Vectorial sequence of mineralization in the turkey leg tendon determined by electron microscopic imaging

Summary Turkey leg tendons were used as a model tissue to study the spatial and temporal relationships of mineral deposition between matrix vesicles and collagen fibrils by various electron microscopic techniques—bright field, selected-area dark field (SADF), and electron spectroscopic imaging (ESI). These latter imaging techniques enabled the direct localization and spatial distributions of both apatite crystals and atomic elements (Ca, P) within matrix vesicles and collagen. In longitudinal planes of section, a consistent vectorial gradient of mineralization was observed which started with the first localization of apatite mineral in matrix vesicles; with further development, the mineral spread from the vesicle to the extravesicular interstices and then into the adjacent collagen fibrils. Once intrafibrillar, the mineral was observed to advance both laterally and axially. The association of vesicle/collagen mineral was examined by ESI analysis of Ca and P elemental maps and appeared as a continuum between the vesicles and the adjacent collagen fibrils. Similarly, an intimate spatial relationship was observed between the mineral of vesicles and collagen in transversely cut sections of tendon. The sequential development of this mineralized matrix is discussed in light of matrix vesicle/collagen interactions.     

Fourier transform infrared microscopy of calcified turkey leg tendon

Fourier transform infrared microscopy (FT-IRMS) was used to monitor spatial variations in the quality and quantity of the mineral phase in calcified turkey tendon. Spectral maps were generated by analysis of 50 μm ×~ 50 μm areas within different regions of the tendon. Spectra of the transitional region, where nonmineralized matrix ends and mineralized matrix begins, revealed marked changes in the spectrally determined mineral-to-matrix ratio, whereas regions deeper into the mineralization front showed a relatively constant ratio. Since spectra of EDTA-demineralized matrix were similar to those of nonmineralized matrix, the nonmineralized regions of the tendon were used for spectral subtraction. The broad, relatively featureless contour of the mineral ν₁,ν₃ phosphate region (900–1200 cm⁻¹) showed only subtle changes at different stages of mineralization. Second derivatives of these spectra were calculated and compared with those of synthetic, poorly crystalline hydroxyapatite (HA). The peak positions seen in second-derivative spectra of the mineral near the transitional region were within ±2 cm⁻¹ of the least mature synthetic HAs whereas spectra of the mineral deeper into the mineralization front were within ±2 cm⁻¹ of the most mature synthetic HAs. Spectra from cross- and longitudinal sections at equivalent positions in the tendon, and polarized FT-IRMS data were analyzed to determine the effect of mineral orientation on the parameters used to characterize the mineral. Spectra of cross- and longitudinal sections of the tendon showed no major differences in either the ν₁,ν₃ phosphate region or the amide I, II, or III components (1200–1800 cm⁻¹). However, polarized FT-IR spectra revealed dramatic differences in both of these regions. Despite these differences, second-derivative analysis of the ν₁,ν₃ regions revealed no significant changes in the positions of the underlying bands used to characterize the environments of the phosphate ion in poorly crystalline HA. The results of this study demonstrate the power of FT-IRMS to monitor spatial variations of the mineral phase in calcified tissue. Also, the incorporation of polarized radiation provides a method capable of assessing the molecular orientation of the mineral phase relative to the collagen matrix. Received: 17 January 1995 / Accepted: 26 May 1995     

Crystal-collagen relationships in calcified turkey leg tendons visualized by selected-area dark field electron microscopy

Summary The distribution and orientation of biological apatite crystals in calcified turkey leg tendons were studied by selected-area dark field electron microscopy. This imaging technique enables the direct visualization of apatite and the specific determination of the crystallographic axes (a, b-axes or c-axis) within calcified collagen fibrils. This study shows that at early stages of mineralization, rod-shaped apatite crystals (5–20 nm in length) were localized and dispersed within gap zones bordering both the collagen molecule C- and N-terminal regions. At later stages of mineral deposition the crystals were more extensive, occupying greater areas of the gap zone and, in addition, apatite crystals were found to occur in the overlap zones. The orientation of apatite crystals was observed to be an alternating and interlocking distribution of a, b-axes and c-axis along the axial period of collagen fibrils. This distribution is interpreted as representing a continuous rotation of apatite axial orientation along the collagen period.     

