TISSUE ENGINEERING
Volume 10, Number 9/10, 2004?Mary Ann Liebert, Inc.
Modulation of In Vitro Angiogenesis in a Three-Dimensional Spheroidal Coculture Model for Bone Tissue Engineering
A. WENGER,1A. STAHL,1H. WEBER,2G. FINKENZELLER,1H.G. AUGUSTIN,2
G.B. STARK,1and U. KNESER 1
ABSTRACT
One of the major challenges in tissue engineering of bone substitutes remains vascularization of the transplant. We have developed a three-dimensional collagen-based coculture system to assess in-teractions between human endothelial cells (hECs) and human osteoblasts (hOBs) in vitro . Human umbilical vein endothelial cells (HUVECs) were grown as three-dimensional multicellular spheroids and seeded in a collagen matrix to assess sprouting of the spheroids, that is, formation of tubelike structures resembling early capillaries. Direct cell contact between hOBs and HUVECs was estab-lished by incorporating hOBs into the EC spheroids, thus forming heterogeneous cospheroids. Spa-tial organization of cospheroids and sprout configuration were assessed by immunohistochemical wholemount staining techniques and confocal laser microscopy. Cumulative sprout length of spher-oids was quantitatively analyzed by digital imaging planimetry. In this model HUVECs and hOBs formed heterogeneous cospheroids with distinct spatial organization. The ability of HUVEC spher-oids to form tubelike structures on angiogenic stimulation with vascular endothelial growth factor and basic fibroblast growth factor was suppressed in heterogeneous HUVEC/hOB cospheroids. The model system introduced in this study may be useful to assess the mechanisms involved in regulat-ing angiogenesis during bone formation and to further investigate the mechanisms by which het-erotypic cell–cell interactions inhibit endothelial tube formation for applications in bone tissue en-gineering.
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INTRODUCTION
A
DVANCES IN TISSUE ENGINEERING
have produced var-ious sasuccessful strategies for fabricating living hu-man tissues for surgical transplantation and reconstruc-tion. Skin, cartilage, and cardiovascular components have entered the human trial stage or have already qualified for clinical application.1–5Low oxygen requirements and limited spatial dimensions of these transplanted tissues allow successful graft survival of tissue-engineered con-structs. In voluminous grafts transplanted without suffi-cient vascular supply, however, the majority of cells fail
to survive through the early postimplantational phase.6In these transplants oxygenation and nutrition are limited by diffusion processes and ingrowth of host vessels. Suf-ficient diffusion, however, is confined to 150 ?m from the next capillary 7and the formation of host vessels within the construct is a prolonged process.
Regarding bone formation, the process of angiogene-sis represents a crucial step both in skeletal development and fracture repair as well as in bone tissue engineering.Unlike soft tissues, which heal predominantly through the generation of scar tissue, healing of bone results in the generation of newly formed bone substance. Thus, bone
1Department of Plastic and Hand Surgery, University of Freiburg Medical Center, Freiburg, Germany.2Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, Freiburg, Germany.
repair can accurately be described as a regenerative pro-cess 8in demand of similar angiogenetic mechanisms present during skeletal development.
During embryonic development a primitive vascular plexus is established before the onset of blood flow.9The circulatory system is initially formed only by endothelial cells (ECs).10From the late fetal phase onward new cap-illaries develop predominantly from preexisting ECs 11,12by elongation of capillary tubes from existing vascular structures. In larger vessels mesenchymal cells are re-cruited to form the vessel wall of mature arteries, arteri-oles, veins, and venules.13–15In capillaries, however, the vessel wall remains exclusively assembled by ECs sur-rounded by a basement membrane and a single layer of pericytes.16Offering a preexisting capillary network in tissue-engineered constructs for host vessels to connect to could improve and accelerate perfusion of implanted constructs and thus diminish hypoxia-related cell damage.
As human endothelial cells (hECs) maintained in stan-dard two-dimensional cultures tend to lose many of their differentiated phenotypic properties 17,18we cocultured human osteoblasts (hOBs) together with hECs as three-dimensional cellular spheroids to induce endothelial cell differentiation and in vitro angiogenesis. Similar spher-oid-based angiogenesis assays have been developed as an in vitro model of vessel maturation 18–20and serve as screening tools for angiogenic and antiangiogenic agents.It has previously been shown that collagen gel-embed-ded EC spheroids give rise to radially outgrowing lum-enized capillary sprouts.19,20We adapted this angiogenic model for bone tissue engineering to develop a three-di-mensional coculture array for hECs and hOBs. Our study aimed at the investigation of early endothelial tube sprouting in the presence of cultured hOBs. We further assessed the role of heterotypic cell–cell interactions and angiogenic stimulation with vascular endothelial growth factor (VEGF)-165 and basic fibroblast growth factor (bFGF) on in vitro angiogenesis.
MATERIALS AND METHODS
Cell culture
Human osteoblasts (hOBs) were isolated from femoral heads kindly donated by the Department for Traumatol-ogy and the Department for Orthopedic Surgery (Uni-versity of Freiburg Medical Center, Freiburg, Germany).The bone material used was obtained with informed con-sent from patients in accordance with the University of Freiburg Medical Center Ethics Committee and would otherwise have been discarded during the operations. Os-teoblasts were cultured in medium 199 with Earle’s salt supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% L -glutamine, and 1% penicillin–streptomycin
MODULATION OF IN VITRO ANGIOGENESIS at 37°C, 5% CO 2in 75–cm 2tissue culture dishes. H u-man umbilical vein endothelial cells (H UVECs) were purchased from Promocell (H eidelberg, Germany) and cells were cultured at 37°C, 5% CO 2in 75-cm 2tissue culture dishes in endothelial cell growth medium (ECGM;Promocell) supplemented with 10% heat-inactivated FCS. Only osteoblasts from passage 2 and HUVECs from passage 2 to 5 were used for experiments.
Generation of endothelial cell, osteoblast, and coculture spheroids
Subconfluent monolayers of H UVECs or osteoblasts were trypsinized. Cells were suspended in endothelial cell basal medium (ECBM; Promocell, containing 20%Methocel (Dow Chemical, Midland, MI) as described in Korff and Augustin.18When seeded into nonadhesive 96-well plates (U shape; Greiner, Frickenhausen, Germany)and cultured at 37°C (5% CO 2, 100% humidity), the sus-pended cells aggregate spontaneously within 4 h to form multicellular spheroids. To generate coculture spheroids,equal amounts of suspended osteoblasts and H UVECs were mixed and seeded in nonadherent U-shaped 96-well plates as described above. Under these conditions all sus-pended cells contribute to the formation of a single spher-oid. The spheroids were harvested after 24 h and used for sprouting experiments or incubated for 1 to 4 days for ultrastructural and immunohistochemical analysis.
Scanning electron microscopy
Spheroids were harvested and fixed in 4% formalde-hyde. After fixation spheroids were washed with deion-ized water and analyzed with an environmental scanning electron microscope (ESEM 2020; Electroscan/Philips Electron Optics, Wilmington, MA) equipped with a gaseous secondary electron detector) to examine the structure of H UVEC/hOB cospheroids. The samples were cooled to 5.0°C and analyzed under water vapor at 787 Pa. This condition prevents the samples from drying during image acquisition.
