内皮祖细胞在再生医学中的应用

随着血管内皮祖细胞(endothelial progenitor cell,EPC)研究的深入,EPC的分离、体外培养、鉴定及在血管相关性疾病(如缺血性疾病)和各种肿瘤发生发展中的作用及功能已成为新的研究热点。EPC具有分化功能,增殖能力极强,体外扩增培养速度快,主要存在于外周血、脐血、骨髓中,且容易获取,是干细胞移植疗法理想的种子细胞来源。目前,EPC在治疗心脑血管、肢体缺血性疾病等方面的研究十分活跃,并初步取得了肯定的成果。本文主要就EPC在再生医学中的细胞治疗及基因治疗上的应用作一综述。     

Acetanilide-loaded injectable hydrogels with enhanced bioactivity and biocompatibility for potential treatment of periodontitis

Chronic periodontitis poses long-term challenges in dentistry, requiring the development of innovative dental composites with biocompatibility, bone regeneration, and antibacterial properties. This study focuses on synthesis of novel injectable thermoresponsive hydrogels composed of chitosan, sodium bicarbonate, bioactive glass (20 and 40% w/w), and acetanilide drug (0.3 and 0.6% w/w). These hydrogels exhibit a sol–gel transition at 37°C, addressing periodontal challenges with reduced gelation time. The smooth flow characteristic was evaluated through 17-22 gauge syringe needles at low temperature. Rheological studies demonstrated pseudoplastic behavior, with viscosity decreasing as shear rate increases. Fourier transform infrared and x-ray diffraction analysis confirmed the bioactivity of hydrogels, forming a bone-like apatite layer in simulated body fluid. The drug-loaded hydrogels demonstrated promising in vitro antibacterial properties against dental pathogens, specifically Staphylococcus aureus and Pseudomonas aeruginosa. Drug dissolution analysis revealed relatively high release rate at 37°C, highlighting its role in rapidly eliminating bacterial colonies at the target site, while the subsequent sustained release contributes to the prevention of infection recurrence. Finally, biocompatibility was assessed with fibroblast, where the cells were observed anchoring into the polymeric chains of hydrogel through extended filopodia.     

Recent advances on injectable nanocomposite hydrogels towards bone tissue rehabilitation

There has been significant interest in the recent past to develop injectable hydrogel scaffolds that follow minimally invasive implantation procedures towards efficient healing and regeneration of defective bone tissues. Such scaffolds offer several advantages, as they can be injected into the irregularly shaped defect and can act as a low-density aqueous reservoir, incorporating necessary components for bone tissue repair and augmentation. Considering that bone is a biocomposite of natural biopolymer and bioapatite nanofiller, there has been a growing trend to develop nanocomposite scaffolds by combining biopolymers and inorganic nanofillers to biomimic the hierarchical nanostructure and composition of natural bone. Furthermore, the nanocomposite scaffolds can be tailored to have patient-specific bone properties, which can lead to better biological responses. The present article begins with the introduction, followed by an overview of polymer matrices, property requirements, and crosslinking techniques employed for injectable hydrogels. Various strategies to develop injectable composites, with emphasis on nanocomposite hydrogels incorporating bioinert and bioactive nanofillers have been discussed. The fundamental challenges related to the development of injectable hydrogel nanocomposite scaffolds and the research efforts directed towards solving these problems have also been reviewed. Finally, future trends and conclusions on new generation injectable hydrogel nanocomposite bone scaffolds have been discussed in this article.     

Chemical synthesis of biomimetic hydrogels for tissue engineering

Owing to their high water content, porous structure, biocompatibility and tissue‐like viscoelasticity, hydrogels have become attractive and promising biomaterials for use in drug delivery, three‐dimensional cell culture and tissue engineering applications. Various chemical approaches have been developed for hydrogel synthesis using monomers or polymers carrying reactive functional groups. For in vivo tissue repair and in vitro cell culture purposes, it is desirable that the crosslinking reactions occur under mild conditions, do not interfere with biological processes and proceed at high yield with exceptional selectivity. Additionally, the crosslinking reaction should allow straightforward incorporation of bioactive motifs or signaling molecules, at the same time providing tunability of the hydrogel microstructure, mechanical properties and degradation rates. In this review, we discuss various chemical approaches applied to the synthesis of complex hydrogel networks, highlighting recent developments from our group. The discovery of new chemistries and novel materials fabrication methods will lead to the development of the next generation of biomimetic hydrogels with complex structures and diverse functionalities. These materials will likely facilitate the construction of engineered tissue models that may bridge the gap between two‐dimensional experiments and animal studies, providing preliminary insight prior to in vivo assessments. © 2017 Society of Chemical Industry     

