Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.     

Osteogenesis of Osteoblast Seeded Polyurethane-Hydroxyapatite Scaffolds in Nude Mice

Summary: Biodegradable porous polyurethane (PU) scaffolds were used in a tissue engineering approach to create new bone. Two groups of elastomeric bioresorbable PU disks were seeded with osteoblasts and implanted into nude mice. One group had disks of pure PU while the other group had disks of PU- hydroxyapatite composite (PU-HA). After 5 weeks both groups showed radiographic and histologic evidence of significant bone formation. As the new bone formed it replaced the PU scaffolds. Although not statistically significant, there was a trend toward more bone formation in the PU-HA group. Bioresorbable PU shows promise for use in bone tissue engineering.     

Intramarrow injection of β-catenin-activated,but not na?ve mesenchymal stromal cells stimulates self-renewal of hematopoietic stem cells in bone marrow

Bone marrow mesenchymal stromal cells (MSCs) have been implicated in the microenvironmental support of hematopoietic stem cells (HSCs) and often co-transplanted with HSCs to facilitate recovery of ablated bone marrows. However, the precise effect of transplanted MSCs on HSC regeneration remains unclear because the kinetics of HSC self-renewal in vivo after co-transplantation has not been monitored. In this study, we examined the effects of intrafemoral injection of MSCs on HSC self-renewal in rigorous competitive repopulating unit (CRU) assays using congenic transplantation models in which stromal progenitors (CFU-F) were ablated by irradiation. Interestingly, naïve MSCs injected into femur contributed to the reconstitution of a stromal niche in the ablated bone marrows, but did not exert a stimulatory effect on the in-vivo self-renewal of co-transplanted HSCs regardless of the transplantation methods. In contrast, HSC self-renewal was four-fold higher in bone marrows intrafemorally injected with β-catenin-activated MSCs. These results reveal that naïve MSCs lack a stimulatory effect on HSC self-renewal in-vivo and that stroma must be activated during recoveries of bone marrows. Stromal targeting of wnt/β-catenin signals may be a strategy to activate such a stem cell niche for efficient regeneration of bone marrow HSCs.     

球状多孔羟基磷灰石生物材料的制备与结构

牛血清白蛋白;药物缓释;球状多孔羟基磷灰石生物材料的制备与结构     

多孔羟基磷灰石纳米球作为抗坏血酸载体的研究

利用超声辅助的化学共沉淀法制备了羟基磷灰石纳米材料, 并实现了其对抗坏血酸的包载. 采用透射电镜、氮气吸附孔径分析仪、X射线粉末衍射仪、傅里叶红外光谱仪和紫外分光光度计等对所得纳米材料的微观形貌、孔径分布、物相、表面官能团和负载抗坏血酸量进行了表征和分析, 并在超声辅助条件下进行了药物释放动力学研究. 结果表明, 在抗坏血酸存在的条件下, 利用声化学方法可以制得多孔的羟基磷灰石纳米球, 其平均直径约为140 nm, 平均孔直径约为15 nm, 可实现对抗坏血酸的包载. 此外, 在超声辅助的条件下, 该纳米球具有持续释药的能力, 不同的超声功率能改变载药粒的释药速度, 表明这种多孔羟基磷灰石纳米球是一种具有很大应用前景的骨组织药物载体.     

Gd3+对原代培养的小鼠骨髓基质细胞成骨分化和成脂分化的影响

本文采用MTT法、碱性磷酸酶活性测定、矿化功能的测定以及油红O的染色和定量测定等手段研究了Gd3+对原代培养的小鼠骨髓基质细胞成骨分化和成脂分化的影响。研究结果表明,浓度为1×10-10和1×10-8 mol.L-1的Gd3+对小鼠骨髓基质细胞的增殖没有影响,其他测试浓度下的Gd3+则抑制小鼠骨髓基质细胞的增殖。当Gd3+与小鼠骨髓基质细胞作用7 d时,其对小鼠骨髓基质细胞成骨分化的影响与作用浓度有关,当Gd3+与小鼠骨髓基质细胞作用14 d时,在全部测试浓度范围内,抑制小鼠骨髓基质细胞成骨分化。除1×10-8和1×10-5 mol.L-1外,其他测试浓度下的Gd3+促进小鼠骨髓基质细胞的矿化功能。当Gd3+与小鼠骨髓基质细胞作用10 d时,其抑制小鼠骨髓基质细胞的成脂分化,当Gd3+与小鼠骨髓基质细胞作用16 d时,除1×10-9mol.L-1外,其他浓度的Gd3+也抑制小鼠骨髓基质细胞的成脂分化。实验结果提示,Gd3+可能通过促进骨髓基质细胞的成骨分化、抑制其成脂分化途径起到对骨的保护作用。Gd3+对原代培养的小鼠骨髓基质细胞成骨分化和成脂分化的影响与作用浓度和时间有关,而且,它们是影响Gd3+对骨是损伤还是保护作用转变的关键因素。     

