An update–tissue engineered nerve grafts for the repair of peripheral nerve injuries

Peripheral nerve injuries（PNI） are caused by a range of etiologies and result in a broad spectrum of disability. While nerve autografts are the current gold standard for the reconstruction of extensive nerve damage, the limited supply of autologous nerve and complications associated with harvesting nerve from a second surgical site has driven groups from multiple disciplines, including biomedical engineering, neurosurgery, plastic surgery, and orthopedic surgery, to develop a suitable or superior alternative to autografting. Over the last couple of decades, various types of scaffolds, such as acellular nerve grafts（ANGs）, nerve guidance conduits, and non-nervous tissues, have been filled with Schwann cells, stem cells, and/or neurotrophic factors to develop tissue engineered nerve grafts（TENGs）. Although these have shown promising effects on peripheral nerve regeneration in experimental models, the autograft has remained the gold standard for large nerve gaps. This review provides a discussion of recent advances in the development of TENGs and their efficacy in experimental models. Specifically, TENGs have been enhanced via incorporation of genetically engineered cells, methods to improve stem cell survival and differentiation, optimized delivery of neurotrophic factors via drug delivery systems（DDS）, co-administration of platelet-rich plasma（PRP）, and pretreatment with chondroitinase ABC（Ch-ABC）. Other notable advancements include conduits that have been bioengineered to mimic native nerve structure via cell-derived extracellular matrix（ECM） deposition, and the development of transplantable living nervous tissue constructs from rat and human dorsal root ganglia（DRG） neurons. Grafts composed of non-nervous tissues, such as vein, artery, and muscle, will be briefly discussed.

Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells fol-lowing induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined speciifc neu-ronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuro-nal-speciifc proteins, includingβIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differen-tiation medium differentiated into a multilayered neural network-like structure with long nerve ifbers that was composed of several parallel microifbers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sec-tioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.

Fabrication of a Novel Hybrid Scaffold for Tissue Engineered Heart Valve

The aim of this study was to fabricate biomatrix/polymer hybrid scaffolds using an electrospinning technique. Then tissue engineered heart valves were engineered by seeding mesenchymal stromal cells （MSCs） onto the scaffolds. The effects of the hybrid scaffolds on the proliferation of seed cells, formation of extracellular matrix and mechanical properties of tissue engineered heart valves were investigated. MSCs were obtained from rats. Porcine aortic heart valves were decellularized, coated with poly（3-hydroxybutyrate-co-4-hydroxybutyrate） using an electrospinning technique, and reseeded and cultured over a time period of 14 days. In control group, the decellularized valve scaffolds were reseeded and cultured over an equivalent time period. Specimens of each group were examined histologically （hematoxylin-eosin [HE] staining, immunohistostaining, and scanning electron microscopy）, biochemically （DNA and 4-hydroxyproline） and mechanically. The results showed that recellularization was comparable to the specimens of hybrid scaffolds and controls. The specimens of hybrid scaffolds and controls revealed comparable amounts of cell mass and 4-hydroxyproline （P〉0.05）. However, the specimens of hybrid scaffolds showed a significant increase in mechanical strength, compared to the controls （P〈0.05）. This study demonstrated the superiority of the hybrid scaffolds to increase the mechanical strength of tissue engineered heart valves. And compared to the decellularized valve scaffolds, the hybrid scaffolds showed similar effects on the proliferation of MSCs and formation of extracellular matrix. It was believed that the hybrid scaffolds could be used for the construction of tissue engineered heart valves.