Visualization of crystal-matrix structure. In situ demineralization of mineralized turkey leg tendon and bone

A technique to correlate the ultrastructural distribution of mineral with its organic material in identical sections of mineralized turkey leg tendon (MTLT) and human bone was developed. Osmium or ethanol fixed tissues were processed for transmission electron microscopy (TEM). The mineralized tissues were photographed at high, intermediate, and low magnifications, making note of section features such as fibril geometry, colloidal gold distribution, or section artifacts for subsequent specimen realignment after demineralization. The specimen holder was removed from the microscope, the tissue section demineralized in situ with a drop of 1 N HCl, then stained with 2% aqueous vanadyl sulfate. The specimen holder was reinserted into the microscope, realigned with the aid of the section features previously noted, and rephotographed at identical magnification used for the mineralized sections. A one to one correspondence was apparent between the mineral and its demineralized crystal ghost in both MTLT and bone. The fine structural periodic banding seen in unmineralized collagen was not observed in areas that were fully mineralized before demineralization, indicating that the axial arrangement of the collagen molecules is altered significantly during mineralization. Regions that had contained extrafibrillar crystallites stained more intensely than the intrafibrillar regions, indicating that the noncollagenous material surrounded the collagen fibrils. The methodology described here may have utility in determining the spatial distribution of the noncollagenous proteins in bone.     

The distribution of water in calcified turkey leg tendon

X-ray diffraction and water sorption data are presented which show that the extracellular water in calcified turkey leg tendon is associated principally with the collagen component.     

Image analysis of collagen-associated mineral distribution in cryogenically prepared turkey leg tendons

Summary This study is concerned with the cryogenic preservation of intrafibrillar apatite distribution in type I collagen of turkey leg tendons. Cryogenic specimen preparations by the rapid freezing of nonfixed and noncryoprotected leg tendons were performed by two different protocols: (1) low temperature substitution, fixation and staining followed by low temperature embedment; (2) frozen hydrated and air-dried cryosections were examined with the electron microscope at −165°C and normal operating temperatures, respectively. These protocols revealed the axial periodicity for mineralized collagen to have a 65–69 nm range with a mean value of 67 nm as determined by point-to-point measurements. Mineral distributions and specific apatite visualization were examined by electron microscopic imaging in bright field and selected-area dark field, respectively. Fourier filtered images and image subtraction were used to separate the axial repeating and nonrepeating intrafibrillar mineral domains of collagen. The removal of these axial repeats revealed an underlying and integrated mineral distribution, demonstrating that apatite is not confined to axial periodicities such as those of the gap zone.     

Stress shielding of patellar tendon: effect on small-diameter collagen fibrils in a rabbit model

The purpose of this study was to assess the effects of stress shielding on the microstructure and ultrastructure of the patellar tendon using 40 mature female Japanese white rabbits. The patellar tendon was completely released from stress by drawing the patella toward the tibial tubercle with a stainless steel wire installed between them. Microstructurally, stress shielding for 3 and 6 weeks increased the number of cells approximately fivefold, to that of the control tendon. Collagen bundles were less well oriented in the stress-shielded tendon than in the control. Ultrastructurally, small collagen fibrils with a diameter of less than 90nm increased in the stress-shielded tendon. The median collagen fibril diameter in 6-week stress-shielded tendon was significantly smaller (P 0.05) than in the control tendon (58.8% of control). The ratio of the total area of collagen fibrils to the whole visualized area in the stress-shielded patellar tendon was significantly smaller at 3 and 6 weeks than that in the control. This study demonstrated that complete stress shielding significantly affects the microstructure and ultrastructure of the patellar tendon     

应用原子力显微镜观察脉冲电磁场对大鼠骨质疏松的治疗后骨组织表面超微结构的变化

目的观察实验性大鼠骨质疏松模型股骨颈超微结构的变化特点,并探讨利用原子力显微镜观察的可行性。方法清洁级雌性SD大鼠(鼠龄3个月)60只,体重(200±20)g。随机将其分成对照组(Sham组)、去卵巢组(OVX组)、阿伦膦酸钠治疗组(ALN组)、PEMFs治疗组(PEMFs组),每组大鼠15只。除Sham组外,对其他各组行双侧卵巢切除术,造模后第30天开始分组干预。ALN组给予阿伦膦酸钠灌胃,PEMFs组给予脉冲电磁场治疗。Sham组和OVX组手术后正常喂养,不予任何处理。治疗后30d取大鼠股骨头切片,通过AFM观察大鼠股骨颈的表面超微结构。结果 Sham组原子力显微镜扫描可显示骨组织的骨陷窝、骨小管及其表面的钙磷晶体沉积,骨组织表面粗糙度为(2.59±0.645)μm。OVX组可见骨陷窝的大小变化及其钙磷晶体的排列紊乱,模型组骨组织表面粗糙度明显高于对照组(P0.01),提示造模成功;ALN组表面粗糙度明显低于OVX组(P0.05),PEMFs组的表面粗糙度与OVX组相比,有明显降低,差异具有统计学意义(P0.05);而PEMFs组与ALN组比较,其表面粗糙度无明显差异(P0.05)。结论通过原子力显微镜能清晰观察到脉冲电磁场对大鼠骨质疏松治疗后的骨组织表面超微结构的变化,其疗效与阿仑膦酸钠相当,为脉冲电磁场治疗骨质疏松提供了一定的理论依据。     