Morphological and Immunohistochemical analysis
For paraffin sections, spheroids were harvested and centrifuged for 3 min at 200?g . Spheroids were fixed in H anks’ balanced saline solution (H BSS) containing 4% paraformaldehyde and processed for paraffin em-bedding as described in Korff and Augustin.18The re-sulting paraffin block was cooled to room temperature and trimmed for sectioning. Paraffin sections (5 ?m)were cut, deparaffinized, rehydrated, and stained accord-ing to standard hematoxylin–eosin protocols. For cryo-sections spheroids were harvested and embedded in tis-sue freezing medium. Cryosections (5 ?m) were obtained
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from the spheroids, using a Leica CM3050 cryomicro-tome (Leica Microsystems, Bensheim, Germany). After washings in phosphate-buffered saline, the sections were incubated for 30 min with blocking solution (10% nor-mal goat serum; Sigma, Deisenhofen, Germany), fol-lowed by incubation with the corresponding primary an-tibody—monoclonal mouse anti-CD31 antibody (diluted 1:25; DakoCytomation, Hamburg, Germany), monoclo-nal mouse anti-osteocalcin antibody (diluted 1:100; Acris, H iddenhausen, Germany), or polyclonal rabbit anti-VEGF antibody (diluted 1:50; Santa Cruz Biotech-nology, Heidelberg, Germany)—in a humid chamber at room temperature for 1 h. After washing they were in-cubated with secondary antibody (ready-to-use biotinyl-ated goat anti-mouse immunoglobulin [DakoCytoma-tion] or alkaline phosphatase [AP]-conjugated goat anti-rabbit immunoglobulin [diluted 1:200; Zymed, South San Francisco, CA] and exposed to streptavidin (DakoCytomation) or fast red substrate (Zytomed, Berlin, Germany). Sections stained for VEGF were weakly coun-terstained with hematoxylin. For alkaline phosphatase evaluation cryosections were stained with an alkaline phosphatase kit from Sigma according to product guide-lines. For in-gel whole-mount stainings collagen gels were fixed with 100% methanol for 20 min at ?20°C. After washing steps in phosphate-buffered saline, the sec-tions were incubated for 30 min with blocking solution (10% normal goat serum; Sigma), followed by incuba-tion with monoclonal mouse anti-CD31 antibody (diluted 1:25; DakoCytomation) on a rocking table for 90 min at room temperature. They were then incubated with a ready-to-use horseradish peroxidase (H RP)-conjugated goat anti-mouse secondary antibody (DakoCytomation) for 30 min and exposed to aminoethyl carbazole (AEC) substrate (DakoCytomation).
Three-dimensional coculture model and in vitro angiogenesis assay
In vitro angiogenesis in collagen gels was quantified with spheroids of endothelial cells and cospheroids of HUVECs and human osteoblasts (hOBs) in adaption of a model for in vitro angiogenesis.19HUVEC spheroids (500 cells per spheroid) or H UVEC/hOB cospheroids (250 HUVECs and 250 hOBs per spheroid) were gener-ated and embedded into collagen gels. The spheroid-con-taining gel was rapidly transferred into prewarmed 24-well suspension plates and allowed to polymerize, after which 0.25 mL of ECBM containing 10% FCS was pipet-ted on top of the gel. After polymerization of the gel 0.25 mL of ECBM containing 10% FCS and the appropriate chemokines for stimulation was pipetted on top of the gel. Human recombinant VEGF-165 from R&D Systems (Wiesbaden, Germany) and human recombinant basic fibroblast growth factor (bFGF) from Promega (Mann-
WENGER ET AL. heim, Germany) were used at a final concentration of 25 ng/mL for angiogenic stimulation. ECBM containing 10% FCS served as control reference. The gels were in-cubated at 37°C in 5% CO2at 100% humidity. Mean sprout diameter was assessed by measuring the diame-ters of the three longest sprouts in 10 spheroids per group in midsprout position. For detailed quantitative analysis of in-gel angiogenesis, the cumulative length of all cap-illary-like sprouts originating from the central plain of an individual spheroid was measured at ?50 magnification, using a digital imaging system (analySIS, Soft Imaging System, Muenster, Germany) connected to an inverted microscope. Ten spheroids per experimental group and experiment were analyzed. At least three independent ex-periments per group were performed, adding up to at least 30 spheroids measured per group. The cumulative anal-ysis takes into consideration that the angiogenic response is most appropriately reflected by the combination of measuring the length of individual capillary-like sprouts and counting the number of capillary-like sprouts.19 Confocal fluorescence microscopy
Spheroid sprout morphology was assessed by confo-cal fluorescence laser microscopy. An LSM 410 laser scanning confocal microscope (Zeiss, Oberkochen, Ger-many) was used with filter set 665 LP allowing mea-surement of DRAQ5 (Biostatus, Shepshed, UK) fluores-cence signals specific for DNA. Optical sections with a thickness of 4 to 6 ?m were obtained under 5-fold mag-nification. Image stacks of 50 to 70 optical sections of one spheroid were processed into one sum projection, us-ing AutoVisualize software (AutoQuant Imaging, Wa-terliet, NY). No other image processing was performed. Plain phase-contrast images as references for the opti-cally sectioned spheroids were obtained by phase-con-trast microscopy at 20-fold magnification.
Statistical analysis and digital imaging
All results are expressed as means?SD. Differences between experimental groups were analyzed by unpaired Student t test. p?0.05 was considered statistically sig-nificant. Apart from optimizations in contrast, brightness, and ?value, using Zeiss Axiovert software, no other im-age processing was performed on microscopic images.
RESULTS
Spheroid morphology
Microstructural analysis by scanning electron mi-croscopy revealed integrity of the spherical cell cluster configuration of a single HUVEC/hOB cospheroid (Fig.
1). Surface cells appear flattened and outstretched, as-sembling into a tightly interwoven cellular conglomerate
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(Fig. 1A). HUVEC/hOB cospheroids displayed a densely integrated pattern of intraspheroidal cell contacts as dem-onstrated on H&E stainings of cospheroids (Fig. 1B). Cell nuclei are detectable in all regions of the spheroid. The spheroidal core appears less organized compared with a more regular curved pattern of cellular arrangement in the outer regions of the spheroid covered by a continu-ous well-defined surface layer of cells.
To examine distribution patterns of H UVECs and hOBs within cospheroids, immunohistochemical investi-gations were performed on cryosections obtained from HUVEC/hOB cospheroids at various time points during spheroid differentiation. Endothelial cells maintained their phenotype throughout the experimental phase as as-sessed by immunohistochemical staining for CD31 as a panendothelial marker (Fig. 2A and B). Interestingly, en-dothelial cells localized more densely near the spheroid surface over time as indicated by different localization patterns of CD31-positive cells on days 1 and 4. On day 1, CD31-positive cells were evenly distributed through-out the whole spheroid (Fig. 2A). On day 4 endothelial cell alignment can be observed in the outer regions of the spheroid, with endothelial cells forming a CD31-positive ringlike structure following the outlines of the spheroid,with the core and the outer rim staining negative for CD31 (Fig. 2B). Only a few CD31-positive cells were unincorporated into the endothelial ring structure.