3D and Porous RGDC‐Functionalized Polyester‐Based Scaffolds as a Niche to Induce Osteogenic Differentiation of Human Bone Marrow Stem Cells

Polyester‐based scaffolds covalently functionalized with arginine‐glycine‐aspartic acid‐cysteine (RGDC) peptide sequences support the proliferation and osteogenic differentiation of stem cells. The aim is to create an optimized 3D niche to sustain human bone marrow stem cell (hBMSC) viability and osteogenic commitment, without reliance on differentiation media. Scaffolds consisting of poly(lactide‐co‐trimethylene carbonate), poly(LA‐co‐TMC), and functionalized poly(lactide) copolymers with pendant thiol groups are prepared by salt‐leaching technique. The availability of functional groups on scaffold surfaces allows for an easy and straightforward method to covalently attach RGDC peptide motifs without affecting the polymerization degree. The strategy enables the chemical binding of bioactive motifs on the surfaces of 3D scaffolds and avoids conventional methods that require harsh conditions. Gene and protein levels and mineral deposition indicate the osteogenic commitment of hBMSC cultured on the RGDC functionalized surfaces. The osteogenic commitment of hBMSC is enhanced on functionalized surfaces compared with nonfunctionalized surfaces and without supplementing media with osteogenic factors. Poly(LA‐co‐TMC) scaffolds have potential as scaffolds for osteoblast culture and bone grafts. Furthermore, these results contribute to the development of biomimetic materials and allow a deeper comprehension of the importance of RGD peptides on stem cell transition toward osteoblastic lineage.     

多巴胺改性海藻酸多孔支架的制备与性能

生物大分子海藻酸(Alg)由于其安全、无毒、可生物降解等特性而被广泛应用于组织工程领域。 受海洋贻贝蛋白结构的启发,多巴胺(DA)具有优异的粘附性能,在碱性水溶液条件下可发生氧化自聚形成聚多巴胺(PDA)。 以Alg为基体,加入PDA纳米粒子复合,并通过冷冻干燥法制备得到Alg/PDA多孔支架材料。 结果表明,Alg/PDA多孔支架材料具有较为规整的内部结构。 改变Alg质量浓度,Alg/PDA支架材料的孔径可控制在60~120 μm之间,孔隙率可控于80%~88%。 所得的支架材料具有适宜大小的孔径和孔隙率,结果表明支架材料对细胞无毒副作用。     

从稀土对骨代谢的影响看稀土的药用及安全性

骨骼的正常代谢涉及多种金属离子,随着稀土的应用,通过环境和经由食物链等渠道进入人体内的稀土量也有所增加, 并且在骨组织中相对富集且难排出;由于稀土与钙离子的相似性,它们将干预和调控骨的形成与再造。一方面,稀土被设想可以用来作为治疗骨疾病的药物, 另一方面,它对骨代谢的影响产生负面作用。因此, 稀土的药用和安全性日益成为人们关注的问题。本文回顾了稀土离子对骨代谢影响的研究进展,探讨了其药用和安全性问题, 主要从以下三个方面进行综述: (1)稀土离子对成骨细胞细胞和破骨细胞的分化和功能表达的影响; (2)稀土离子对骨髓基质细胞成骨分化和成脂分化的影响; (3)稀土离子对动物骨矿物相的影响。此外,还指出了该研究领域的不足以及今后应该加强的研究方向。     

利用溶剂热压方法制备羟基磷灰石多孔纳米固体

利用溶剂热压方法,以羟基磷灰石（HAP）纳米颗粒为原料制备了一种介于纳米粉体和纳米陶瓷体之间的过渡态——体块HAP多孔纳米固体,并用X射线衍射（XRD）、扫描电子显微镜（SEM）、压汞仪和热分析（DSC和TGA）等方法对样品进行了表征,初步研究了溶剂的种类、分布均匀性等因素对HAP多孔纳米固体的孔容和孔径分布的影响,并对HAP多孔纳米固体的热稳定性进行了分析.     

A Copolymer Scaffold Functionalized with Nanodiamond Particles Enhances Osteogenic Metabolic Activity and Bone Regeneration

Functionalizing polymer scaffolds with nanodiamond particles (nDPs) has pronounced effect on the surface properties, such as improved wettability, an increased active area and binding sites for cellular attachment and adhesion, and increased ability to immobilize biomolecules by physical adsorption. This study aims to evaluate the effect of poly(l ‐lactide‐co‐ε‐caprolactone) (poly(LLA‐co‐CL)) scaffolds, functionalized with nDPs, on bone regeneration in a rat calvarial critical size defect. Poly(LLA‐co‐CL) scaffolds functionalized with nDPs are also compared with pristine scaffolds with reference to albumin adsorption and seeding efficiency of bone marrow stromal cells (BMSCs). Compared with pristine scaffolds, the experimental scaffolds exhibit a reduction in albumin adsorption and a significant increase in the seeding efficiency of BMSCs (p = 0.027). In the calvarial defects implanted with BMSC‐seeded poly(LLA‐co‐CL)/nDPs scaffolds, live imaging at 12 weeks discloses a significant increase in osteogenic metabolic activity (p = 0.016). Microcomputed tomography, confirmed by histological data, reveals a substantial increase in bone volume (p = 0.021). The results show that compared with conventional poly(LLA‐co‐CL) scaffolds those functionalized with nDPs promote osteogenic metabolic activity and mineralization capacity. It is concluded that poly(LLA‐co‐CL) composite matrices functionalized with nDPs enhance osteoconductivity and therefore warrant further study as potential scaffolding material for bone tissue engineering.