Applications of atomic force microscopy for the assessment of nanoscale morphological and mechanical properties of bone

Scanning probe microscopy (SPM) has been in use for 30 years, and the form of SPM known as atomic force microscopy (AFM) has been around for 25 of those years. AFM has been used to produce high resolution images of a variety of samples ranging from DNA to carbon nanotubes. Type I collagen and many collagen-based tissues (including dentin, tendon, cartilage, skin, fascia, vocal cords, and cornea) have been studied with AFM, but comparatively few studies of bone have been undertaken. The purpose of this review is to introduce the general principles of AFM operation, demonstrate what AFM has been used for in bone research, and discuss the new directions that this technique can take the study of bone at the nanoscale.     

小腿筋膜皮瓣及小腿内侧逆行轴形皮瓣在下肢创伤修复时的选择及评估

目的:探讨两种小腿皮瓣(小腿筋膜皮瓣及小腿内侧逆行轴形皮瓣)在下肢创伤后皮肤软组织缺损修复时的选择及评估。方法:①在小腿设计局部随意筋膜皮瓣,皮瓣大小范围4cm×6cm～7cm×15cm,转移修复同侧或对侧小腿皮肤软组织缺损并骨外露;②以胫后动脉内踝上发出的皮动脉穿支为轴,在小腿内侧设计逆行轴形皮瓣,皮瓣大小范围4cm×12cm～6cm×15cm,转移修复同侧或对侧足底、踝部、小腿皮肤软组织缺损并肌腱或骨外露。结果:临床共选用11个皮瓣,其中6例为筋膜皮瓣,5例为小腿内侧逆行轴形皮瓣,修复同侧或对侧下肢皮肤软组织缺损并肌腱或骨外露。11例皮瓣术后均成活良好,创面修复效果良好,随访6～12个月,伤肢外形及功能恢复满意。结论:小腿筋膜皮瓣,设计灵活,切取便利,不损伤主要血管,血供丰富,尤其在局部主要血管受损时是修复下肢严重创伤的有效方法之一。小腿内侧逆行轴形皮瓣可切取皮瓣距离长,面积大,血供恒定可靠,成活率高,是修复足底、踝部、小腿严重创伤的最佳选择。此两种皮瓣的应用应针对小腿创面的部位、面积及其创伤特点进行合适的选择,可达到满意的修复效果。     

Regional membrane specialization in the thin limbs of Henle's loops as seen by freeze-fracture electron microscopy

Rat thin limbs of Henle were studied by freeze-fracture electron microscopy. Thin limb segments in both short- and long-looped nephrons were identified by previously developed ultrastructural criteria, continuity with known thick segments, and architectural relationships in the outer medulla. Intramembrane particle (IMP) density and the number of intramembrane fibrils comprising the zonula occludens were determined for each morphologically identifiable thin limb segment. The IMP density on the protoplasmic faces of both the luminal and abluminal membranes of the upper portion of the descending thin limb (DTL) of the long-looped nephron is quantitatively greater than in the short-looped thin limb, lower portion of the long-looped DTL, and in the ascending thin limb. The zonulae occludens in the long-looped upper DTL consists of a single fibril; the long-looped lower DTL contains 3.13 +/- 0.14 fibrils; the ascending thin limb contains 1.31 +/- 0.09 fibrils; and the short-looped DTL contains 3.75 +/- 0.19 fibrils. These studies further support the contention that there is anatomic heterogeneity among the thin limb segments. Because direct physiologic studies in the thin limbs are incomplete and conflicting, the need for correlative physiologic studies on anatomically characterized structures is indicated.     

