In vitro study on hemocompatibility and cytocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Samples of polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 4–20% (mol/mol) 3-hydroxyhexanoate (3HHx) were characterized as potential components of blood-contact biomaterials. In an erythrocyte contact hemolysis assay, all tested PHBHHx films had substantially reduced reactivity, typically displaying about 2-fold less hemolytic activity compared with that of PHBV. Both 12% and 20% containing PHBHHx also bound less platelets than other films. After a 120-min exposure to platelet-rich plasma (PRP), few platelets adhered to the 12% and 20% containing PHBHHx films, while numerous platelets were seen on PHBV. Surface properties investigation suggested along with increasing 3HHx content, PHBHHx co-polymer films became smoother and smoother, which may contribute to lower platelet adhesion of PHBHHx containing high HHx content in a short-term contact to platelet-rich plasma. In a long-term contact to PRP, the difference in crystallization of PHBVand PHBHHx can be a critical parameter for platelet adhesion. Human umbilical vein endothelial cells (HUVECs) grew well on PHBHHx containing high content of 3HHx, indicating that both had good biocompatibility with HUVECs. While gelatin-coated or lipase-treated polyesters improved HUVECs proliferation compared with that on uncoated films, platelet adhesion was also decreased on gelatin-coated polyester. The hemocompatibility and biocompatibility of PHBHHx film were markedly improved. Thus, PHBHHx, particularly the surface-modified PHBHHx film, is promising for blood-contact materials.     

Investigations into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) surface properties causing delayed osteoblast growth

Osteoblast proliferation is sensitive to material surface properties. In this study, the proliferation of MC3T3 E1-S14 osteoblastic cells on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films with different surface characteristics was investigated with the aim of evaluating the cause of a lag in cell growth previously observed. The solvent-cast films were prepared using three different solvents/solvent mixtures which produced PHBV films with both a rough (at the air interface) and smooth (at the glass interface) surface. Investigation of the surface roughness by scanning electron and scanning probe microscopy revealed that the surfaces had features that were different in both average lateral size and average amplitude (R _a 20–200 nm). Water contact angles showed that all surfaces were hydrophobic in nature ( _A in the range 69–82°). The lateral distribution of surface crystallinity of the films was evaluated by use of micro-attenuated total reflectance Fourier transform infrared (ATR–FT-IR) by determining the surface crystallinity index (CI) which was found to differ between samples. MC3T3-E1-S14 osteoblasts were cultured on the six surfaces and proliferation was determined. After 2 days, cell proliferation on all surfaces was significantly less than on the control substrate; however, after 4 days cell proliferation was optimal on three surfaces. It was concluded that the initial lag on all substrates was due to the hydrophobic nature of the substrates. The ability of the cells to recover on the materials was attributed to the degree of heterogeneity of the crystallinity and surface roughness: samples with a roughness of 80 nm were found to support cell proliferation. In addition, the lateral surface features influenced the proliferation of osteoblasts on the PHBV film surface.     

PHBV/HA骨修复材料的研究

利用硝酸钙与磷酸钠的水溶液制备羟基磷灰石(HA)，同时在HA生成过程中与聚羟基丁酸一戊酸酯(PHBV)复合，探索了HA增强PHBV使之适于作为骨修复材料的一种新途径。结果表明，HA的均匀分散增进了复合材料中两相问的相互结合，能明显地提高复合材料的力学性能。     

PHBV在玻璃化转变温度以上的物理老化行为

研究了聚（3-羟基丁酸酯-3-羟基戊酸酯） （简称PHBV）和含成核剂氮化硼（BN）的PHBV在30 ℃等温条件下老化过程中的热性能和弹性性能随老化时间的变化规律。根据聚合物三相模型理论,通过调制式差示扫描量热法（MDSC）计算得到PHBV的可动无定形相含量（X_MA）、刚直无定形相含量（X_RA）和结晶相含量（X_c）。结果表明,成核剂的加入对抑制PHBV的老化有明显效果。通过热性能、弹性性能和晶体形貌分析进一步证实了这一结果。     