Stem cell bioprocessing for regenerative medicine

Stem cell‐derived products have the potential to represent promising therapeutic approaches for the treatment of a wide range of conditions. Neurodegenerative diseases, like Parkinson's disease or Huntington's disease, neurological disorders, cardiac failure, and blood disorders, among others, may one day be treated using cellular therapies and regenerative medicine approaches based on stem cells. Furthermore, owing to the potential positive impact in healthcare systems, translation of stem cell technologies into clinical applications will bring a broad social and economic advantage worldwide. However, to fully realize this potential, advanced bioprocessing systems are needed to deliver sufficient numbers of cells in compliance with stringent regulatory landscapes and that can be used in a safe and effective manner. This review presents and summarizes recent advancements in the field of stem cell engineering, in particular novel technologies for the interrogation of stem cell fate and systems for the robust manufacturing of cells under standardized, reproducible and strictly controlled conditions. © 2013 Society of Chemical Industry     

Matricryptic peptide-inspired hydrogels for promoting osteogenic differentiation

In the natural process of bone repair, matricryptic peptides were activated by up-regulated ECM-degrading enzymes and properly released at bone injury sites, which plays critical roles in bone self-healing. Inspired by this natural process, here, matricryptic peptide-inspired (MPI) hydrogels were designed to promote osteogenic differentiation. MPI hydrogels can be degraded by enzymes and during this time, the masked bioactive components were released to promote osteogenic differentiation. These MPI hydrogels which normally serve as structural support for cell proliferation while promote osteogenic differentiation using embedded osteogenic BFP-1 peptides after degradation may provide a novel design for bone regeneration materials.     

Design of artificial extracellular matrices for tissue engineering

The design of artificial extracellular matrix (ECM) is important in tissue engineering because artificial ECM regulates cellular behaviors, including proliferation, survival, migration, and differentiation. Artificial ECMs have several functions in tissue engineering, including provision of cell-adhesive substrate, control of three-dimensional tissue structure, and presentation of growth factors, cell-adhesion signals, and mechanical signals. Design criteria for artificial ECMs vary considerably depending on the type of the engineered tissue. This article reviews the materials and methods that have been used in fabrication of artificial ECMs for engineering of specific tissues, including liver, cartilage, bone, and skin. This article also reviews artificial ECMs used for modulation of stem cell behaviors for tissue engineering applications.     

Functionalized nanostructures with application in regenerative medicine

In the last decade, both regenerative medicine and nanotechnology have been broadly developed leading important advances in biomedical research as well as in clinical practice. The manipulation on the molecular level and the use of several functionalized nanoscaled materials has application in various fields of regenerative medicine including tissue engineering, cell therapy, diagnosis and drug and gene delivery. The themes covered in this review include nanoparticle systems for tracking transplanted stem cells, self-assembling peptides, nanoparticles for gene delivery into stem cells and biomimetic scaffolds useful for 2D and 3D tissue cell cultures, transplantation and clinical application.     

Cardiac Extracellular Matrix Hydrogel Enriched with Polyethylene Glycol Presents Improved Gelation Time and Increased On-Target Site Retention of Extracellular Vesicles

