组织再生：梦想、希望和挑战

组织再生是21世纪生物学和医学领域研究的重点和热点.在回顾再生医学研究历史的基础上,概要介绍了组织再生所涉及的关键科学问题、重大需求以及部分已经取得的重要进展,并对将来的发展进行了展望.     

Enhanced mesenchymal stromal cell recruitment via natural killer cells by incorporation of inflammatory signals in biomaterials

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an ‘ideal’ inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell–MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies.     

Bone regeneration using coculture of mesenchymal stem cells and angiogenic cells

Cellular strategies remain a crucial component in bone tissue engineering （BTE）. So far, the outcome of cell-based strategies from initial clinical trials is far behind compared to animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the Ussue-engineered constructs. Cocultures, by introducing angiogenic cells into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, pre-clinical studies demonstrated that cocultures enhance vascularization and bone formation compared to monocultures. However, there has been no report on the application of cocultures in clinics. Therefore, this mini-review aims to provide an overview regarding （i） critical parameters in cocultures and the outcomes of cocultures compared to monocultures in the currently available pre-clinical studies using human mesenchymal stem cells implanted in orthotopic animal models; and （ii） the usage of monocultures in clinical application in BTE.     

Use of adipose stem cells and polylactide discs for tissue engineering of the temporomandibular joint disc

There is currently no suitable replacement for damaged temporomandibular joint (TMJ) discs after discectomy. In the present study, we fabricated bilayer biodegradable polylactide (PLA) discs comprising a non-woven mat of poly(L/D)lactide (P(L/D)LA) 96/4 and a P(L/DL)LA 70/30 membrane plate. The PLA disc was examined in combination with adipose stem cells (ASCs) for tissue engineering of the fibrocartilaginous TMJ disc in vitro. ASCs were cultured in parallel in control and chondrogenic medium for a maximum of six weeks. Relative expression of the genes, aggrecan, type I collagen and type II collagen present in the TMJ disc extracellular matrix increased in the ASC-seeded PLA discs in the chondrogenic medium. The hypertrophic marker, type X collagen, was moderately induced. Alcian blue staining showed accumulation of sulphated glycosaminoglycans. ASC differentiation in the PLA discs was close to that observed in pellet cultures. Comparison of the mRNA levels revealed that the degree of ASC differentiation was lower than that in TMJ disc-derived cells and tissue. The pellet format supported the phenotype of the TMJ disc-derived cells under chondrogenic conditions and also enhanced their hyalinization potential, which is considered part of the TMJ disc degeneration process. Accordingly, the combination of ASCs and PLA discs has potential for the development of a tissue-engineered TMJ disc replacement.     

Autologous adipose stem cells and polylactide discs in the replacement of the rabbit temporomandibular joint disc

The temporomandibular joint (TMJ) disc lacks functional replacement after discectomy. We investigated tissue-engineered bilayer polylactide (PLA) discs and autologous adipose stem cells (ASCs) as a potential replacement for the TMJ disc. These ASC discs were pre-cultured either in control or in differentiation medium, including transforming growth factor (TGF)-β1 for one week. Prior to implantation, expression of fibrocartilaginous genes was measured by qRT-PCR. The control and differentiated ASC discs were implanted, respectively, in the right and left TMJs of rabbits for six (n = 5) and 12 months (n = 5). Thereafter, the excised TMJ areas were examined with cone beam computed tomography (CBCT) and histology. No signs of infection, inflammation or foreign body reactions were detected at histology, whereas chronic arthrosis and considerable condylar hypertrophy were observed in all operated joints at CBCT. The left condyle treated with the differentiated ASC discs appeared consistently smoother and more sclerotic than the right condyle. The ASC disc replacement resulted in dislocation and morphological changes in the rabbit TMJ. The ASC discs pre-treated with TGF-β1 enhanced the condylar integrity. While adverse tissue reactions were not shown, the authors suggest that with improved attachment and design, the PLA disc and biomaterial itself would hold potential for TMJ disc replacement.     

Tissue Engineering Strategies Designed to Realize the Endogenous Regenerative Potential of Peripheral Nerves

http://doi.wiley.com/10.1002/adma.v21:32/33 Bridging peripheral nerve gaps without the use of autografts has significant clinical importance. But in order to rationally design novel scaffolds, a good understanding of the nerve regeneration process is vital. Appropriate amount of structural and chemical cues are required to stimulate the endogenous mechanisms of repair and functional recovery. Synthetic and natural materials present various opportunities to induce the growth of supporting cells as well as promote axon regeneration. An overview of tissue engineering strategies currently being explored that stimulate the different steps of the regenerative sequence is presented.     