     

粒子沥滤法制备组织工程用多孔支架的研究进展

粒子沥滤法作为常用的制备组织工程支架的方法,这种方法对实验设备和实验条件要求较低,孔隙率、孔径可控。但传统的溶液浇铸/粒子沥滤法也存在一些缺点,只适合制备薄层支架,孔-孔连通性不好,聚合物有可能保留致孔剂和/或有毒溶剂。本文总结了近几年科研工作者对这种方法所进行的改进工作,并探讨了改进的效果和不足。     

急/慢性粒细胞白血病患者骨髓血细胞的FTIR研究

研究了急性粒细胞白血病（AML）和慢性粒细胞白血病（CML）患者的骨髓淋巴细胞、粒细胞和红细胞的傅立叶红外光谱（FTIR）。结果发现：（1）这三种细胞FTIR存在着差异性。（2）AML与CML之间淋巴细胞和粒细胞的谱图差异性不明显，但红细胞的谱图却存在可识别的差异性。与CML红细胞相比，AML红细胞酰胺Ⅰ带较宽，AML红外细胞蛋白质二级结构中α-螺旋结构的含量较低；1540cm^-1与1084cm^-1吸收峰面积的比值较低，范围较窄，该结果提示，AML和CML骨髓血红细胞中核红细胞的数量分布不同，同时提示骨髓红细胞的成熟可能与白血病细胞的增殖有关。     

以大蓟髓芯为模板制备复合孔生物活性玻璃及其体外成骨性能

以天然植物大蓟髓芯为大孔模板, 以嵌段共聚物为介孔软模板, 制备了孔径为60~100 μm、孔壁为介孔相的高度有序多级复合孔生物活性玻璃. 用扫描电子显微镜(SEM)、粉末X射线衍射仪(XRD)、高分辨率透射电镜(HRTEM)及N2吸附-脱附等测试手段对合成的样品进行了表征. 结果表明, 合成的材料精确地复制了植物模板的形貌, 同时具有较高的比表面积和较大的孔容. 通过体外模拟生理体液测试表明, 这种复合孔生物活性玻璃可诱导羟基磷灰石晶体在其表面形成, 具有良好的体外成骨性能, 因而在骨组织修复方面具有潜在应用前景.     

Structural Requirement of hA5G18 Peptide (DDFVFYVGGYPS) from Laminin α5 Chain for Amyloid-like Fibril Formation and Cell Adhesion

The hA5G18 peptide (DDFVFYVGGYPS) identified from the human laminin α5 chain G domain promotes cell attachment and spreading when directly coated on a plastic plate, but does not show activity when it is conjugated on a chitosan matrix. Here, we focused on the structural requirement of hA5G18 for activity. hA5G18 was stained with Congo red and formed amyloid-like fibrils. A deletion analysis of hA5G18 revealed that FVFYV was a minimum active sequence for the formation of amyloid-like fibrils, but FVFYV did not promote cell attachment. Next, we designed functional fibrils using FVFYV as a template for amyloid-like fibrils. When we conjugated an integrin binding sequence Arg-Gly-Asp (RGD) to the FVFYV peptide with Gly-Gly (GG) as a spacer, FVFYVGGRGD promoted cell attachment in a plate coat assay, but a negative control sequence RGE conjugated peptide, FVFYVGGRGE, also showed activity. However, when the peptides were conjugated to Sepharose beads, the FVFYVGGRGD beads showed cell attachment activity, but the FVFYVGGRGE beads did not. These results suggest that RGD and RGE similarly contribute to cell attachment activity in amyloid-like fibrils, but only RGD contributes the activity on the Sepharose beads. Further, we conjugated a basic amino acid (Arg, Lys, and His) to the FVFYV peptide. Arg or Lys-conjugated FVFYV peptides, FVFYVGGR and FVFYVGGK, showed cell attachment activity when they were coated on a plate, but a His-conjugated FVFYV peptide FVFYVGGH did not show activity. None of the basic amino acid-conjugated peptides showed cell attachment in a Sepharose bead assay. The cell attachment and spreading on FVFYVGGR and FVFYVGGK were inhibited by an anti-integrin β1 antibody. These results suggest that the Arg and Lys residues play critical roles in the interaction with integrins in amyloid-like fibrils. FVFYV is useful to use as a template for amyloid-like fibrils and to develop multi-functional biomaterials.