Expression of matrix metalloproteinase-9 was effected by epoxy chloropropan on creating tissue engineered heart valves

Objectives To investigate the effects of epoxy chloropropan on the expression of matrix metalloproteinases-9 （MMP-9）in creating tissue engineered heart valves（TEHV）,on the tissue structures of TEHV,and to study the effects of epoxy chloropropan on the calcification of TEHV.Methods The porcine aortic valve leaflets were digested and decellularized by using detergent and trypsin.Those treated with 0.3% glutaraldehyde for 48 hours were the control group;those treated with 3% epoxy choloropropan for 24 hours were the experimental group.The cultured human bone marrow mesenchymal stem cells（hBMSCs）were seeded onto the decellularized scaffolds of TEHV.The histological studies were done with pathological sections and scanning electron microscopy and reverse transcriptase-polymerase chain reaction（RT-PCR）were used to detect the expression of MMP-9.Results In the experimental group.the histology showed that the BMSCs grew well into the pores and formed a confluent layer in decellularized scaffolds;RT-PCR indicated significantly attenuated expressions of MMP-9,compared with the control（P〈0.05）.Conclusion The decellularized porcine aortic valves treated with 3% epoxy chloropropan may inhibit the expression of MMP-9;therefore epoxy chloropropan may prevent the calcification of tissue engineered heart valves.

Repair of sciatic nerve defects using tissue engineered nerves

In this study, we constructed tissue-engineered nerves with acellular nerve allografts in Sprague-Dawley rats, which were prepared using chemical detergents-enzymatic digestion and mechanical methods, in combination with bone marrow mesenchymal stem cells of Wistar rats cultured in vitro, to repair 15 mm sciatic bone defects in Wistar rats. At postoperative 12 weeks, electrophysiological detection results showed that the conduction velocity of regenerated nerve after repair with tissue-engineered nerves was similar to that after autologous nerve grafting, and was higher than that after repair with acellular nerve allografts. Immunohistochemical staining revealed that motor endplates with acetylcholinesterase-positive nerve fibers were orderly arranged in the middle and superior parts of the gastrocnemius muscle; regenerated nerve tracts and sprouted branches were connected with motor endplates, as shown by acetylcholinesterase histochemistry combined with silver staining. The wet weight ratio of the tibialis anterior muscle at the affected contralateral hind limb was similar to the sciatic nerve after repair with autologous nerve grafts, and higher than that after repair with acellular nerve allografts. The hind limb motor function at the affected side was significantly improved, indicating that acellular nerve allografts combined with bone marrow mesenchymal stem cell bridging could promote functional recovery of rats with sciatic nerve defects.

Restoring nervous system structure and function using tissue engineered living scaffolds

Neural tissue engineering is premised on the integration of engineered living tissue with the host nervous system to directly restore lost function or to augment regenerative capacity following ner- vous system injury or neurodegenerative disease. Disconnection of axon pathways - the long-distance fibers connecting specialized regions of the central nervous system or relaying peripheral signals - is a common feature of many neurological disorders and injury. However, functional axonal regenera- tion rarely occurs due to extreme distances to targets, absence of directed guidance, and the presence of inhibitory factors in the central nervous system, resulting in devastating effects on cognitive and sensorimotor function. To address this need, we are pursuing multiple strategies using tissue engi- neered ＂living scaffolds＂, which are preformed three-dimensional constructs consisting of living neural cells in a defined, often anisotropic architecture. Living scaffolds are designed to restore function by serving as a living labeled pathway for targeted axonal regeneration - mimicking key developmental mechanisms- or by restoring lost neural circuitry via direct replacement of neurons and axonal tracts. We are currently utilizing preformed living scaffolds consisting of neuronal dusters spanned by long axonal tracts as regenerative bridges to facilitate long-distance axonal regeneration and for targeted neurosurgical reconstruction of local circuits in the brain. Although there are formidable challenges in predinical and clinical advancement, these living tissue engineered constructs represent a promising strategy to facilitate nervous system repair and functional recovery.