Stem-cell-derived extracellular vesicles (EVs) have demonstrated multiple beneficial effects in preclinical models of cardiac diseases. However, poor retention at the target site may limit their therapeutic efficacy. Cardiac extracellular matrix hydrogels (cECMH) seem promising as drug-delivery materials and could improve the retention of EVs, but may be limited by their long gelation time and soft mechanical properties. Our objective was to develop and characterize an optimized product combining cECMH, polyethylene glycol (PEG), and EVs (EVs–PEG–cECMH) in an attempt to overcome their individual limitations: long gelation time of the cECMH and poor retention of the EVs. The new combined product presented improved physicochemical properties (60% reduction in half gelation time, p < 0.001, and threefold increase in storage modulus, p < 0.01, vs. cECMH alone), while preserving injectability and biodegradability. It also maintained in vitro bioactivity of its individual components (55% reduction in cellular senescence vs. serum-free medium, p < 0.001, similar to EVs and cECMH alone) and increased on-site retention in vivo (fourfold increase vs. EVs alone, p < 0.05). In conclusion, the combination of EVs–PEG–cECMH is a potential multipronged product with improved gelation time and mechanical properties, increased on-site retention, and maintained bioactivity that, all together, may translate into boosted therapeutic efficacy.     

Halloysite clay nanotube in regenerative medicine for tissue and wound healing

The vital necessity of effective treatment at damaged tissue or wound site has resulted in emerging tissue engineering and regenerative medicine. Tissue engineering has been introduced as an alternative approach for common available therapeutic strategies in the terms of restoring deformed tissue structure and its functionality via the developing of new bio-scaffold. Designed three-dimensional (3D) scaffolds, alone or in combination with bioactive agents, should be able to stimulate and accelerate the development of engineered tissues and provide proper mechanical support during in-vivo implantation and later regeneration process. To cover it up, a series of new bio-structures with higher mechanical strength were designed through the combination of halloysite nanotubes (HNTs) into 3D bio-polymeric networks. HNTs clay mineral with its unique rod-like structure and distinctive chemical surface features, exhibits excellent biocompatibility and biosafety for doping into regenerative scaffolds to enhance their mechanical stiffness and biological performance. In this paper, the ongoing procedures of bone/cartilage tissue engineering and wound healing strategies focusing on the designing of 3D-HNTs bio-composites and their multi-cellular interactions in-vitro and in-vivo preclinical studies are reviewed. Furthermore, the challenges and prospects of 3D-HNTs and HNTs-based functional bio-devices for regenerative medicine are also discussed.     

Ammonium perchlorate–binding poly(allylamine hydrochloride) hydrogels for wastewater remediation

Systems that are capable of removing highly toxic anions from wastewater effluents, even at extremely low concentrations, are a major need in the defense industry. This study reports on the features of two new batch and continuous‐flow sorption processes with regard to ultimate removal and recovery of the perchlorate (ClO) anion from ammonium perchlorate (NH₄ClO₄) wastewater. The sorbent developed is a crosslinked poly(allylamine hydrochloride) (PAA·HCl) polymeric hydrogel. The pH‐sensitive PAA·HCl hydrogels were synthesized by chemically crosslinking a solution of linear PAA·HCl chains with epichlorohydrin (EPI). The perchlorate‐binding capacity of the polymer gels was measured in standard solutions and studied as a function of gel synthesis parameters. Equilibrium perchlorate loadings of 5770 ± 870 mg ClO/g gel were calculated from measurement of the decrease in perchlorate concentration in aqueous standard solutions using UV‐Vis spectrophotometry. Batch experiments in wastewater originating from the Naval Surface Warfare Center (NSWC) Indian Head Division showed that perchlorate concentrations decreased by 85%. Preliminary lab‐scale packed‐column experiments in wastewater achieved up to 40% reduction in total perchlorate content. The regeneration ability of the gels was demonstrated by release of the bound perchlorate anions, upon washing with a 1N NaOH solution, providing opportunities to recover and reuse the hydrogel over multiple regeneration cycles. The PAA·HCl hydrogels are demonstrated to be appropriate materials for treating wastewaters that contain ammonium perchlorate. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 2073–2083, 2001     

Modern Approaches to Acellular Therapy in Bone and Dental Regeneration

An approach called cell-free therapy has rapidly developed in regenerative medicine over the past decade. Understanding the molecular mechanisms and signaling pathways involved in the internal potential of tissue repair inspires the development of new strategies aimed at controlling and enhancing these processes during regeneration. The use of stem cell mobilization, or homing for regeneration based on endogenous healing mechanisms, prompted a new concept in regenerative medicine: endogenous regenerative medicine. The application of cell-free therapeutic agents leading to the recruitment/homing of endogenous stem cells has advantages in overcoming the limitations and risks associated with cell therapy. In this review, we discuss the potential of cell-free products such as the decellularized extracellular matrix, growth factors, extracellular vesicles and miRNAs in endogenous bone and dental regeneration.     