Simultaneous chronic rupture of quadriceps tendon and contra-lateral patellar tendon in a patient affected by tertiary hyperparatiroidism

Spontaneous ruptures of the extensor mechanism of the knee are very rare. They tend to increase considerably in patients with metabolic diseases such as chronic renal failure, hyperparathyroidism, diabetes, gout, and systemic lupus erythematosus. The reported case regards a 48-year-old man with chronic, spontaneous and simultaneous quadriceps, and contra-lateral patellar tendon rupture. The patient suffered from chronic renal failure and for the past year from tertiary hyperparathyroidism. Ruptured tendons were repaired and both knee were evaluated monthly for the next 12 months. Good functional recovery was achieved on both knees without relapse. This case emphasizes the importance of long-term high parathyroid hormone level in the etiology of tendons ruptures.     

Evaluating adhesion reduction efficacy of type I/III collagen membrane and collagen-GAG resorbable matrix in primary flexor tendon repair in a chicken model

Background

Reduction of peritendinous adhesions after injury and repair has been the subject of extensive prior investigation. The application of a circumferential barrier at the repair site may limit the quantity of peritendinous adhesions while preserving the tendon’s innate ability to heal. The authors compare the effectiveness of a type I/III collagen membrane and a collagen-glycosaminoglycan (GAG) resorbable matrix in reducing tendon adhesions in an experimental chicken model of a “zone II” tendon laceration and repair.

Methods

In Leghorn chickens, flexor tendons were sharply divided using a scalpel and underwent repair in a standard fashion (54 total repairs). The sites were treated with a type I/III collagen membrane, collagen-GAG resorbable matrix, or saline in a randomized fashion. After 3 weeks, qualitative and semiquantitative histological analysis was performed to evaluate the “extent of peritendinous adhesions” and “nature of tendon healing.” The data was evaluated with chi-square analysis and unpaired Student’s t test.

Results

For both collagen materials, there was a statistically significant improvement in the degree of both extent of peritendinous adhesions and nature of tendon healing relative to the control group. There was no significant difference seen between the two materials. There was one tendon rupture observed in each treatment group. Surgical handling characteristics were subjectively favored for type I/III collagen membrane over the collagen-GAG resorbable matrix.

Conclusion

The ideal method of reducing clinically significant tendon adhesions after injury remains elusive. Both materials in this study demonstrate promise in reducing tendon adhesions after flexor tendon repair without impeding tendon healing in this model.     

The effect of different mineral frames on ectopic bone formation in mouse hind leg muscles induced by native reindeer bone morphogenetic protein

Introduction Bone morphogenetic proteins (BMPs) require carrier material for slow release and framing material for osteoconduction.Materials and methods The effect of a frame on early bone formation induced by partially purified native reindeer BMP in composite implants containing 3 mg of BMP, type IV collagen and tricalcium phosphate (TCP/Col/BMP) or hydroxyapatite (HA/Col/BMP) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA/Col/BMP) or biocoral (NC/Col/BMP) was evaluated using a mouse hind leg muscle pouch model. Collagen with native reindeer BMP (Col/BMP) and corresponding implants without native reindeer BMP served as controls. Evaluation was done by incorporation of ⁴⁵Ca, radiographically and histologically 3 weeks after the implantation.Results None of the implants without native reindeer BMP were able to induce new bone visible on radiographs. The area of new bone formation in the Col/BMP (p=0.026) and TCP/HA/Col/BMP (p=0.012) groups was significantly greater than in the TCP/Col/BMP group. The optical density of the new bone area was significantly greater in the TCP/HA/Col/BMP group than in the TCP/Col/BMP (p=0.036) or Col/BMP (p=0.02) groups. ⁴⁵Ca incorporation was many times greater in all the groups containing native reindeer BMP than in the corresponding groups without BMP. In the Col/BMP (p=0.046) and TCP/HA/Col/BMP (p=0.046) groups, ⁴⁵Ca incorporation was significantly greater than in the TCP/Col/BMP group. No significant differences were found in any parameters between HA/Col/BMP and NC/Col/BMP groups and the other BMP-containing groups.Conclusions Hydroxyapatite, biocoral and biphasic tricalciumphosphate-hydroxyapatite are equally good as framing material for native reindeer BMP, while tricalciumphosphate is somewhat worse. Osteoinduction of native reindeer BMP works well with collagen alone.     