Tissue engineered indigenous pericardial patch urethroplasty: A promising solution to a nagging problem

Objective:Urethral stricture is a highly prevalent disease and has a continued ris-ing incidence.The global burden of disease keeps rising as there are significant rates of recur-rence with the existing management options with the need for additional repeat procedures.Moreover,the existing treatment options are associated with significant morbidity in the pa-tient.Long segment urethral strictures are most commonly managed by augmentation urethro-plasty.We explored the potential for the application of an acellular tissue engineered bovine pericardial patch in augmentation urethroplasty in a series of our patients suffering from ure-thral stricture disease.The decreased morbidity due to the avoidance of harvest of buccal mu-cosa,decreased operative time and satisfactory postoperative results make it a promising option for augmentation urethroplasty.Methods:Nine patients with long segment anterior urethral strictures(involving penile and/or bulbar urethra and stricture length>4 cm)were included in the study after proper informed consent was obtained.Acellular tissue engineered indigenous bovine pericardial patch was used for urethroplasty using dorsal onlay technique.Results:A total of nine patients underwent tissue engineered indigenous pericardial patch ur-ethroplasty for long segment urethral strictures,mostly catheter injury induced or associated with balanitis xerotica obliterans.Median follow-up was 8 months(range:2-12 months).Out of nine patients,eight(88.9%)were classifed as success and one(11.1%)was classified as fail-ure.Conclusion:Our study brings a product of tissue engineering,already being used in the cardio-vascular surgery domain,into the urological surgery operating room with satisfactory results achieved using standard operating techniques of one stage urethroplasty.

The Experimental Study of Constructing Tissue Engineered Bone by Compounding Zinc-sintered Bovine Cancellous Bone with Marrow Stromal Cells

To study the osteogenic ability of tissue-engineered bone constructed by compounding zinc-sintered bovine cancellous bone with rabbit marrow stromal cells (MSCs) in vivo,the zinc-sintered bovine cancellous bone of beta-tricalcium phosphate (TCP) type was prepared by sintering the fresh calf cancellous bone twice and then loading it with zinc-ion.The rabbit MSCs were cultured,induced and seeded onto the zinc-sintered bovine cancellous bones.The tissue-engineered bones were then implanted into the rabbits' back muscles.The newly formed bone tissues were observed by histological methods and the areas of new osseous tissues were measured at the end of the 4th and 8th week.The zinc-sintered bovine cancellous bones alone were implanted on the other side as control.The osteogenic activity of MSCs was identified by alkaline phosphatase (ALP) staining and calcification nod chinalizarin staining.At the end of 4th week,a small amount of new bone tissues was observed.At the end of 8th week,there were many newly formed bone mature tissues.Moreover,the area of the latter was significantly larger than that of the former(P<0.01),while in the control group there was no new bone formation.The tissue-engineered bone,which was constructed by combining zinc-sintered bovine cancellous bone with MSCs,has satisfactory osteogenic capabilities in vivo.

Application of modified polyethylene glycol hydrogels in the construction of tissue engineered heart valve

To enhance the adhesion of seeding-cells to the biomaterial scaffolds, the PEG-hydrogels were modified. Porcine aortic valves were decellularized with Triton X-100 and trypsin. The cells were encapsulated into the PEG-hydrogels to complete the process of the cells attaching to the acellular porcine aortic valves. Herein, the autologous mesenchymal stem cells （MSCs） of goats were selected as the seeding-cells and the tendency of MSCs toward differentiation was observed when the single semilunar TEHV had been implanted into their abdominal aortas. Furthermore, VEGF, TGF-β1, and the cell adhesive peptide motif RGD were incorporated. Light and electron microscopy observations were performed. Analysis of modified PEG-hydrogels TEHV＇s （PEG-TEHV） tensile strength, and the ratio of reendothelial and mural thrombosis revealed much better improvement than the naked acellular porcine aortic valve （NAPAV）. The data illustrated the critical importance of MSC differentiation into endothelial and myofibroblast for remodeling into native tissue. Our results indicate that it is feasible to reconstruct TEHV efficiently by combining modified PEG-hydrogels with acellular biomaterial scaffold andautologous MSCs cells.