Degradable natural polymer hydrogels for articular cartilage tissue engineering

Articular cartilage has poor ability to heal once damaged. Tissue engineering with scaffolds of polymer hydrogels is promising for cartilage regeneration and repair. Polymer hydrogels composed of highly hydrated crosslinked networks mimic the collagen networks of the cartilage extracellular matrix and thus are employed as inserts at cartilage defects not only to temporarily relieve the pain but also to support chondrocyte proliferation and neocartilage regeneration. The biocompatibility, biofunctionality, mechanical properties, and degradation of the polymer hydrogels are the most important parameters for hydrogel‐based cartilage tissue engineering. Degradable biopolymers with natural origin have been widely used as biomaterials for tissue engineering because of their outstanding biocompatibility, low immunological response, low cytotoxicity, and excellent capability to promote cell adhesion, proliferation, and regeneration of new tissues. This review covers several important natural proteins (collagen, gelatin, fibroin, and fibrin) and polysaccharides (chitosan, hyaluronan, alginate and agarose) widely used as hydrogels for articular cartilage tissue engineering. The mechanical properties, structures, modification, and structure–performance relationship of these hydrogels are discussed since the chemical structures and physical properties dictate the in vivo performance and applications of polymer hydrogels for articular cartilage regeneration and repair. © 2012 Society of Chemical Industry     

Periostin Is Required for the Maintenance of Muscle Fibers during Muscle Regeneration

Skeletal muscle regeneration is a well-organized process that requires remodeling of the extracellular matrix (ECM). In this study, we revealed the protective role of periostin, a matricellular protein that binds to several ECM proteins during muscle regeneration. In intact muscle, periostin was localized at the neuromuscular junction, muscle spindle, and myotendinous junction, which are connection sites between muscle fibers and nerves or tendons. During muscle regeneration, periostin exhibited robustly increased expression and localization at the interstitial space. Periostin-null mice showed decreased muscle weight due to the loss of muscle fibers during repeated muscle regeneration. Cultured muscle progenitor cells from periostin-null mice showed no deficiencies in their proliferation, differentiation, and the expression of Pax7, MyoD, and myogenin, suggesting that the loss of muscle fibers in periostin-null mice was not due to the impaired function of muscle stem/progenitor cells. Periostin-null mice displayed a decreased number of CD31-positive blood vessels during muscle regeneration, suggesting that the decreased nutritional supply from blood vessels was the cause of muscle fiber loss in periostin-null mice. These results highlight the novel role of periostin in maintaining muscle mass during muscle regeneration.     

Polymerizable Skin Hydrogel for Full Thickness Wound Healing

The skin is the largest organ in the human body, comprising the main barrier against the environment. When the skin loses its integrity, it is critical to replace it to prevent water loss and the proliferation of opportunistic infections. For more than 40 years, tissue-engineered skin grafts have been based on the in vitro culture of keratinocytes over different scaffolds, requiring between 3 to 4 weeks of tissue culture before being used clinically. In this study, we describe the development of a polymerizable skin hydrogel consisting of keratinocytes and fibroblast entrapped within a fibrin scaffold. We histologically characterized the construct and evaluated its use on an in vivo wound healing model of skin damage. Our results indicate that the proposed methodology can be used to effectively regenerate skin wounds, avoiding the secondary in vitro culture steps and thus, shortening the time needed until transplantation in comparison with other bilayer skin models. This is achievable due to the instant polymerization of the keratinocytes and fibroblast combination that allows a direct application on the wound. We suggest that the polymerizable skin hydrogel is an inexpensive, easy and rapid treatment that could be transferred into clinical practice in order to improve the treatment of skin wounds.     