用复合诱导型骨修复材料修复兔桡骨缺损的实验研究

以不同的载药方式构建4种壳聚糖/聚羟基丁酸酯-羟基戊酸酯(PHBV)复合诱导型骨修复材料,检测并比较4种支架材料对兔桡骨缺损的修复效果,筛选出最佳骨修复材料并确定最佳药物控释方式。以淫羊藿苷为诱导因子,采用两相混合冷冻干燥技术以微球载药、改性药物微球(W/O法制得并表征)、改性药物与材料共价结合等药物添加方式及不加药制得4种支架材料,并对其进行显微结构以及载药支架药物缓释表征,后将4种材料分别植入兔桡骨缺损处,于1、3、6个月进行X射线及三维CT观察支架材料对兔桡骨缺损的修复情况,HE,Masson染色观察其诱导成骨效果。结果表明,支架材料呈网络状串珠状的显微结构,载药微球粒径分布在3～11 μm,载药支架材料有着良好的药物缓释,其中共价结合组药物释放峰值时间较其他组推迟,为72 h,且峰值后药物缓释量迅速平稳为75 μg左右。X射线及三维CT观察显示,最终共价结合组支架材料骨缺损处连通,且骨密度高于其他3组。HE、Masson染色结果显示,共价结合组成骨效果优于其他组。共价结合的药物添加方式能使支架具有良好的药物缓释效果,进而对兔桡骨缺损表现出良好的修复效果。     

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-based nanofibrous scaffolds to support functional esophageal epithelial cells towards engineering the esophagus

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV–gelatin were electrospun to obtain defect-free nanofibers by optimizing various process and solution parameters. Tensile strength, Young’s modulus, and wettability of PHBV–gelatin nanofibrous scaffold were determined and compared with PHBV nanofibrous scaffold. Our results demonstrate that PHBV–gelatin nanofibers exhibited higher tensile strength and Young’s modulus than the PHBV nanofibers. Human esophageal epithelial cells (HEEpiC) were cultured on PHBV and PHBV–gelatin nanofiber showed better cell proliferation in PHBV nanofibrous scaffold than the PHBV–gelatin scaffold after 7?days of culture. HEEpiC cultured on PHBV and PHBV–gelatin nanofibrous scaffold exhibited characteristic epithelial cobblestone morphology after 3 days of culture. Further, the HEEpiC extracellular matrix (ECM) proteins (collagen type IV and laminin) and phenotypic marker proteins (cytokeratin-4 and 14) expressions were significantly higher in PHBV–gelatin nanofibrous scaffold than the PHBV nanofiber scaffold. However, the long-term stability and functional state of the cells on the PHBV scaffold give it an edge over the blend scaffolds. Thus, PHBV-based nanofibrous scaffolds could be explored further as ECM substitutes for the regeneration of esophageal tissue.     

Electrospun Poly(Hydroxybutyrate-co-Hydroxyvalerate) Fibrous Membranes Consisting of Parallel-Aligned Fibers or Cross-Aligned Fibers: Characterization and Biological Evaluation

Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) fibrous membranes consisting of parallel-aligned fibers or cross-aligned fibers were fabricated through electrospinning with the help of a rotating cylinder as fiber collector and auxiliary electrodes, and their application as tissue-engineering scaffolds was assessed. First, these membranes were characterized in terms of fiber diameter, spacing between adjacent aligned fibers or interstitial pore diameter, degree of fiber alignment, wettability and tensile properties. Then, human osteoblast-like cells (SaOS-2) were seeded and cultured on these membranes for up to 14 days. The cell morphology and proliferation were evaluated at different cell culture times. Membranes consisting of random fibers or parallel-aligned fibers were obtained when the rotational speed of the cylinder was 500 rpm or 3000 rpm, respectively. A very high rotational speed of 15 000 rpm resulted in the formation of parallel-aligned fibers having low or no spacing between the aligned fibers. Membranes consisting of cross-aligned fibers were made at the rotational speed of 3000 rpm and micrometer-sized fiber spacing was observed in these membranes. The alignment of fibers led to enhanced wettability of fibrous membranes. Tensile testing revealed that the parallel-aligned fibrous membranes were strong in the longitudinal direction but weak in the transverse direction. The cross-aligned fibrous membranes did not exhibit particularly weak tensile properties in any direction. In vitro biological evaluation showed that SaOS-2 cells spread randomly on membranes of random fibers but elongated in membranes of aligned fibers. All membranes supported cell proliferation in spite of the differences in cell morphology.     

Effect of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanofiber matrices cocultured with hair follicular epithelial and dermal cells for biological wound dressing

Abstract:  We tested the effects on the early-stage wound healing of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBV) nanofiber matrices cultured with hair follicular cells. PHBV only, PHBV/collagen, and PHBV/gelatin at a 7/3 weight ratio were produced by electrospinning, and their in vitro cell culture and in vivo wound healing as biological dressings were examined. In cell attachment and growth on matrices, dermal sheath (DS) cells attached to hydrophilic PHBV/collagen and PHBV/gelatin faster than hydrophobic PHBV at the early incubation stage (up to 6 h). From 6- to 24-h incubation, PHBV/collagen showed the best results in cell culture. Furthermore, PHBV/collagen cocultured for 3–5 days with DS and epithelial outer root sheath (ORS) cells expressed more extracellular materials, such as type I collagen, elastin, and α-smooth muscle actin, than cocultured PHBV with the same cells. However, there was no significant difference between PHBV and PHBV/collagen in the amounts of cytokeratin 8 expressed. Grafting of PHBV and PHBV/collagen matrices cocultured with ORS/DS cells for 3–5 days showed that PHBV promoted wound closure and re-epithelization more obviously than PHBV/collagen in both cocultured matrices and matrices alone. Cocultured matrices would heal wounds better than the corresponding matrices alone. Thus, PHBV cocultured with ORS/DS cells could be used as a cell-seeded biological dressing, thereby reducing preparation time as well as regenerating the epidermis efficiently during the early stage of wound healing.     

以PHBV为支架构建组织工程化软骨

[目的]探讨聚（羟基丁酸酯．羟基戊酸酯）（PHBV）多孔材料作为软骨组织工程支架的可行性以及体内外培养方式对软骨形成的影响。[方法]采用“压片-热处理-粒子析出”技术制备PHBV多孔支架。体外分离培养软骨细胞后接种到PHBV支架体外培养2周，期间扫描电镜观察细胞在支架上的生长情况，然后与单纯PHBV支架同植入裸鼠皮下继续培养4、8周后取材，与体外培养至6、10周的细胞一支架复合物同行组织学观察。[结果]电镜观察示软骨细胞在支架上黏附、增殖良好并能分泌细胞外基质；组织学观察示PHBV浅层有新生软骨组织形成，且皮下培养的软骨组织比体外培养的更为成熟。单纯PHBV支架皮下培养没有软骨组织形成。[结论]PHBV可以作为软骨组织工程支架材料。体内培养较体外更有利于组织工程化软骨的形成。     

新型纳米仿生骨植入材料体内降解性能的初步研究

目的 研究新型纳米仿生骨植入材料的体内降解性.方法 制备犬胫骨骨缺损模型,实验组植入材料,空白对照组不作处理.术后2,4,8,12 周取材,观察该材料的体内降解性及骨再生的效果.结果 材料植入体内2周已开始降解,12周时材料基本完全降解,光镜下及电镜下均未见材料颗粒的残留,骨缺损区新骨充填,骨再生量明显优于对照组.结论 新型纳米仿生骨植入材料体内降解速度快,材料降解后为新生骨组织所替代.