纳米羟基磷灰石仿生骨材料的研究进展

纳米羟基磷灰石仿生骨材料因其与天然骨结构和成分相近,已成为组织工程领域研究的热点之一。介绍了纳米羟基磷灰石的各种制备方法及其复合材料的合成方法,并对纳米羟基磷灰石复合材料的特性进行了说明。通过将纳米羟基磷灰石表面修饰改性后,其复合材料有着广阔的应用前景,可用于修复骨缺损,也可以作为药物载体治疗肿瘤。对近年来纳米羟基磷灰石仿生骨材料的研究进展进行了综述。     

Strain in UHMWPE for orthopaedic use studied by Raman microprobe spectroscopy

Ultra-high-molecular-weight polyethylene (UHMWPE) has been the most popular bearing material against both metal and ceramic counter-faces in total hip and knee joint replacements. Therefore, it is desirable to clarify the complex phenomena occurring both in vivo and in vitro, using highly sensitive analytical techniques. However, conventional analytical techniques used so far suffer from destructive measurements, lack of precision and/or intricate techniques. In the present study, the physical and chemical properties of both conventional UHMWPE (PE) and highly cross-linked UHMWPE (CLPE) were investigated by Raman microprobe spectroscopy, which combines the advantages of high precision and non-destructive measurements. It was found that the strain of UHMWPE can be evaluated by a change in the full width at half maximum (FWHM) of a selected Raman band (located at around 1127 cm^?1), and that these spectroscopic strain coefficients were (0.42±0.01) × 10^?2 cm^?1/% elongation and (0.48±0.01) × 10^?2cm^?1/% elongation for PE and CLPE (100 kGy), respectively. The difference in the crystalline nature between PE and CLPE was also confirmed by Raman microprobe spectroscopy. In addition, the Raman microprobe spectroscopy technique enabled us to obtain hyperspectral images of strain and crystallinity on a microscopic scale. Thus, Raman microprobe spectroscopy is a very effective method for analyzing UHMWPE for or thopaedic use.     

Strain in UHMWPE for orthopaedic use studied by Raman microprobe spectroscopy

Ultra-high-molecular-weight polyethylene (UHMWPE) has been the most popular bearing material against both metal and ceramic counter-faces in total hip and knee joint replacements. Therefore, it is desirable to clarify the complex phenomena occurring both in vivo and in vitro, using highly sensitive analytical techniques. However, conventional analytical techniques used so far suffer from destructive measurements, lack of precision and/or intricate techniques. In the present study, the physical and chemical properties of both conventional UHMWPE (PE) and highly cross-linked UHMWPE (CLPE) were investigated by Raman microprobe spectroscopy, which combines the advantages of high precision and non-destructive measurements. It was found that the strain of UHMWPE can be evaluated by a change in the full width at half maximum (FWHM) of a selected Raman band (located at around 1127 cm(-1)), and that these spectroscopic strain coefficients were (0.42 +/- 0.01) x 10(-2) cm(-1)/% elongation and (0.48 +/- 0.01) x 10(-2) cm(-1)/% elongation for PE and CLPE (100 kGy), respectively. The difference in the crystalline nature between PE and CLPE was also confirmed by Raman microprobe spectroscopy. In addition, the Raman microprobe spectroscopy technique enabled us to obtain hyperspectral images of strain and crystallinity on a microscopic scale. Thus, Raman microprobe spectroscopy is a very effective method for analyzing UHMWPE for orthopaedic use.     

Electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue engineering

The development of bioinspired or biomimetic materials is essential and has formed one of the most important paradigms in today's tissue engineering research. This paper reports a novel biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan (HAp/CTS) prepared by combining an in situ co-precipitation synthesis approach with an electrospinning process. A model HAp/CTS nanocomposite with the HAp mass ratio of 30wt% was synthesized through the co-precipitation method so as to attain homogenous dispersion of the spindle-shaped HAp nanoparticles (ca. 100x30nm) within the chitosan matrix. By using a small amount (10wt%) of ultrahigh molecular weight poly(ethylene oxide) (UHMWPEO) as a fiber-forming facilitating additive, continuous HAp/CTS nanofibers with a diameters of 214+/-25nm had been produced successfully and the HAp nanoparticles with some aggregations were incorporated into the electrospun nanofibers. Further SAED and XRD analysis confirmed that the crystalline nature of HAp remains and had survived the acetic acid-dominant solvent system. Biological in vitro cell culture with human fetal osteoblast (hFOB) cells for up to 15 days demonstrated that the incorporation of HAp nanoparticles into chitosan nanofibrous scaffolds led to significant bone formation oriented outcomes compared to that of the pure electrospun CTS scaffolds. The electrospun nanocomposite nanofibers of HAp/CTS, with compositional and structural features close to the natural mineralized nanofibril counterparts, are of potential interest for bone tissue engineering applications.     

Strengthening mechanisms of bone bonding to crystalline hydroxyapatite in vivo

The formation and strengthening mechanisms of bone bonding of crystalline hydroxyapatite (HA) has been investigated using high-resolution transmission electron microscope (HRTEM) and energy-dispersive X-ray (EDX) analysis. A series of results were obtained: (i) a layer of amorphous HA, which has almost the same chemistry as the implanted HA, was formed on the surface of crystalline HA particles prior to dissolution; (ii) at 3 months a bone-like tissue formed a bonding zone between mature bone and the HA implant, composed of nanocrystalline and amorphous apatite; and (iii) at 6 months, mature bone was in direct contact with HA particles, and collagen fibres were perpendicularly inserted into the surface layer of implanted HA crystals. Findings (i) and (ii) indicated the following dissolution-precipitation process. (i) The crystalline HA transforms into amorphous HA; (ii) the amorphous HA dissolves into the surrounding solution, resulting in over-saturation; and (iii) the nanocrystallites are precipitated from the over-saturated solution in the presence of collagen fibres. A preliminary analysis indicated several conclusions: (i) the transition from crystalline to amorphous HA might be the controlling step in the bone bonding of crystalline HA; (ii) biological interdigitation (or incorporation) of collagen fibres with HA and chemical bonding of a apatite layer were both necessary to strengthen and toughen a bone bond, not only for the bonding between bone and HA at 6 months, but also for the bonding zone at 3 months, which would otherwise be very fragile due to the inherited brittleness of polycrystalline ceramics; and (iii) perpendicular interdigitation is an effective way for collagen fibres to impart their unique combination of flexibility and strength to the interface which they are keying.     

Kerr-gated time-resolved Raman spectroscopy of equine cortical bone tissue

Picosecond time-resolved Raman spectroscopy in equine cortical bone tissue is demonstrated. Using 400-nm pulsed laser excitation (1 ps at 1 kHz) it is shown that Kerr cell gating with a 4-ps window provides simultaneously time-resolved rejection of fluorescence and time-resolved Raman scatter enabling depth profiling through tissue. The Raman shifts are the same as those observed by conventional cw Raman spectroscopy using deep-red or near-infrared lasers. The time decay of Raman photons is shown to fit an inverse square root of time function, suggesting propagation by a diffusive mechanism. Using polystyrene behind a bone specimen, it is shown that the 400-nm laser light penetrates at least 0.31 mm below the surface of a fully mineralized bone tissue specimen and generates observable bone Raman scatter (approximately 415 to 430 nm) through most of this depth. These novel results demonstrate great promise for in vivo applications for studying diseased bone tissue, and ways to optimize the setup are discussed.     

The effect of glass-ceramic bone implant materials on the in vitro formation of hydroxyapatite

The extracts of four glass-ceramic bone implant materials were investigated for dissolved material, for effects on in vitro formation of hydroxyapatite, and for surface morphology of glass-ceramic particles in scanning electron microscopy. In vitro leaching released substances that affected in vitro formation of hydroxyapatite, i.e., initiation time and growth of crystals. Leaching also changed the surface morphology of the materials. The ability of the materials to bond to bone did not correlate with the inhibition of hydroxyapatite formation by the released substances. Surface morphology and other factors at present not yet known are probably involved in controlling the bonding to bone of these ceramics.     