Osteocalcin and alkaline phosphatase stains were per-formed on cryosections of HUVEC/hOB cospheroids in
order to identify osteoblasts and assess differentiation (Fig. 2C and D). Osteocalcin is a protein secreted by hOBs as a component of bone extracellular matrix. Ac-cordingly osteocalcin was immunohistochemically de-tected in uniform distribution throughout the cospheroid (Fig. 2C). Expression patterns for osteocalcin did not change during spheroidal maturation from day 1 to day 4. Alkaline phosphatase as a marker for osteoblast dif-ferentiation remained restricted to the inner regions of the cospheroid. The spheroidal periphery stained alkaline phosphatase negative (Fig. 2D). This pattern was equally consistent throughout spheroid maturation.
Immunohistochemical stainings for VEGF revealed that HUVEC/hOB cospheroids express VEGF in a uni-form distribution in all regions throughout the cospher-oid (Fig. 2E and F). This homogeneous pattern of VEGF expression was constant from day 1 (Fig. 2E) to day 4(Fig. 2F).
Morphological analysis of gel-immobilized spheroids
HUVEC spheroids, hOB spheroids, and HUVEC/hOB cospheroids were seeded into collagen gels to assess the sprouting ability of spheroids, that is, the radial outgrowth of cells from the spheroids after 1 day of incubation and angiogenic stimulation with VEGF or bFGF (Fig. 3A–C).HUVEC spheroid sprouts have previously been shown to form lumenized capillary-like structures.19Cellular pro-
FIG. 1.(A ) Scanning electron microscopy image of a HUVEC/hOB cospheroid on day 1. Surface cells appear flattened and outstretched, assembling into a confluent cellular conglomerate with a continuous well-defined surface layer. (B ) H&E staining of paraffin section of a HUVEC/hOB cospheroid on day 1 (original magnification, ?400). A densely integrated pattern of in-traspheroidal cell contacts is detectable in all regions of the spheroid. The spheroidal core appears less organized compared with a more regular curved pattern of cellular arrangement in the outer regions of the spheroid. The spheroid surface is covered by a continuous layer of cells.
WENGER ET AL.
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F I
G . 2.(A a n d B ) I m m u n o h i s t o c h e m i c a l s t a i n i n g o f
H U V E C /h O B c o s p h e r o i d s f o r C D 31 d i s p l a y s a h e t e r o g e n e o u s d i s t r i b u t i o n o f C D 31-p o s i t i v e e n d o t h e l i a l c e l l s t h r o u g h -o u t t h e c o -s p h e r o i d o n d a y 1 (A ;o r i g i n a l m a g n i f i c a t i o n , ?200). E n d o t h e l i a l c e l l s a l i g n i n t o a c i r c u l a r c o n s t e l l a t i o n t o f o r m a C D 31-p o s i t i v e r i n g l i k e s t r u c t u r e f o l l o w i n g t h e o u t l i n e s o f t h e s p h e r o i d , w i t h t h e c o r e a n d a n o u t e r r i m s t a i n i n g n e g a t i v e f o r C D 31 o n d a y 4 (B ;m a g n i f i c a t i o n , ?400). (C )
I m m u n o h i s t o c h e m i c a l s t a i n i n g o f H U V E C /h O B c o s p h e r o i d s f o r o s t e o c a l c i n a s a p r o t e i n o f b o n e e x t r a c e l l u l a r m a t r i x c o n f i r m s o s t e o b l a s t i d e n t i t y . U n i f o r m d i s t r i b u t i o n o f o s t e o c l a c i n a c r o s s t h e c o s p h e r o i d (C ,d a y 3; o r i g i -n a l m a g n i f i c a t i o n ?400) d i d n o t c h a n g e t h r o u g h o u t s p h e r o i d a l m a t u r a t i o n f r o m d a y 1 t o d a y 4. (D ) H i s t o c h e m i s t r y o f H U V E C /h O B c o s p h e r o i d s f o r a l k a l i n e p h o s p h a t a s e a s a m a r k e r f o r o s t e o b l a s t d i f f e r e n t i a t i o n d i s p l a y s a l k a l i n e p h o s p h a t a s e a c t i v i t y i n t h e i n n e r r e g i o n s o f t h e c o s p h e r o i d , s p a r i n g a n o u t e r r i m f o r m e d b y t w o o r t h r e e l a y e r s o f a l -k a l i n e p h o s p h a t a s e -n e g a t i v e c e l l s (D ,d a y 1; o r i g i n a l m a g n i f i c a t i o n , ?400). T h i s p a t t e r n w a s c o n s i s t e n t f r o m d a y 1 t o 4. (E a n d F ) I m m u n o h i s t o c h e m i c a l s t a i n i n g o f H U -V E C /h O B c o s p h e r o i d s f o r V E G F d i s p l a y s V E G F e x p r e s s i o n i n a u n i f o r m d i s t r i b u t i o n i n a l l r e g i o n s t h r o u g h o u t t h e c o s p h e r o i d i n a c o n s i s t e n t p a t t e r n f r o m d a y 1 (E ) t o d a y 4 (F ) (E a n d F :o r i g i n a l m a g n i f i c a t i o n , ?400).
trusions originating from spheroids consisting exclu-sively of hOBs as well as from HUVEC/hOB cospher-oids were abundant and more disorganized compared with the ordered architecture of HUVEC spheroid sprouts (Fig. 3B and C). They were also markedly thinner com-pared with HUVEC sprouts (Fig. 4).
Sprouts emerging from H UVEC spheroids as well as cellular protrusions from hOB spheroids and H U-VEC/hOB cospheroids were investigated by confocal laser microscopy using fluorescence labeling for DRAQ5as a specific marker for DNA and compared with phase-contrast microscopy images of the same spheroids (Fig.5A–C). Sumview projections of optical sections through H UVEC spheroids revealed distinct DRAQ5 signals within HUVEC spheroid sprouts, with linear alignment of DRAQ-positive cell nuclei (Fig. 5A). Sprouts emerg-ing from hOB and HUVEC/hOB cospheroids, however,
displayed only few if any DRAQ5-positive signals within spheroid sprouts (Fig. 5B and C). Immunohistochemical collagen gel-based whole-mount staining for CD31 dem-onstrated HUVEC spheroids and sprouts emerging from HUVEC spheroids to be CD31 positive (Fig. 5D). Plain hOB spheroids and cellular protrusions emerging from hOB spheroids were negative for CD31 (Fig. 5E). In HUVEC/hOB cospheroids the cospheroid itself displayed an irregular staining pattern for CD31, with morpholog-ically thin spheroidal protrusions staining negative for CD31 (Fig. 5F).
Quantification of spheroidal sprout length in gel-immobilized HUVEC spheroids
The influence of angiogenic stimulation on H UVEC spheroid sprouting and H UVEC/hOB cospheroids was assessed by measuring the cumulative sprout length of
FIG. 3.Phase contrast microscopy of sprouting patterns in HUVEC spheroids (A ), hOB spheroids (B ), and HUVEC/hOB cos-pheroids (C ) in collagen gels. H UVEC spheroid sprouts have previously been shown to form lumenized capillary-like struc-tures.19Cellular protrusions also originate from spheroids consisting exclusively of hOBs as well as from H UVEC/hOB cos-pheroids (B and C ). These protrusions, however, are markedly thinner compared with HUVEC sprouts and more abundant and disorganized compared with the ordered architecture of single HUVEC spheroid sprouts (original magnification, ?200).
all sprouts originating from 10 randomly selected spher-oids per well, using digital imaging planimetry (Fig. 6).In HUVEC spheroids, baseline sprouting was 293?115?m in nonstimulated control groups. On VEGF stimula-tion HUVEC spheroid sprouting was increased to 776?174 ?m and in groups stimulated with bFGF cumulative spheroidal sprout length attained 827?21 ?m. Spher-oidal protrusion length from H UVEC/hOB cospheroids was 564?194 ?m in control groups and remained at 580?225 ?m on VEGF stimulation and 561?177 ?m on stimulation with bFGF.