The role of the macrophage in tendinopathy and tendon healing

The role of the macrophage is an area of emerging interest in tendinopathy and tendon healing. The macrophage has been found to play a key role in regulating the healing process of the healing tendon. The specific function of the macrophage depends on its functional phenotype. While the M1 macrophage phenotype exhibits a phagocytic and proinflammatory function, the M2 macrophage phenotype is associated with the resolution of inflammation and tissue deposition. Several studies have been conducted on animal models looking at enhancing or suppressing macrophage function, targeting specific phenotypes. These studies include the use of exogenous biological and pharmacological substances and more recently the use of transgenic and genetically modified animals. The outcomes of these studies have been promising. In particular, enhancement of M2 macrophage activity in the healing tendon of animal models have shown decreased scar formation, accelerated healing, decreased inflammation and even enhanced biomechanical strength. Currently our understanding of the role of the macrophage in tendinopathy and tendon healing is limited. Furthermore, the roles of therapies targeting macrophages to enhance tendon healing is unclear. Clinical Significance: An increased understanding of the significance of the macrophage and its functional phenotypes in the healing tendon may be the key to enhancing tendon healing. This review will present the current literature on the function of macrophages in tendinopathy and tendon healing and the potential of therapies targeting macrophages to enhance tendon healing.     

原子力显微镜对角质形成细胞与黑素细胞共培养模型的观察研究

目的：用原子力显微镜观察人表皮黑素细胞（MC）、角质形成细胞（KC）单独培养和共培养的表面形态以及α-促黑素（α-MSH）对细胞表面形态的影响。方法：分别纯化培养来自人包皮的表皮MC和KC,MC以自配的添加MC生长物质的MCDB153培养基培养,KC以KC无血清培养基（K-SFM）常规培养。传第2代后以1：10的比例将两细胞接种到3Cm×3Cm的小培养皿中,以K-SFM培养基继续培养,单独或混合培养的细胞经添加含或不含100nMα-MSH的培养基干预3天后,0．5％戊二醛固定10min,原子力显微镜常温常压下,触摸式扫描。结果：正常人表皮MC有3个树突,每个树突有明显的二级分枝,除主干和分支见到膨出的颗粒物质,我们在树突的侧缘底侧和顶端还发现有丝状伪足结构,经α-MSH刺激后树突明显变长、变细,主干和分支表面膨出颗粒物质更为密集,许多已脱离枝干,丝状伪足则未有明显变化。表皮KC表面可见许多片状或钩状突起。共培养后,KC与MC接触部位可见明显的丝状伪足样结构,未连接部位则未见到丝状伪足样结构,添加α-MSH后,两细胞连接处的丝状伪足样结构明显增多。结论：通过胞吐和丝状伪足输送可能是黑素小体从MC向KC传递的两种主要方式,α-MSH可能通过促进这两种结构的发生而发挥促黑素传递的作用。     

闭合性跟腱断裂微创腱皮缝合的远期疗效观察

[目的]通过对长期疗效和可能并发症的观察,介绍并评价微创腱皮缝合术治疗急性闭合性跟腱断裂的临床效果。[方法]自1996年1月～2005年4月间,随机选取跟腱断裂的患者28名,经术前查体和MRI证实,均为新鲜完全性闭合性跟腱断裂。首先微创暴露跟腱断端,清除断端的瘢痕和血凝块,梳理对合两断端,在皮外经跟腱断端以远健康部位做减张缝合。患者常规术后随访,平均随访4年（1～7年）。随访包括常规的临床评价和术后MRI检查,并应用Arner-Lindholm疗效评定标准对术后效果进行评定。其中2例未能按时随访。[结果]对所有26例患者,按Arner-Lindholm疗效评定标准进行评定,其中优19例,良6例,差1例,优良率为97%,无感染发生,再断裂1例,为长期局部类固醇封闭患者。术后MRI显示,全部患者的跟腱得到了良好的修复,跟腱断端连续性好,疤痕小。[结论]通过长期随访,微创腱皮缝合是一种修复急性闭合性跟腱断裂较好的方法,具有创伤小,有效保护跟腱血运,术后并发症少的优点。     

The distribution of trace metal ions in bone and tendon

The natural trace metal compositions of human bone mineral and demineralized bone were measured by emission spectroscopy and compared with those of the original whole bone and human tendon. Cu, Fe and Zn remained in the collagenous matrix of bone and were found in similar quantities in tendon. It is suggested, therefore, that these ions are chemically bound to the collagen matrix in these tissuesin vivo, and that the binding has a part in their activity. Most of the Pb, Si, Sr, and V in bone remained with the mineral portion, probably as substituted or interstitial ions. Zn is divided between the organic and mineral phases.This work was supported in part by Grant No. AMO 7626, National Institutes of Health, United States Public Health Service and by the Veterans Administration Research Service.