Transcutaneous fiber optic Raman spectroscopy of bone using annular illumination and a circular array of collection fibers

Transcutaneous bone Raman spectroscopy with an exciting annulus of 785-nm laser light surrounding the field of view of a circular array of collection fibers is demonstrated. The configuration provides distributed laser light. The annulus is located 2 to 3 mm beyond the edge of the field of view of the collection fibers to reject contributions from skin and other overlying tissues. Data are presented for rat and chicken tissue. For rat tibia, the carbonate/phosphate ratio measured at a depth of 1 mm below the skin is in error by 2.3% at an integration time of 120 s and within 10% at a 30-s integration time. For chicken tibia 4 mm below the skin surface, the error is less than 8% with a 120-s integration time.     

Optical clearing in transcutaneous Raman spectroscopy of murine cortical bone tissue

The effect of optical clearing with glycerol on the Raman spectra of bone tissue acquired transcutaneously on right and left tibiae from four mice is studied. Multiple transcutaneous measurements are obtained from each limb; glycerol is then applied as an optical clearing agent, and additional transcutaneous measurements are taken. Glycerol reduces the noise in the raw spectra (p=0.0037) and significantly improves the cross-correlation between the recovered bone factor and the exposed bone measurement in a low signal-to-noise region of the bone spectra (p=0.0245).     

Identifying compositional and structural changes in spongy and subchondral bone from the hip joints of patients with osteoarthritis using Raman spectroscopy

Raman microspectroscopy was used to examine the biochemical composition and molecular structure of extracellular matrix in spongy and subchondral bone collected from patients with clinical and radiological evidence of idiopathic osteoarthritis of the hip and from patients who underwent a femoral neck fracture, as a result of trauma, without previous clinical and radiological evidence of osteoarthritis. The objectives of the study were to determine the levels of mineralization, carbonate accumulation and collagen quality in bone tissue. The subchondral bone from osteoarthritis patients in comparison with control subject is less mineralized due to a decrease in the hydroxyapatite concentration. However, the extent of carbonate accumulation in the apatite crystal lattice increases, most likely due to deficient mineralization. The alpha helix to random coil band area ratio reveals that collagen matrix in subchondral bone is more ordered in osteoarthritis disease. The hydroxyapatite to collagen, carbonate apatite to hydroxyapatite and alpha helix to random coil band area ratios are not significantly changed in the differently loaded sites of femoral head. The significant differences also are not visible in mineral and organic constituents' content in spongy bone beneath the subchondral bone in osteoarthritis disease.     

Identification of abnormal stem cells using Raman spectroscopy

The clinical use of stem cells in cell-based therapeutics for degenerative diseases requires development of criteria for defining normal stem cells to ensure safe transplantation. Currently, identification of abnormal from normal stem cells is based on extensive ex vivo and in vivo testing. Raman microscopy is a label-free method for rapid and sensitive detection of changes in cells' bio-molecular composition. Here, we report that by using Raman spectroscopy, we were able to map the distribution of different biomolecules within 2 types of stem cells: adult human bone marrow-derived stromal stem cells and human embryonic stem cells and to identify reproducible differences in Raman's spectral characteristics that distinguished genetically abnormal and transformed stem cells from their normal counterparts. Raman microscopy can be prospectively employed as a method for identifying abnormal stem cells in ex vivo cultures prior to clinical transplantation.     

Formation and adhesion of biomimetic hydroxyapatite deposited on titanium substrates

This study has been carried out to investigate the bioactivity of rutile and to deposit hydroxyapatite (HA) on heat-treated titanium through a biomimetic method. Biomimetic deposition of HA has gained large interest because of its low deposition temperature and good step coverage; however, it demands a substrate with bioactive properties. Commercially pure titanium is not bioactive but it can acquire bioactive properties through various surface treatments. In the present study, titanium plates were heat-treated at 800 °C to achieve rutile TiO₂ surfaces. These samples were immersed in a phosphate-buffered saline solution for seven days in order to deposit a HA layer on the surface. The rutile TiO₂ surfaces were found to be highly bioactive: after seven days of immersion, a layer of HA several micrometers thick covered the plates. The HA surfaces were confirmed by electron microscopy and X-ray diffraction. A scratch test was used to assess the adhesion of the HA coatings. This is a standard method to provide a measure of the coating-to-substrate adhesion and was found to be a useful method to test the thin HA coatings deposited on the bioactive surfaces. The critical pressure of the layer was estimated to be 2.4 ± 0.1 GPa.     