DISCUSSION
Coculture models of heterogenic cell types more closely represent in vivo conditions in an e x vivo envi-ronment than do homotypic cell culture systems and thus represent valuable tools for understanding the complex mechanisms of cell interactions both during development and tissue regeneration. In this study we adapted a mul-ticellular spheroidal sprouting model established for an-giogenic investigations and applied it to assess hetero-genic cell–cell interactions between human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs).The initial process of vessel formation in vivo begins with the association of ECs into elongated cords con-
necting with one another, thus generating a primitive net-work.20,21,22From the late fetal phase onward, tubelike structures develop predominantly from preexisting ECs of the capillaries and pre- or postcapillary vessels.11,12In our in vitro model, spheroidal sprouts from endothelial cell spheroids resemble primitive blood vessels regard-ing cordlike alignment of ECs and lumen formation.H owever, the complex development of mature vessels with mural cell recruitment and connection to a perfused blood system cannot be mimicked in this setting, but needs to be assessed under experimental in vivo condi-tions.
For applications in tissue engineering, the inosculation of preformed capillary structures to host vessels is cru-cial for graft survival after transplantation. HUVEC-de-rived cords formed in a three-dimensional collagen ma-trix have been shown to survive and inosculate with mouse microvessels after transplantation.15Stromal cells of the tissue to be engineered must be brought into close proximity with spheroid-derived endothelial tubes in or-der to benefit from a preformed vascular plexus. Thus,analyzing the role of heterogenic cell contacts is crucial for understanding the process of tissue neovasculariza-tion and for successfully establishing a primordial capil-lary plexus within tissue-engineered constructs.Conditions suitable for cocultivation of HUVECs and hOBs meeting the needs of both cell types regarding ma-trix properties and nutritional demands were established
FIG. 4.Mean sprout diameter in H UVEC spheroids, hOB spheroids, and H UVEC/hOB cospheroids. *Significantly thinner cellular protrusions from hOB spheroids and from HUVEC/hOB cospheroids compared with HUVEC spheroid sprouts (p ?0,05).
to develop a spheroidal coculture system of HUVECs and hOBs, with both cell types contributing to one heteroge-neous cospheroid. Endothelial cells cultured as HUVEC spheroids gain the ability to form lumenized capillary-like structures.19Osteoblasts cultured both in homoge-neous hOB spheroids as well as in heterogeneous HUVEC/hOB cospheroids express markers for osteoblast differentiation such as osteocalcin and alkaline phospha-
tase. Consistent with earlier investigations,19we ob-served sprouting from HUVEC spheroids that was sen-sitive to angiogenic stimulation with VEGF or bFGF. In-terestingly, radial finger-like protrusions emerged also from hOB spheroids and H UVEC/hOB cospheroids.These protrusions, however, seem not to correspond to endothelial lumenized structures, as is discussed below.Earlier experiments with heterogeneous cospheroids
FIG. 5.(A–C ) Phase-contrast images (original magnification, ?200) of HUVEC spheroids (A ), hOB spheroids (B ), and HU-VEC/hOB cospheroids (C ) with corresponding confocal laser microscopy images (original magnification, ?50) of the same spher-oids labeled with DRAQ5 as a specific marker for DNA. Sumview projections of optical sections through HUVEC spheroids re-veal distinct DRAQ5 signals within HUVEC spheroid sprouts with linear alignment of DRAQ5-positive cell nuclei (A ). Sprouts emerging from hOB and HUVEC/hOB co-spheroids display only few if any DRAQ5-positive signals within spheroid sprouts (B and C ). (D–F ) Immunohistochemical collagen-based whole-mount staining for CD31 as a panendothelial marker. HUVEC spher-oids and sprouts emerging from HUVEC spheroids stain CD31 positive (D:original magnification, ?320). Plain hOB spheroids and cellular protrusions emerging from hOB spheroids were negative for CD31 (E:original magnification, ?320). In HUVEC/hOB cospheroids the cospheroid itself shows an irregular staining pattern for CD31, with morphologically thin cellular protrusions staining negative for CD31 (F:original magnification, ?400). Scale bars: 100 ?m.
consisting of HUVECs and smooth muscle cells (SMCs)showed polar patterns of spheroid organization, with HU-VECs covering the spheroid surface and SMCs lying un-derneath.20We observed a similar, but in some respects,distinct pattern of spatial organization in H UVEC/hOB cospheroids. Starting from a homogeneous mixture of both cell types, HUVEC/hOB cospheroids display align-ment of H UVECs in a circular structure after 4 days.Whether this alignment is due to active migration of HU-VECs within the spheroid or is the result of cells within the spheroid center undergoing apoptosis 18remains to be investigated. One possible factor regulating endothelial cell migration and organization is VEGF. Immunohisto-chemical investigations showed VEGF expression in HU-VEC/hOB cospheroids througout the process of spher-oidal organization. As a potent angiogenic mediator VEGF is capable of inducing hEC migration.9VEGF might also act on hOBs during the process of cospheroid organization. Expression of VEGFR-1 and VEGFR-2 on osteoblasts is still a matter of debate,23but osteoblasts do express the VEGF receptor neuropilin-1,24and it has been
shown that VEGF enhances osteoblast differentiation.25Furthermore, endothelial cells have been shown to stim-ulate differentiation of osteoblasts through endothelial-derived growth factors such as endothelin-1.26,27Thus,VEGF and other cytokines might be possible mediators directing EC alignment and hOB differentiation in HU-VEC/hOB cospheroids.
In HUVEC/hOB cospheroids osteoblasts and endothe-lial cells are cultured in direct contact with each other to provide a maximum degree of heterogenic cell inter-actions. When assessing the effect of H UVEC/hOB cell–cell interactions on the modulation of sprout forma-tion in heterotypic cospheroids we observed cellular pro-trusions emerging both from HUVEC spheroids and HU-VEC/hOB cospheroids as well as from plain hOB spheroids in the control group. However, different bio-logical processes seem to be responsible for spheroid sprout formation, as sprouting in HUVEC spheroids dif-fered markedly from protrusions emerging from H U-VEC/hOB cospheroids and hOB spheroids. Sprouts in H UVEC spheroids develop through migration of cells
FIG. 6.Cumulative sprout length of all sprouts originating from 10 randomly selected spheroids per well. In HUVEC spher-oids (gray columns), baseline sprouting was 293?115 ?m in nonstimulated control groups. On VEGF stimulation H UVEC spheroid sprouting was increased to 776?174 ?m and in groups stimulated with bFGF cumulative spheroidal sprout length at-tained 827?21 ?m. Spheroidal protrusion length from HUVEC/hOB cospheroids (open columns) was 564?194 ?m in con-trol groups and remained at 580?225 ?m on VEGF stimulation and 561?177 ?m on stimulation with bFGF. *Cumulative sprout length in stimulated HUVEC spheroids (VEGF and FGF) significantly higher (p ?0.05) compared with HUVEC control spheroids (CTR).
from the spheroid and linear alignment of CD31-positive cells within the sprouts as demonstrated by DRAQ5 and immunohistochemical whole-mount staining. Sprouts emerging from hOB spheroids and H UVEC/hOB co-spheroids, however, contained only few if any cell nu-clei. These findings suggest that cellular protrusions from hOB spheroids and HUVEC/hOB cospheroids differ sig-nificantly from the lumenized sprouts observed in endo-thelial cell spheroids. Protrusions from hOB spheroids and H UVEC/hOB cospheroids may correspond to cell filopodia emerging from the spheroid to invade the sur-rounding collagen matrix.
