Sensitivity of spatially offset Raman spectroscopy (SORS) to subcortical bone tissue

The development of spatially offset Raman spectroscopy (SORS) has enabled deep, non‐invasive chemical characterization of turbid media. Here, we use SORS to measure subcortical bone tissue and depth‐resolved biochemical variability in intact, exposed murine bones. We also apply the technique to study a mouse model of the genetic bone disorder osteogenesis imperfecta. The results suggest that SORS is more sensitive to disease‐related biochemical differences in subcortical trabecular bone and marrow than conventional Raman measurements.     

Differentiation of histochemically visualized mercury and silver

Summary Accumulations of silver and mercury can be visualized in tissue sections by a technique called autometallography or physical development. In order to make a histological differentiation between mercury and silver in tissue exposed to both metals, it is necessary to remove one of the metals while leaving the other untouched. The present paper describes a technique by which silver accumulations in histological sections can be removed by potassium cyanide, yet leaving mercury accumulations intact to be developed autometallographically.     

The origin of electrokinetic potentials in bone tissue: the organic phase

The purpose of this study was to determine the relative contributions of the organic and mineral phases of cow cortical bone to its electrokinetic response at room temperature. The technique of particle electrophoresis permitted electrokinetic (zeta) potentials to be calculated and plotted as a function of pH. Control and demineralized bone particles exhibited similar isoelectric points at pH approximately 5.1 (pH at which the zeta potential is zero), well below the isoelectric point of the bone mineral (pH approximately 8.6). In addition, the use of phosphate-containing buffers resulted in a zeta potential sign reversal of the bone mineral but had no effect on both the control and demineralized bone. These key results form the basis from which we suggest that the bone mineral lies within the organic phase (e.g., the mineral is not exposed to the fluid phase) and that the electrokinetic behavior of bone tissue is dominated by its organic ultrastructure.     

Segmental bone replacement via patient‐specific,three‐dimensional printed bioresorbable graft substitutes and their use as templates for the culture of mesenchymal stem cells under mechanical stimulation at various frequencies

The treatment of large segmental bone defects remains a challenge as infection, delayed union, and nonunion are common postoperative complications. A three‐dimensional printed bioresorbable and physiologically load‐sustaining graft substitute was developed to mimic native bone tissue for segmental bone repair. Fabricated from polylactic acid, this graft substitute is novel as it is readily customizable to accommodate the particular size and location of the segmental bone of the patient to be replaced. Inspired by the structure of the native bone tissue, the graft substitute exhibits a gradient in porosity and pore size in the radial direction and exhibit mechanical properties similar to those of the native bone tissue. The graft substitute can serve as a template for tissue constructs via seeding with stem cells. The biocompatibility of such templates was tested under in vitro conditions using a dynamic culture of human mesenchymal stem cells. The effects of the mechanical loading of cell‐seeded templates under in vitro conditions were assessed via subjecting the tissue constructs to 28 days of daily mechanical stimulation. The frequency of loading was found to have a significant effect on the rate of mineralization, as the alkaline phosphatase activity and calcium deposition were determined to be particularly high at the typical walking frequency of 2 Hz, suggesting that mechanical stimulation plays a significant role in facilitating the healing process of bone defects. Utilization of such patient‐specific and biocompatible graft substitutes, coupled with patient’s bone marrow cells seeded and exposed to mechanical stimulation of 2 Hz have the potential of reducing significant volumes of cadaveric tissue required, improving long‐term graft stability and incorporation, and alleviating financial burdens associated with delayed or failed fusions of long bone defects.     