Identification of monoclinic calcium pyrophosphate dihydrate and hydroxyapatite in human sclera using Raman microspectroscopy

Raman microspectroscopy was first used to determine the composition of a calcified plaque located at the pterygium-excision site of a 51-year-old female patient's left nasal sclera after surgery. It was unexpectedly found that the Raman spectrum of the calcified sample at 1149, 1108, 1049, 756, 517, 376 and 352/cm was similar to the Raman spectrum of monoclinic form of calcium pyrophosphate dihydrate (CPPD) crystal, but differed from the Raman spectrum of triclinic form of CPPD. An additional peak at 958/cm was also observed in the Raman spectrum of the calcified plaque, which was identical to the characteristic peak at 958/cm of hydroxyapatite (HA). This is the first study to report the spectral biodiagnosis of both monoclinic CPPD and HA co-deposited in the calcified plaque of a patient with sclera dystrophic calcification using Raman microspectroscopy.     

In vivo chemical investigation of human skin using a confocal Raman fiber optic microprobe

To evaluate the potential of a new in vivo confocal Raman microprobe, we undertake a pilot study in human skin. A fiber optic probe is operated with a 633-nm laser and trials are conducted in healthy volunteers. We examine changes in molecular composition and structure of the stratum corneum, from different volunteers, from different anatomical sites and skin layers. Main spectral variations are detected in the following regions: 800 to 900 cm(-1) (amino acids); 1200 to 1290 cm(-1) (proteins); and 1030 to 1130 cm(-1), 1300 to 1450 cm(-1), and 2800 to 2900 cm(-1) (lipids). Curve fitting of the amide 1 region performs in detail protein secondary structural variations of the amide 1 band. Protein conformation is also found to vary depending on the anatomical site and volunteer. Similar analysis of the 730- to 1170-cm(-1) spectral window reveals a different organization of lamellar lipids: gel for forearm and palm, and liquid-crystalline phase for fingertips. All these variations result from changes in the stratum corneum components such as natural moisturizing factor (NMF), lipids (namely ceramides), and water. Hierarchical clustering classification is also performed to sort out Raman data obtained from different subjects. Further improvement of the confocal probe would be to adapt a 360-deg configuration enabling access to other anatomical sites.     

Assessment of thermal coagulation in ex-vivo tissues using Raman spectroscopy

Raman spectroscopy is used to study the effects of heating on specific molecular bonds present in albumen-based coagulation phantoms and ex-vivo tissues. Thermal coagulation is induced by submerging albumen-based phantoms in a 75°C water bath to achieve target temperatures of 45, 55, 65, and 75°C. Laser photocoagulation is performed on ex-vivo bovine muscle samples, yielding induced temperatures between 46 and 90°C, as reported by implanted microthermocouples. All phantoms and tissue samples are cooled to room temperature, and Raman spectra are acquired at the microthermocouple locations. Shifts in major Raman bands are observed with laser heating in bovine muscle, specifically from the amide-1 α-helix group (～1655 cm(-1)), the CH(2)/CH(3) group (～1446 cm(-1)), the Cα-H stretch group (～1312 cm(-1)), and the CN stretch group (～1121cm(-1)). Raman bands at 1334 cm(-1) (tryptophan), 1317 cm(-1) [ν(Cα-H)], and 1655 cm(-1) (amide-1 α-helix) also show a decrease in intensity following heating. The results suggest that Raman band locations and relative intensities are affected by thermal denaturation of proteins, and hence, may be a useful tool for monitoring the onset and progression of coagulation during thermal therapies.     

