,and discuss different microfabricated platforms designed for these purposes.