Small extracellular vesicles secreted by urine-derived stem cells enhanced wound healing in aged mice by ameliorating cellular senescence

Regeneration of chronic skin wounds in tissue is still a key challenge in regenerative medicine because of the accumulation of senescent cells and increasing secretion of s¨enescence-associated secretory phenotype(SASP)in the wound site.Recently,some studies have reported that small extracellular vesicles(sEVs)derived from stem cells can alleviate cellular senescence with very low risk of tumorigenesis and immune responses.As our previous studies have shown that urine-derived stem cells(USCs)can be obtained easily and noninvasively and sEVs derived from USCs(USC-sEVs)have capabilities of regenerating tissue injuries,using USC-sEVs to enhance chronic skin wound healing in aged tissue might be a feasible and efficient strategy.Therefore,in this study,the USC-sEVs were collected and firstly loaded in a human acellular amniotic membrane(HAAM)for controlled releasing and locating the USC-sEVs in the wound site before they were implanted into a chronic skin wound in aged mice.In vivo results showed that the USC-sEVs in HAAM could effectively accelerate the wound healing by ameliorating cellular senescence and reducing the secretion of SASP in the aged skin wounds.To elucidate the mechanism,USC-sEVs were used to in vitro culture human dermal fibroblasts(HDFs)and results showed that USC-sEVs could rejuvenate senescent fibroblasts by reversing the aging phenotypes of senescent HDFs and efficiently reducing the secretion of SASP after they activated the Sirt1 pathway.Therefore,USC-sEVs are efficient for enhancing wound healing in aged mice by ameliorating cellular senescence.     

Manipulation of live mouse embryonic stem cells using holographic optical tweezers

We report the ability to move and arrange patterns of live embryonic stem cells using holographic optical tweezers. Single cell suspensions of mouse embryonic stem cells were manipulated with holographic optical tweezers into a variety of patterns including lines, curves and circles. Individual cells were also lifted out of the sample plane highlighting the potential for 3D positional control. Trypan blue dye exclusion and Live/Dead? staining (CMFDA^?1, EthHD^?1) showed that the cells were still viable after manipulation with the optical tweezers. The ability to move individual stem cells into specific, pre-defined patterns provides a method to study how arrangement and associated small-scale interactions occur between neighbouring cells.     

Targeting the stem cell niche with regenerative biomaterials

Regenerative medicine aims to restore form and function to aged, injured, and diseased tissues. One strategy that is gaining traction is in vivo regenerative medicine. Many adult tissues possess stem and progenitor cells that are enlisted to repair minor tissue damage after insult. However, in the setting of disease, aging, or criticalsized injuries, the microenvironment may lack structural elements, physical, and/or chemical cues required to drive repair to completion. Natural and synthetic materials offer an opportunity to facilitate the repair process by restoring the natural reparative capacity of adult tissues. Using design criteria selected based upon an understanding of the inductive niche cues that naturally instruct stem and progenitor cell behavior and fate, it is possible to elicit tissue repair by implementing a cell-free approach. This review highlights recent studies that assess biomaterials for in vivo regenerative medicine applications and demonstrate a capacity for controlling stem and progenitor cell behavior to restore tissue form and function.     

In vivo bone regeneration with injectable chitosan/hydroxyapatite/collagen composites and mesenchymal stem cells

For reconstruction of irregular bone defects, injectable biomaterials are more appropriate than the preformed biomaterials. We herein develop a biomimetic in situ-forming composite consisting of chitosan (CS) and mineralized collagen fibrils (nHAC), which has a complex hierarchical structure similar to natural bone. The CS/nHAC composites with or without mesenchymal stem cells (MSCs) are injected into cancellous bone defects at the distal end of rabbit femurs. Defects are assessed by radiographic, histological diagnosis and Raman microscopy until 12 weeks. The results show that MSCs improve the biocompatibility of CS/nHAC composites and enhance new bone formation in vivo at 12 weeks. It can be concluded that the injectable CS/nHAC composites combined with MSCs may be a novel method for reconstruction of irregular bone defects.     

A Micro‐Ark for Cells: Highly Open Porous Polyhydroxyalkanoate Microspheres as Injectable Scaffolds for Tissue Regeneration

To avoid large open surgery using scaffold transplants, small‐sized cell carriers are employed to repair complexly shaped tissue defects. However, most cell carriers show poor cell adherences and viability. Therefore, polyhydroxyalkanoate (PHA), a natural biopolymer, is used to prepare highly open porous microspheres (OPMs) of 300–360 µm in diameter, combining the advantages of microspheres and scaffolds to serve as injectable carriers harboring proliferating stem cells. In addition to the convenient injection to a defected tissue, and in contrast to poor performances of OPMs made of polylactides (PLA OPMs) and traditional less porous hollow microspheres (PHA HMs), PHA OPMs present suitable surface pores of 10–60 µm and interconnected passages with an average size of 8.8 µm, leading to a high in vitro cell adhesion of 93.4%, continuous proliferation for 10 d and improved differentiation of human bone marrow mesenchymal stem cells (hMSCs). PHA OPMs also support stronger osteoblast‐regeneration compared with traditional PHA HMs, PLA OPMs, commercial hyaluronic acid hydrogels, and carrier‐free hMSCs in an ectopic bone‐formation mouse model. PHA OPMs protect cells against stresses during injection, allowing more living cells to proliferate and migrate to damaged tissues. They function like a micro‐Noah's Ark to safely transport cells to a defect tissue.     