Deep-ultraviolet Raman spectroscopy study of the effect of aging on human cortical bone

The age-related deterioration in bone quality and consequent increase in fracture incidence is an obvious health concern that is becoming increasingly significant as the population ages. Raman spectroscopy with deep-ultraviolet excitation (244 nm) is used to measure vibrational spectra from human cortical bone obtained from donors over a wide age range (34-99 years). The UV Raman technique avoids the fluorescence background usually found with visible and near-infrared excitation and, due to resonance Raman effects, is particularly sensitive to the organic component of bone. Spectral changes in the amide I band at 1640 cm(-1) are found to correlate with both donor age and with previously reported fracture toughness data obtained from the same specimens. These results are discussed in the context of possible changes in collagen cross-linking chemistry as a function of age, and are deemed important to further our understanding of the changes in the organic component of the bone matrix with aging.     

仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原的制备及其细胞相容性

背景：目前骨组织工程常用的支架材料主要有无机材料、有机高分子材料及天然衍生材料等,上述材料各有优缺点,为了充分发挥各类材料的优势,弥补其不足,目前多采用联合材料制备复合支架。 目的：制备新型仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原,并观察其对骨髓间充质干细胞增殖、黏附及分化的影响。 方法：制备壳聚糖/纳米羟基磷灰石/胶原复合支架材料,扫描电镜观察支架材料表面微观形貌;采用真空吸附法将骨形态发生蛋白7多肽与支架材料复合,高效液相色谱仪检测骨形态发生蛋白7多肽在体外的释放规律;将骨髓间充质干细胞接种到复合骨形态发生蛋白7多肽的仿生支架材料上,以未复合多肽的支架材料作为对照,检测支架材料表面细胞增殖、黏附率、生长形态及碱性磷酸酶活性。 结果与结论：壳聚糖/纳米羟基磷灰石/胶原支架材料呈多孔状,孔径10~100 µm;骨形态发生蛋白7多肽可以从支架材料中缓慢释出;在复合多肽的仿生支架材料表面,骨髓间充质干细胞的黏附及向成骨细胞方向分化能力均明显强于对照组(P < 0.05),而增殖能力与对照组差异无显著性意义(P > 0.05)。说明新型仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原是一种理想的骨组织工程支架材料,具有良好的细胞相容性。     

Remote temperature monitoring in ocular tissue using confocal Raman spectroscopy

We demonstrated the feasibility of Raman spectroscopy for remote temperature monitoring within the aqueous humor of the rabbit eye in vivo. Using a confocal Raman spectroscopy system, Raman spectra from 2580 to 3800 cm(-1) were recorded in HPLC-grade water and in the aqueous humor of the rabbit eye under in vivo and ex vivo conditions within a temperature range of 14-34 degrees C. The ratio between the integrated Raman intensities of two temperature dependent OH-vibrational regions (OH2/OH1) in the spectra of water showed high linear dependence on temperature both in pure water [0.0049(+/-1.2%)T+0.4522(+/-0.5%), R2=0.99, n=50, p<0.05], as well as in the rabbit aqueous humor [0.0036(+/-2.8%)T+0.4966(+/-0.6%), R2=0.98, n=162, p<0.05] with a high degree of reproducibility and sensitivity ( approximately 0.2-0.7 degrees C). Raman spectroscopy can be used for high resolution and remote monitoring of temperature in the aqueous humor under in vivo conditions.     

生物医学拉曼光谱质控方法研究

目的评价生物医学拉曼光谱设备的关键性能参数,为开展不同厂家产品的一致性比对和建立拉曼光谱类医疗器械的检测规范做准备。方法开发通用的拉曼光谱实验平台,测量化学参考物质,开发光谱分析方法,对实验系统的分辨率、波长校正、信噪比、系统响应等进行分析,并使用几种常见的生物分子进行测试验证。结果根据化学参考物质的拉曼光谱提取了实验系统的光谱分辨率、波长校正、信噪比、系统响应等信息,在测试中可有效地将生物分子的原始拉曼谱还原为可比对的标准拉曼谱。结论本文方法可有效提取和评价拉曼系统的性能参数,这对于拉曼光谱和其他相关光谱医疗器械的质控具有积极意义。