Extracellular Environment-Controlled Angiogenesis,and Potential Application for Peripheral Nerve Regeneration

Endothelial cells acquire different phenotypes to establish functional vascular networks. Vascular endothelial growth factor (VEGF) signaling induces endothelial proliferation, migration, and survival to regulate vascular development, which leads to the construction of a vascular plexuses with a regular morphology. The spatiotemporal localization of angiogenic factors and the extracellular matrix play fundamental roles in ensuring the proper regulation of angiogenesis. This review article highlights how and what kinds of extracellular environmental molecules regulate angiogenesis. Close interactions between the vascular and neural systems involve shared molecular mechanisms to coordinate developmental and regenerative processes. This review article focuses on current knowledge about the roles of angiogenesis in peripheral nerve regeneration and the latest therapeutic strategies for the treatment of peripheral nerve injury.     

Controlled Liposome Delivery from Chitosan-Based Thermosensitive Hydrogel for Regenerative Medicine

This work describes the development of an injectable nanocomposite system based on a chitosan thermosensitive hydrogel combined with liposomes for regenerative medicine applications. Liposomes with good physicochemical properties are prepared and embedded within the chitosan network. The resulting nanocomposite hydrogel is able to provide a controlled release of the content from liposomes, which are able to interact with cells and be internalized. The cellular uptake is enhanced by the presence of a chitosan coating, and cells incubated with liposomes embedded within thermosensitive hydrogels displayed a higher cell uptake compared to cells incubated with liposomes alone. Furthermore, the gelation temperature of the system resulted to be equal to 32.6 °C; thus, the system can be easily injected in the target site to form a hydrogel at physiological temperature. Given the peculiar performance of the selected systems, the resulting thermosensitive hydrogels are a versatile platform and display potential applications as controlled delivery systems of liposomes for tissue regeneration.     

Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin’s influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.     

Bacterial Cellulose and ECM Hydrogels: An Innovative Approach for Cardiovascular Regenerative Medicine

Cardiovascular diseases are considered the leading cause of death in the world, accounting for approximately 85% of sudden death cases. In dogs and cats, sudden cardiac death occurs commonly, despite the scarcity of available pathophysiological and prevalence data. Conventional treatments are not able to treat injured myocardium. Despite advances in cardiac therapy in recent decades, transplantation remains the gold standard treatment for most heart diseases in humans. In veterinary medicine, therapy seeks to control clinical signs, delay the evolution of the disease and provide a better quality of life, although transplantation is the ideal treatment. Both human and veterinary medicine face major challenges regarding the transplantation process, although each area presents different realities. In this context, it is necessary to search for alternative methods that overcome the recovery deficiency of injured myocardial tissue. Application of biomaterials is one of the most innovative treatments for heart regeneration, involving the use of hydrogels from decellularized extracellular matrix, and their association with nanomaterials, such as alginate, chitosan, hyaluronic acid and gelatin. A promising material is bacterial cellulose hydrogel, due to its nanostructure and morphology being similar to collagen. Cellulose provides support and immobilization of cells, which can result in better cell adhesion, growth and proliferation, making it a safe and innovative material for cardiovascular repair.     

Synthesis and characterization of electrospun cerium-doped bioactive glass/chitosan/polyethylene oxide composite scaffolds for tissue engineering applications

In this study, a series of electrospun chitosan/polyethylene oxide (PEO) nanofibrous scaffolds containing different amount of cerium-doped bioactive glasses (Ce-BGs) have been fabricated and proposed for tissue engineering applications. On a biological level, higher 8Ce-BG content significantly improved cytocompatibility of the scaffolds. Moreover, results of fibroblast cell culture study showed that greater 8Ce-BG content could enhance cell attachment and cell expansion on fiber mesh. Characterization of the scaffolds revealed that increasing 8Ce-BG content caused bioactive glass nanoparticles to agglomerate at a higher rate. The SEM mapping revealed thorough dispersion of submicrometric clusters in all areas of the polymeric matrix. Contact angle measurements showed that increasing 8Ce-BG/CH ratio from 0 to 10 (wt.%) improved wettability of the scaffold significantly. However, by increasing the ratio beyond 10 (wt.%), the wettability values decreased gradually. In conclusion, it was found that increasing 8Ce-BG/CH weight ratio up to 40 (wt.%) in the scaffold system was practical and useful for soft tissue engineering applications.