Radial protrusion of filopodia is characteristic of nor-mal bone cells in vivo and a distinctive feature of os-teoblasts in two-dimensional cell cultures. Osteoblast filopodia physiologically gain contact to capillaries within the bone matrix and play important roles in mo-lecular and ionic transport as well as homotypic inter-cellular gap junction communication.28Differences in sprout morphology, lack of cell nuclei, and CD31-nega-tive staining of cospheroid sprouts suggest that HUVECs contribute little if anything to sprout formation in H U-VEC/hOB cospheroids. Protrusion of osteoblast filopo-dia seems to override endothelial cell sprouting in het-erogeneous HUVEC/hOB cospheroids, with cospheroid sprouts representing hOB cell filopodia rather than lum-enized endothelial structures. Accordingly, cumulative sprout length in H UVEC/hOB cospheroids is nonre-sponsive to angiogenic stimulation with VEGF and bFGF. Thus, our investigations indicate that co-cultiva-tion of HUVECs and hOBs in heterogeneous cospheroids renders H UVECs quiescent regarding sprouting ability on angiogenic VEGF or bFGF stimulation.
Inhibition of cell migration and proliferation through direct cell–cell contact in two-dimensional coculture ar-rays has been described for cells of homotypic as well as heterotypic origin.29,30Regarding endothelial cell–osteo-blast interactions it has been demonstrated that endothe-lial cells modulate differential gene expression of os-teoblasts.31This modulation involves the gap junction protein connexin43 and is thus considered to be, at least in part, cell contact dependent.32Contact between ECs and mural cell precursors has been shown to downregu-late VEGF receptor 2 (VEGFR-2) expression on ECs as a result of TGF-?1activation,33and cocultivation of smooth muscle cells (SMCs) with HUVECs forming het-erogeneous HUVEC/SMC cospheroids has been demon-strated to inhibit formation of lumenized structures by HUVECs.20The process of rendering H UVECs nonre-sponsive to angiogenic stimuli by co-cultivation with SMCs has been considered to be due to a stabilizing ef-fect of SMCs on immature vasculature, mimicking the physiological course of vessel maturation toward the qui-escent EC phenotype of mature blood vessels.20In em-
MODULATION OF IN VITRO ANGIOGENESIS bryonic development the acquisition of a pericyte coat-ing marks the end of a plasticity window during which oxygen-regulated and VEGF-mediated fine tuning of vas-cular density takes place.34As discussed above, we ob-served corresponding effects in H UVEC/hOB cospher-oids. These findings indicate that SMCs are not the only cells capable of rendering HUVECs quiescent in regard to angiogenic stimulation. In our model hOBs incorpo-rated into HUVEC/hOB cospheroids impede HUVEC re-sponsiveness to angiogenic stimulation and inhibit the formation of tubelike structures.
Physiologically, osteoblasts and capillary ECs come into close contact with each other in the bone marrow.35Interestingly, stromal cells lining the endothelium of the marrow vasculature are related to pericytes but also pos-sess the potential to differentiate into skeletal components such as bone.36In our model mature hOBs proved to have similar stabilizing effects on early capillary vessels as de-scribed earlier for pericytes and SMCs.20,29,30The mech-anism of contact inhibition between hOBs and hECs dem-onstrated here in vitro may correspond to important regulatory components in bone vascularization in vivo .It is well documented that hOBs express VEGF.25The observed inhibition of H UVEC spheroid sprouting by hOBs seems contradictory to hOBs producing angiogenic factors. Direct contact or close proximity between hOBs and H UVECs, however, seems to override any angio-genic stimulation provided by soluble angiogenic factors (VEGF or bFGF, respectively). As various junctions via cadherins and gap junctions are thought to mediate het-erotypic signaling between the endothelium and mural cells,37,38similar ways of direct cell–cell communication may play a role in HUVEC/hOB interactions. Osteoblast-induced alterations of extracellular matrix conditions may also contribute to a modification of H UVEC sprouting patterns on contact or close proximity with hOBs. As hOBs cultured in collagen gels synthesize osteocalcin, a component of bone extracellular matrix, H UVECs cul-tured in close proximity to hOBs might be influenced by an altered compilation of extracellular matrix proteins.Taken together, we have demonstrated that the multi-cellular spheroidal sprouting model can be adapted for supplying a preformed capillary network for tissues en-gineered ex vivo , using endothelial cell spheroids to form lumenized sprouts in a collagen matrix similar to that cur-rently used in FDA-approved tissue substitutes.39The co-culture system introduced in this study will be useful in elucidating the mechanisms involved in regulating an-giogenesis in an osteoblast environment and investiga-tions continue to further characterize the heterotypic hEC/hOB interactions. Enhanced graft vascularization will be assessed under experimental in vivo conditions for future application in tissue-engineered bone substi-tutes.
1545
ACKNOWLEDGMENTS
This work was supported by funding through the Kom-petenznetz Biomaterialien Baden-Wuerttemberg (Ger-many). We gratefully acknowledge R. Landers (Freiburg Materials Research Center, FMF) for scanning electron micrographs and M. Follow (Core Facility ZKF Freiburg) for use of the confocal laser microscope. This work con-tains parts of Andreas Stahl’s medical thesis.
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Address reprint requests to:
A. Wenger, M.D.
Department of Plastic and Hand Surgery
University Hospital Freiburg
Hugstetter Strasse 5579106 Freiburg, Germany E-mail:wenger@https://www.doczj.com/doc/2a3179814.html,l.uni-freiburg.de
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比较级与最高级 1.as...as 与(not) as(so)...as as...as...句型中，as的词性 第一个as是副词，用在形容词和副词的原级前，常译为“同样地”。第二个as是连词，连接与前面句子结构相同的一个句子(相同部分常省略)，可译为“同..... He is as tall as his brother is (tall) . (后面的as 为连词) 只有在否定句中，第一个as才可换为so 改错: He is so tall as his brother.（X） 2.在比较状语从句中，主句和从句的句式结构一般是相同的 与as...as 句式中第二个as一样，than 也是连词。as和than这两个连词后面的从句的结构与前面的句子大部分情况下结构是相同的，相同部分可以省略。 He picked more apples than she did. 完整的表达为: He picked more apples than she picked apples. 后而的picked apples和前面相同，用did 替代。 He walked as slowly as she did.完整表达为: He walked as slowly as she walked slowly. she后面walked slowly与前面相同，用did替代。
3.谓语的替代 在as和than 引导的比较状语从句中，由于句式同前面 主句相同，为避免重复，常把主句中出现而从句中又出现的动词用do的适当形式来代替。 John speaks German as fluently as Mary does. 4.前后的比较对象应一致 不管后面连词是than 还是as,前后的比较对象应一致。The weather of Beijing is colder than Guangzhou. x than前面比较对象是“天气”，than 后面比较对象是“广州”，不能相比较。应改为: The weather of Bejing is colder than that of Guangzhou. 再如: His handwriting is as good as me. 应改为: His handwriting is as good as mine. 5.可以修饰比较级的词 常用来修饰比较级的词或短语有: Much,even,far,a little,a lot,a bit,by far,rather,any,still,a great deal等。 by far的用法: 用于强调，意为“...得多”“最最...”“显然”等，可修饰形容词或副词的比较级和最高级，通常置于其后，但是若比较级或最高级前有冠词，则可置于其前或其后。
The way 的用法 Ⅰ常见用法： 1)the way+ that 2)the way + in which（最为正式的用法） 3)the way + 省略（最为自然的用法） 举例：I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法： 在当代美国英语中，the way用作为副词的对格，“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2）The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3）The way =how/ how much No one can imagine the way he missed her. 4）The way =because
人教版（新目标）初中英语形容词与副词的比较级与最高级 （一）规则变化： 1．绝大多数的单音节和少数双音节词，加词尾-er ，-est tall—taller—tallest 2．以不发音的e结尾的单音节词和少数以-le结尾的双音节词只加-r，-st nice—nicer—nicest , able—abler—ablest 3．以一个辅音字母结尾的重读闭音节词或少数双音节词，双写结尾的辅音字母，再加-er，-est big—bigger—biggest 4．以辅音字母加y结尾的双音节词，改y为i再加-er，-est easy—easier—easiest 5．少数以-er，-ow结尾的双音节词末尾加-er，-est clever—cleverer—cleverest, narrow—narrower—narrowest 6．其他双音节词和多音节词，在前面加more，most来构成比较级和最高级 easily—more easily—most easily （二）不规则变化 常见的有： good / well—better—best ; bad (ly)/ ill—worse—worst ; old—older/elder—oldest/eldest many / much—more—most ; little—less—least ; far—farther/further—farthest/furthest
用法： 1．原级比较：as + adj./adv. +as（否定为not so/as + adj./adv. +as）当as… as中间有名字时，采用as + adj. + a + n.或as + many / much + n. This is as good an example as the other is . I can carry as much paper as you can. 表示倍数的词或其他程度副词做修饰语时放在as的前面 This room is twice as big as that one. 倍数+as+adj.+as = 倍数+the +n.+of Your room is twice as larger as mine. = Your room is twice the size of mine. 2．比较级+ than 比较级前可加程度状语much, still, even, far, a lot, a little, three years. five times,20%等 He is three years older than I (am). 表示“（两个中）较……的那个”时，比较级前常加the（后面有名字时前面才能加冠词） He is the taller of the two brothers. / He is taller than his two brothers. Which is larger, Canada or Australia? / Which is the larger country, Canada or Australia? 可用比较级形式表示最高级概念，关键是要用或或否定词等把一事物（或人）与其他同类事物（或人）相分离 He is taller than any other boy / anybody else.
大多数形容词有三种形式,原级,比较级和最高级, 以表示形容词说明的性质在程度上的不同。 形容词的原级: 形容词的原级形式就是词典中出现的形容词的原形。例如: poor tall great glad bad 形容词的比较级和最高级: 形容词的比较级和最高级形式是在形容词的原级形式的基础上变化的。分为规则变化和不规则变化。 规则变化如下: 1) 单音节形容词的比较级和最高级形式是在词尾加 -er 和 -est 构成。 great (原级) (比较级) (最高级) 2) 以 -e 结尾的单音节形容词的比较级和最高级是在词尾加 -r 和 -st 构成。wide (原级) (比较级) (最高级) 3)少数以-y, -er, -ow, -ble结尾的双音节形容词的比较级和最高级是在词尾加 -er 和 -est 构成。 clever(原级) (比较级) (最高级) 4) 以 -y 结尾,但 -y 前是辅音字母的形容词的比较级和最高级是把 -y 去掉,加上 -ier 和-est 构成. happy (原形) (比较级) (最高级) 5) 以一个辅音字母结尾其前面的元音字母发短元音的形容词的比较级和最高级是双写该辅音字母然后再加 -er和-est。 big (原级) (比较级) (最高级) 6) 双音节和多音节形容词的比较级和最高级需用more 和 most 加在形容词前面来构成。 beautiful (原级) (比较级) (比较级) difficult (原级) (最高级) (最高级) 常用的不规则变化的形容词的比较级和最高级: 原级------比较级------最高级 good------better------best many------more------most much------more------most bad------worse------worst far------farther, further------farthest, furthest 形容词前如加 less 和 least 则表示"较不"和"最不 形容词比较级的用法: 形容词的比较级用于两个人或事物的比较,其结构形式如下: 主语+谓语(系动词)+ 形容词比较级+than+ 对比成分。也就是, 含有形容词比较级的主句+than+从句。注意从句常常省去意义上和主句相同的部分, 而只剩下对比的成分。
The way的用法及其含义（二） 二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语： 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势，忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中
英语比较级和最高级的用法归纳 在学习英语过程中，会遇到很多的语法问题，比如比较级和最高级的用法，对于 这些语法你能够掌握吗？下面是小编整理的英语比较级和最高级的用法，欢迎阅读！ 英语比较级和最高级的用法 一、形容词、副词的比较级和最高级的构成规则 1.一般单音节词和少数以-er，-ow结尾的双音节词，比较级在后面加-er，最高级 在后面加-est; (1)单音节词 如：small→smaller→smallest short→shorter→shortest tall→taller→tallest great→greater→greatest (2)双音节词 如：clever→cleverer→cleverest narrow→narrower→narrowest 2.以不发音e结尾的单音节词，比较在原级后加-r，最高级在原级后加-st; 如：large→larger→largest nice→nicer→nicest able→abler→ablest 3.在重读闭音节(即：辅音+元音+辅音)中，先双写末尾的辅音字母，比较级加-er，最高级加-est; 如：big→bigger→biggest hot→hotter→hottest fat→fatter→fattest 4.以“辅音字母+y”结尾的双音节词，把y改为i，比较级加-er，最高级加-est; 如：easy→easier→easiest heavy→heavier→heaviest busy→busier→busiest happy→happier→happiest 5.其他双音节词和多音节词，比较级在前面加more，最高级在前面加most; 如：bea utiful→more beautiful→most beautiful different→more different→most different easily→more easily→most easily 注意：(1)形容词最高级前通常必须用定冠词 the，副词最高级前可不用。 例句： The Sahara is the biggest desert in the world. (2) 形容词most前面没有the，不表示最高级的含义，只表示"非常"。 It is a most important problem. =It is a very important problem.
定冠词the的用法： 定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸 或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...