Three-dimensional printing of biomaterials for bone tissue engineering: a review

Processing biomaterials into porous scaffolds for bone tissue engineering is a critical and a key step in defining and controlling their physicochemical, mechanical, and biological properties. Biomaterials such as polymers are commonly processed into porous scaffolds using conventional processing techniques, e.g., salt leaching. However, these traditional techniques have shown unavoidable limitations and several shortcomings. For instance, tissue-engineered porous scaffolds with a complex three-dimensional (3D) geometric architecture mimicking the complexity of the extracellular matrix of native tissues and with the ability to fit into irregular tissue defects cannot be produced using the conventional processing techniques. 3D printing has recently emerged as an advanced processing technology that enables the processing of biomaterials into 3D porous scaffolds with highly complex architectures and tunable shapes to precisely fit into irregular and complex tissue defects. 3D printing provides computer-based layer-by-layer additive manufacturing processes of highly precise and complex 3D structures with well-defined porosity and controlled mechanical properties in a highly reproducible manner. Furthermore, 3D printing technology provides an accurate patient-specific tissue defect model and enables the fabrication of a patient-specific tissue-engineered porous scaffold with pre-customized properties.     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Abstract

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

中枢神经再生材料研究进展

首先介绍了目前中枢神经再生面临的问题和应对策略,然后系统地综述了脑再生和脊髓再生修复材料的发展。研究发现,成人中枢神经系统内存的神经干细胞和具有特定分化方向的前体细胞具有潜在的、巨大的修复功能;生物支架材料与神经干细胞的联合使用能够较好地控制细胞微环境,有望提高细胞移植后的存活状况,促进中枢神经再生。最后,结合现在中枢神经再生的研究热点——神经干细胞,阐述了中枢神经再生材料调控干细胞的研究进展和潜能,为联合应用生物材料与干细胞促进中枢再生提供了参考。     

矿化柞蚕丝胶膜表面粗糙度的调控及其对骨髓间充质干细胞生长行为的影响

生物材料表面的粗糙度是影响细胞行为的重要因素之一。为了调控丝蛋白生物材料表面的粗糙度,并评价材料表面粗糙度对细胞生长行为的影响,首先,通过湿化学共沉淀法,以柞蚕丝胶(AS)溶液为模板,诱导了羟基磷灰石(HAp)晶体成核,进而调控了AS膜表面的粗糙度。然后,采用SEM、粗糙仪、FTIR及EDX等对HAp/AS复合膜表面形貌、粗糙度及成分进行了表征。最后,通过SEM和CellTiter 96?AQueous单溶液细胞增殖检测试剂盒(MTS)检测了骨髓间充质干细胞(BMSCs)在HAp/AS复合膜表面的形貌及增殖率。结果表明:纯AS膜的表面粗糙度为0.15μm,矿化1、8及24h后,表面粗糙度分别为0.38、0.46和1.20μm;矿化24h后,在HAp/AS复合膜表面可观察到直径为30~80nm的球状复合物,生成的矿化物为HAp;HAp/AS复合膜具有良好的细胞相容性,表面粗糙度为1.20μm的复合膜能够显著促进BMSCs的增殖,粗糙度对BMSCs在HAp/AS复合膜表面的粘附和形貌有着重要的影响。因此,可通过矿化的方法在生物大分子表面诱导HAp晶体的成核与生长,从而调控材料的表面粗糙度,研究材料界面上的细胞行为。     

The Role of Bone Morphogenetic Proteins in Tissue Engineering Particulate Bone Grafts

Bone defects of various causes are important medical and socioeconomical problems because of the impossibility of spontaneous healing, difficult treatment, and long healing period. There are multiple, varied, and relatively complicated ways of solving these problems. Over time, numerous investigations carried out have shown failures arising from the use of autografts and homografts. The disadvantages of these methods prompted a search for other methods of bone reconstruction. Bone substitutes can play an important role in bone reconstructive surgery. In this context, tissue engineering bone grafts has offered an alternative. The aim of our research was to evaluate the feasibility of creating a tissue-engineered bone using mesenchymal stem cells seeded on a scaffold obtained from the red deer deciduous horn. We tried to demonstrate the advantages of using bone morphogenetic proteins (BMP-2) and transforming growth factor β (TGF-β) as promoters of the differentiation process. Our study was carried out on animal model, an outbred CD 1 mouse strain. Our research demonstrated that supplementation with growth factors (BMP-2, TGF-β) in osteogenic medium improved and accelerated bony-line differentiation and mineralization process. The same factors accelerated and stabilized the osteoblast differentiation and inhibit different lineage appearance such as myeloid metaplasia.