The Experimental Study on Constructing the Tissue Engineered Myocardium-like Tissue in vitro with Bone Mesenchymal Stem Cells

Objective:Unlike other tissues,myocardium has not substitute whick can be used to repair damaged cardiac tissue.This paper proposes engineering 3-D myocardium-like tissue constructs in vitro with bone mesenchymal stem cells(BMSCs) of infant and poly-lactic-co-glycolic acid(PLGA)in vitro.Methods:Bone marrow was obtained from the sternal marrow cavum outflow of infant with congenital heart disease (CHD)undergoing cardiac operation.BMSCs were obtained by density gradient centrifugation.The cells in passages two were induced in DMED with 10 umol/L 5- Azacytidine(5-Aza)for 24 h.When the induced BMSCS were cultured nearly into filled,the cells were planted in the scaffold of PLGA in 5.5×106 cells/cm2.The cell- scaffold complex has been cultured in the shake cultivation for 1 week,then the complex has been planted in the dorse of the nude mouse.When the experiment had been finished,the histology,immunology,real time PCR and so on were done.Results: The BMSCs of infant with congenital heart disease have the properties of the stable growth and the rapid proliferation.The immunohistochemistry showed that tissue engineered myocardium constructed in vitro expressed some cardiac related proteins such asα-actin,Cx-43,Desmine,cTNI and so on.The transparent myofilaments,gap junctions and intercalated disk-like structure formation could be observed in the 3D tissue-like constructs by transmission electron microscope(TEM).The engineered myocardium-like tissue had the auto-myocardial property as assessed by real time- PCR and so on.Conclusion:The engineered myocardial tissue-like constructs could be built with infant BMSCs and PLGA in vitro.Our results may provide the first step on the long road toward engineering myocardial material for repairing the defect or augmenting the tract in CHD,such as ventricular septal defect,tetralogy of Fallot and so on.

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.

Biomaterials and tissue engineering in traumatic brain injury:novel perspectives on promoting neural regeneration

Traumatic brain injury is a serious medical condition that can be attributed to falls, motor vehicle accidents, sports injuries and acts of violence, causing a series of neural injuries and neuropsychiatric symptoms. However, limited accessibility to the injury sites, complicated histological and anatomical structure, intricate cellular and extracellular milieu, lack of regenerative capacity in the native cells, vast variety of damage routes, and the insufficient time available for treatment have restricted the widespread application of several therapeutic methods in cases of central nervous system injury. Tissue engineering and regenerative medicine have emerged as innovative approaches in the field of nerve regeneration. By combining biomaterials, stem cells, and growth factors, these approaches have provided a platform for developing effective treatments for neural injuries, which can offer the potential to restore neural function, improve patient outcomes, and reduce the need for drugs and invasive surgical procedures. Biomaterials have shown advantages in promoting neural development, inhibiting glial scar formation, and providing a suitable biomimetic neural microenvironment, which makes their application promising in the field of neural regeneration. For instance, bioactive scaffolds loaded with stem cells can provide a biocompatible and biodegradable milieu. Furthermore, stem cells-derived exosomes combine the advantages of stem cells, avoid the risk of immune rejection, cooperate with biomaterials to enhance their biological functions, and exert stable functions, thereby inducing angiogenesis and neural regeneration in patients with traumatic brain injury and promoting the recovery of brain function. Unfortunately, biomaterials have shown positive effects in the laboratory, but when similar materials are used in clinical studies of human central nervous system regeneration, their efficacy is unsatisfactory. Here, we review the characteristics and properties of various bioactive materials, followed by the introduction of applications based on biochemistry and cell molecules, and discuss the emerging role of biomaterials in promoting neural regeneration. Further, we summarize the adaptive biomaterials infused with exosomes produced from stem cells and stem cells themselves for the treatment of traumatic brain injury. Finally, we present the main limitations of biomaterials for the treatment of traumatic brain injury and offer insights into their future potential.

Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds

There is a high demand for bespoke grafts to replace damaged or malformed bone and cartilage tissue.Three-dimensional(3D)printing offers a method of fabricating complex anatomical features of clinically relevant sizes.However,the construction of a scaffold to replicate the complex hierarchical structure of natural tissues remains challenging.This paper reports a novel biofabrication method that is capable of creating intricately designed structures of anatomically relevant dimensions.The beneficial properties of the electrospun fibre meshes can finally be realised in 3D rather than the current promising breakthroughs in two-dimensional(2D).The 3D model was created from commercially available computer-aided design software packages in order to slice the model down into many layers of slices,which were arrayed.These 2D slices with each layer of a defined pattern were laser cut,and then successfully assembled with varying thicknesses of 100μm or 200μm.It is demonstrated in this study that this new biofabrication technique can be used to reproduce very complex computer-aided design models into hierarchical constructs with micro and nano resolutions,where the clinically relevant sizes ranging from a simple cube of 20 mm dimension,to a more complex,50 mm-tall human ears were created.In-vitro cell-contact studies were also carried out to investigate the biocompatibility of this hierarchal structure.The cell viability on a micromachined electrospun polylactic-co-glycolic acid fibre mesh slice,where a range of hole diameters from 200μm to 500μm were laser cut in an array where cell confluence values of at least 85%were found at three weeks.Cells were also seeded onto a simpler stacked construct,albeit made with micromachined poly fibre mesh,where cells can be found to migrate through the stack better with collagen as bioadhesives.This new method for biofabricating hierarchical constructs can be further developed for tissue repair applications such as maxillofacial bone injury or nose/ear cartilage replacement in the future.

Protein-spatiotemporal partition releasing gradient porous scaffolds and anti-inflammatory and antioxidant regulation remodel tissue engineered anisotropic meniscus

Meniscus is a wedge-shaped fibrocartilaginous tissue,playing important roles in maintaining joint stability and function.Meniscus injuries are difficult to heal and frequently progress into structural breakdown,which then leads to osteoarthritis.Regeneration of heterogeneous tissue engineering meniscus(TEM)continues to be a scientific and translational challenge.The morphology,tissue architecture,mechanical strength,and functional applications of the cultivated TEMs have not been able to meet clinical needs,which may due to the negligent attention on the importance of microenvironment in vitro and in vivo.Herein,we combined the 3D(three-dimensional)-printed gradient porous scaffolds,spatiotemporal partition release of growth factors,and anti-inflammatory and anti-oxidant microenvironment regulation of Ac2-26 peptide to prepare a versatile meniscus composite scaffold with heterogeneous bionic structures,excellent biomechanical properties and anti-inflammatory and anti-oxidant effects.By observing the results of cell activity and differentiation,and biomechanics under anti-inflammatory and anti-oxidant microenvironments in vitro,we explored the effects of anti-inflammatory and anti-oxidant microenvironments on construction of regional and functional heterogeneous TEM via the growth process regulation,with a view to cultivating a high-quality of TEM from bench to bedside.

Biomimetic Erythrocyte-Like Particles from Microfluidic Electrospray for Tissue Engineering

Microparticles have demonstrated value for regenerative medicine.Attempts in this field tend to focus on the development of intelligent multifunctional microparticles for tissue regeneration.Here,inspired by erythrocytes-associated self-repairing process in damaged tissue,we present novel biomimetic erythrocyte-like microparticles(ELMPs).These ELMPs,which are composed of extracellular matrix-like hybrid hydrogels and the functional additives of black phosphorus,hemoglobin,and growth factors(GFs),are generated by using a microfluidic electrospray.As the resultant ELMPs have the capacity for oxygen delivery and near-infrared-responsive release of both GFs and oxygen,they would have excellent biocompatibility and multifunctional performance when serving as microscaffolds for cell adhesion,stimulating angiogenesis,and adjusting the release profile of cargoes.Based on these features,we demonstrate that the ELMPs can stably overlap to fill a wound and realize controllable cargo release to achieve the desired curative effect of tissue regeneration.Thus,we consider our biomimetic ELMPs with discoid morphology and cargo-delivery capacity to be ideal for tissue engineering.