More than的用法 A. “More than+名词”表示“不仅仅是” 1)Modern science is more than a large amount of information. 2)Jason is more than a lecturer; he is a writer, too. 3) We need more than material wealth to build our country.建设我们国家，不仅仅需要物质财富. B. “More than+数词”含“以上”或“不止”之意，如： 4)I have known David for more than 20 years. 5)Let's carry out the test with more than the sample copy. 6) More than one person has made this suggestion. 不止一人提过这个建议. C. “More than+形容词”等于“很”或“非常”的意思，如： 7)In doing scientific experiments, one must be more than careful with the instruments. 8)I assure you I am more than glad to help you. D. more than + (that)从句，其基本意义是“超过（=over）”，但可译成“简直不”“远非”.难以，完全不能（其后通常连用情态动词can） 9) That is more than I can understand . 那非我所能懂的. 10) That is more than I can tell. 那事我实在不明白。 11) The heat there was more than he could stand. 那儿的炎热程度是他所不能忍受的 此外，“more than”也在一些惯用语中出现，如： more...than 的用法 1. 比……多，比……更 He has more books than me. 他的书比我多。 He is more careful than the others. 他比其他人更仔细。 2. 与其……不如 He is more lucky than clever. 与其说他聪明，不如说他幸运。 He is more （a）scholar than （a）teacher. 与其说他是位教师，不如说他是位学者。 注：该句型主要用于同一个人或物在两个不同性质或特征等方面的比较，其中的比较级必须用加more 的形式，不能用加词尾-er 的形式。 No more than/not more than 1. no more than 的意思是“仅仅”“只有”“最多不超过”，强调少。如： --This test takes no more than thirty minutes. 这个测验只要30分钟。 --The pub was no more than half full. 该酒吧的上座率最多不超过五成。-For thirty years，he had done no more than he （had）needed to. 30年来，他只干了他需要干的工作。 2. not more than 为more than （多于）的否定式，其意为“不多于”“不超过”。如：Not more than 10 guests came to her birthday party. 来参加她的生日宴会的客人不超过十人。 比较： She has no more than three hats. 她只有3顶帽子。（太少了） She has not more than three hats. 她至多有3顶帽子。（也许不到3顶帽子） I have no more than five yuan in my pocket. 我口袋里的钱最多不过5元。（言其少） I have not more than five yuan in my pocket. 我口袋里的钱不多于5元。（也许不到5元） more than, less than 的用法 1. （指数量）不到，不足 It’s less than half an hour’s drive from here. 开车到那里不到半个钟头。 In less than an hour he finished the work. 没要上一个小时，他就完成了工作。 2. 比……（小）少 She eats less than she should. 她吃得比她应该吃的少。 Half the group felt they spent less than average. 半数人觉得他们的花费低于平均水平。 more…than,/no more than/not more than (1)Mr．Li is ________ a professor; he is also a famous scientist． (2)As I had ________ five dollars with me, I couldn’t afford the new jacket then． (3)He had to work at the age of ________ twelve． (4)There were ________ ten chairs in the room．However, the number of the children is twelve． (5)If you tel l your father what you’ve done, he’ll be ________ angry． (6)－What did you think of this novel? －I was disappointed to find it ________ interesting ________ that one． 倍数表达法 1. “倍数+形容词（或副词）的比较级+than+从句”表示“A比B大（长、高、宽等）多少倍” This rope is twice longer than that one.这根绳是那根绳的三倍（比那根绳长两倍）。The car runs twice faster than that truck.这辆小车的速度比那辆卡车快两倍（是那辆卡车的三倍）。 2. “倍数+as+形容词或副词的原级+as+从句”表示“A正好是B的多少倍”。
“tｈｅwａｙ+从句”结构的意义及用法 首先让我们来看下面这个句子: Ｒead the foｌloｗｉnｇpassagｅａnd talkａbout ｉt wi ｔh your classmaｔes．Try tｏｔeｌl whaｔyｏu thiｎk ｏf Tom aｎd ｏｆｔhe ｗay the chiｌｄrenｔrｅated ｈim. 在这个句子中，ｔhe waｙ是先行词,后面是省略了关系副词that或in whｉch的定语从句。 下面我们将叙述“the ｗaｙ+从句”结构的用法。 1.tｈe wａy之后，引导定语从句的关系词是tｈat而不是hoｗ,因此，<<现代英语惯用法词典＞＞中所给出的下面两个句子是错误的：Ｔhｉs is ｔｈeｗayｈowｉtｈａppened. This is the way how he ａlwａｙs treats me. 2．在正式语体中,thaｔ可被in which所代替;在非正式语体中，ｔhat则往往省略。由此我们得到theｗay后接定语从句时的三种模式：1) tｈe ｗaｙ＋ｔhａt-从句２）the way +ｉn whiｃh－从句3) the ｗay +从句 例如：Ｔｈe way(in whiｃh ，thａt) ｔhｅｓｅｃomｒade ｓloｏｋａｔｐrobｌems is wrong．这些同志看问题的方法
不对。 Ｔｈｅｗａy(that ,ｉn ｗhiｃh)yoｕ’ｒe doinｇit is comple ｔeｌy crａzy.你这么个干法,简直发疯。 Wｅａdmiｒｅd him for thｅｗay ｉｎｗhｉch he fａｃeｓdiｆficultieｓ． Waｌlａｃe ａｎd Ｄarwiｎｇreed ｏn the way ｉｎwhi ｃh ｄｉfｆｅrｅnt forms ｏf life had ｂegｕn.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 Thｉs is ｔｈe ｗaｙ（tｈａｔ) hｅdｉd it. I lｉkeｄｔhe waｙ(ｔhat) ｓｈeｏｒganized ｔhe ｍeetｉng． 3.theｗay(tｈat)有时可以与hｏw(作“如何”解）通用。例如： That’s tｈe ｗaｙ(tｈaｔ) shｅspoke. = Tｈat’s how shｅspoke.
初中英语比较级和最高级讲解与练习 形容词比较级和最高级 一．绝大多数形容词有三种形式,原级,比较级和最高级, 以表示形容词说明的性质在程度上的不同。 1. 形容词的原级: 形容词的原级形式就是词典中出现的形容词的原形。例如: poor tall great glad bad 2. 形容词的比较级和最高级: 形容词的比较级和最高级形式是在形容词的原级形式的基 础上变化的。分为规则变化和不规则变化。 二．形容词比较级和最高级规则变化如下: 1) 单音节形容词的比较级和最高级形式是在词尾加-er 和-est 构成。 great (原级) greater(比较级) greatest(最高级) 2) 以-e 结尾的单音节形容词的比较级和最高级是在词尾加-r 和-st 构成。 wide (原级) wider (比较级) widest (最高级) 3) 少数以-y, -er, -ow, -ble结尾的双音节形容词的比较级和最高级是在词尾加 -er 和-est构成。 clever(原级) cleverer(比较级) cleverest(最高级)， slow(原级) slower(比较级) slowest (最高级) 4) 以-y 结尾,但-y 前是辅音字母的形容词的比较级和最高级是把-y 去掉,加上-ier 和-est 构成. happy (原形) happier (比较级) happiest (最高级) 5) 以一个辅音字母结尾其前面的元音字母发短元音的形容词的比较级和最高级是双写该 辅音字母然后再加-er和-est。 原形比较级最高级原形比较级最高级 big bigger biggest hot hotter hottest red redder reddest thin thinner thinnest 6) 双音节和多音节形容词的比较级和最高级需用more 和most 加在形容词前面来构 成。 原形比较级最高级 careful careful more careful most careful difficult more difficult most difficult delicious more delicious most delicious 7)常用的不规则变化的形容词的比较级和最高级: 原级比较级最高级 good better best 好的 well better best 身体好的 bad worse worst 坏的 ill worse worst 病的 many more most 许多 much more most 许多 few less least 少数几个 little less least 少数一点儿 （little littler littlest 小的） far further furthest 远（指更进一步，深度。亦可指更远） far farther farthest 远（指更远，路程）
表示“方式”、“方法”，注意以下用法： 1.表示用某种方法或按某种方式，通常用介词in(此介词有时可省略)。如： Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法，其后可接不定式或of doing sth。 如： It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句，但是其后的从句不能由how 来引导。如： 我不喜欢他说话的态度。 正：I don’t like the way he spoke. 正：I don’t like the way that he spoke. 正：I don’t like the way in which he spoke. 误：I don’t like the way how he spoke. 4.注意以下各句the way 的用法： That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看，你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题： ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A，而不选其他几项，则要弄清选项中含way的四个短语的不同意义和用法，下面我们就对此作一归纳和小结。 一、in a way的用法 表示：在一定程度上，从某方面说。如： In a way he was right.在某种程度上他是对的。注：in a way也可说成in one way。 二、on the way的用法 1、表示：即将来(去)，就要来(去)。如： Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示：在路上，在行进中。如： He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示：(婴儿)尚未出生。如： She has two children with another one on the way.她有两个孩子，现在还怀着一个。 She's got five children，and another one is on the way.她已经有5个孩子了，另一个又快生了。 三、by the way的用法
形容词比较级和最高级的形式 一、形容词比较级和最高级的构成 形容词的比较级和最高级变化形式规则如下 构成法原级比较级最高级 ①一般单音节词末尾加 er 和 est strong stronger strongest ②单音节词如果以 e结尾，只加 r 和 st strange stranger strangest ③闭音节单音节词如末尾只有一个辅音字母， 须先双写这个辅音字母，再加 er和 est sad big hot sadder bigger hotter saddest biggest hottest ④少数以 y, er(或 ure), ow, ble结尾的双音节词， 末尾加 er和 est(以 y结尾的词，如 y前是辅音字母， 把y变成i，再加 er和 est，以 e结尾的词仍 只加 r和 st) angry Clever Narrow Noble angrier Cleverer narrower nobler angriest cleverest narrowest noblest ⑤其他双音节和多音节词都在前面加单词more和most different more different most different 1) The most high 〔A〕mountain in 〔B〕the world is Mount Everest，which is situated 〔C〕in Nepal and is twenty nine thousand one hundred and fourty one feet high 〔D〕 . 2) This house is spaciouser 〔A〕than that 〔B〕white 〔C〕one I bought in Rapid City，South Dakota 〔D〕last year. 3) Research in the social 〔A〕sciences often proves difficulter 〔B〕than similar 〔C〕work in the physical 〔D〕sciences. 二、形容词比较级或最高级的特殊形式：
比较级和最高级 1.在形容词词尾加上―er‖ ―est‖ 构成比较级、最高级： bright（明亮的）—brighter—brightest broad（广阔的）—broader—broadest cheap（便宜的）—cheaper—cheapest clean（干净的）—cleaner—cleanest clever（聪明的）—cleverer—cleverest cold（寒冷的）—colder—coldest cool（凉的）—cooler—coolest dark（黑暗的）—darker—darkest dear（贵的）—dearer—dearest deep（深的）—deeper—deepest fast（迅速的）—faster—fastest few（少的）—fewer—fewest great（伟大的）—greater—greatest hard（困难的,硬的）—harder—hardest high（高的）—higher—highest kind（善良的）—kinder—kindest light（轻的）—lighter—lightest long（长的）—longer—longest loud（响亮的）—louder—loudest low（低的）—lower—lowest near（近的）—nearer—nearest new（新的）—newer—newest poor（穷的）—poorer—poorest quick（快的）—quicker—quickest quiet（安静的）—quieter—quietest rich（富裕的）—richer—richest short（短的）—shorter—shortest slow（慢的）—slower—slowest small（小的）—smaller—smallest smart（聪明的）—smarter—smartest soft（柔软的）—softer—softest strong（强壮的）—stronger—strongest sweet（甜的）—sweeter—sweetest tall（高的）-taller-tallest thick（厚的）—thicker—thickest warm（温暖的）—warmer—warmest weak（弱的）—weaker—weakest young（年轻的）—younger—youngest 2.双写最后一个字母,再加上―er‖ ―est‖构成比较级、最高级： big（大的）—bigger—biggest fat（胖的）—fatter—fattest hot（热的）—hotter—hottest red（红的）—redder—reddest sad（伤心的）—sadder—saddest thin（瘦的）—thinner—thinnest wet（湿的）—wetter—wettest mad（疯的）—madder—maddest 3.以不发音的字母e结尾的形容词,加上―r‖ ―st‖ 构成比较级、最高级：able（能干的）—abler—ablest brave（勇敢的）—braver—bravest close（接近的）—closer—closest fine（好的,完美的）—finer—finest large（巨大的）—larger—largest late（迟的）—later—latest nice（好的）—nicer—nicest ripe（成熟的）—riper—ripest
The way的用法及其含义（一） 有这样一个句子：In 1770 the room was completed the way she wanted. 1770年，这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么？其意义如何？在阅读时，学生经常会碰到一些含有the way 的句子，如：No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中，the way之后的部分都是定语从句。第一句的意思是，“没人知道他是怎样发明这台机器的。”the way的意思相当于how；第二句的意思是，“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中，the way也是as的含义。随着现代英语的发展，the way的用法已越来越普遍了。下面，我们从the way的语法作用和意义等方面做一考查和分析： 一、the way作先行词，后接定语从句 以下3种表达都是正确的。例如：“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如：“火灾如何发生的，有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.
英语语法---比较级和最高级的用法 在英语中通常用下列方式表示的词:在形容词或副词前加more(如 more natural,more clearly )或加后缀 -er(newer,sooner )。典型的是指形容词或副词所表示的质、量或关系的增加。英语句子中，将比较两个主体的方法叫做“比较句型”。其中，像“A比B更……”的表达方式称为比较级；而“A最……”的表达方式则称为最高级。组成句子的方式是将形容词或副词变化成比较级或最高级的形态。 一、形容词、副词的比较级和最高级的构成规则 1．一般单音节词和少数以-er，-ow结尾的双音节词，比较级在后面加-er，最高级在后面加-est； （1）单音节词 如：small→smaller→smallest short→shorter→shortest tall→taller→tallest great→greater→greatest （2）双音节词 如：clever→cleverer→cleverest narrow→narrower→narrowest 2．以不发音e结尾的单音节词，比较在原级后加-r，最高级在原级后加-st； 如：large→larger→largest nice→nicer→nicest able→abler→ablest 3．在重读闭音节（即：辅音＋元音＋辅音）中，先双写末尾的辅音字母，比较级加-er，最高级加-est； 如：big→bigger→biggest hot→hotter→hottest fat→fatter→fattest 4．以“辅音字母＋y”结尾的双音节词，把y改为i，比较级加-er，最高级加-est； 如：easy→easier→easiest heavy→heavier→heaviest busy→busier→busiest happy→happier→happiest 5．其他双音节词和多音节词，比较级在前面加more，最高级在前面加most； 如：beautiful→more beautiful→most beautiful different→more different→most different easily→more easily→most easily