Mesenchymal progenitor cells in adult human dental pulp and their ability to form bone when transplanted into immunocompromised mice

The technique of tissue engineering is developing for the restoration of lost tissues. This new technique requires cells that fabricate tissue. Mesenchymal stem cells in bone marrow have been used as the cell source for this technique; however, dental pulp cells have recently been shown to possess stem-cell-like properties. We earlier demonstrated that dental pulp cells proliferate and produce an extracellular matrix that subsequently becomes mineralized in vitro. We now report that such dental pulp cells (first to eighth passage) produced bone instead of dentin when those cells were implanted into subcutaneous sites in immunocompromised mice with HA/TCP powder as their carrier. This evidence shows that dental pulp cells are the common progenitors of odontoblasts and osteoblasts, or dental pulp cells are mesenchymal stem cells themselves. It is expected that dental pulp cells can be a useful candidate cell source for tissue engineering, and contain the potential of new therapeutic approaches for the restoration of damaged or diseased tissue.     

Autometallography: tissue metals demonstrated by a silver enhancement kit

In biological tissue, minute accumulations of gold, silver, mercury and zinc can be visualized by a technique whereby metallic silver is precipitated on tiny accumulations of the two noble metals, or on selenites or sulphides of all four metals. In the present study a silver enhancement kit, primarily intended for the amplification of colloidal gold particles, has been used to demonstrate these catalytic tissue metals. Sections from animals exposed intravitally to aurothiomalatate, silver lactate, mercury chloride, sodium selenite or perfused with sodium sulphide were subjected to a commercial silver enhancement kit (IntenSE, Janssen Pharmaceutica). It was found that the kit performs adequately to the silver lactate gum arabic developer and to the photographic emulsion technique. The kit can be used as a silver enhancement medium for the demonstration of zinc by the Neo-Timm and selenium methods and for demonstration of gold, silver, and mercury in tissues from animals intravitally exposed to these metals. It can also be used for counterstaining silver treated osmium fixed tissues embedded in plastic.     

Advances in 3D printing of composite scaffolds for the repairment of bone tissue associated defects

The conventional methods of using autografts and allografts for repairing defects in bone, the osteochondral bone, and the cartilage tissue have many disadvantages, like donor site morbidity and shortage of donors. Moreover, only 30% of the implanted grafts are shown to be successful in treating the defects. Hence, exploring alternative techniques such as tissue engineering to treat bone tissue associated defects is promising as it eliminates the above-mentioned limitations. To enhance the mechanical and biological properties of the tissue engineered product, it is essential to fabricate the scaffold used in tissue engineering by the combination of various biomaterials. Three-dimensional (3D) printing, with its ability to print composite materials and with complex geometry seems to have a huge potential in scaffold fabrication technique for engineering bone associated tissues. This review summarizes the recent applications and future perspectives of 3D printing technologies in the fabrication of composite scaffolds used in bone, osteochondral, and cartilage tissue engineering. Key developments in the field of 3D printing technologies involves the incorporation of various biomaterials and cells in printing composite scaffolds mimicking physiologically relevant complex geometry and gradient porosity. Much recently, the emerging trend of printing smart scaffolds which can respond to external stimulus such as temperature, pH and magnetic field, known as 4D printing is gaining immense popularity and can be considered as the future of 3D printing applications in the field of tissue engineering.     

Iliac crest bone graft harvesting techniques: a comparison

This study was undertaken to compare the morbidity of traditional iliac bone graft harvesting techniques for grafting alveolar clefts to minimally invasive techniques. Fifty-five age-matched patients, ages 6.5 to 16 years (mean, 11.2 years), 22 girls and 33 boys, were divided into three groups. The traditional bone window open harvesting technique served as the control group. Two different minimally invasive techniques, one that used a bone grinder and another that used a trephine, for bone harvesting were compared with the control. Both invasive techniques were statistically superior, p < 0.05, in terms of total time pain medication was necessary (mean of 12.0 hours for bone grinder, 17.6 hours for trephine, 26.0 hours for control), operative time for bone harvest (mean of 11 minutes for bone grinder and trephine, 20 minute for control), and mean incision length (2 cm for bone grinder and trephine, 5 cm for control). Patients exposed to the minimally invasive techniques had fewer complications, a trend toward earlier ambulation, and shorter hospital stays when compared with the bone grinder technique. The patients exposed to the bone grinder demonstrated earlier ambulation and fewer requirements for analgesia when compared with the trephine technique, although these results did not reach statistical significance. The trephine technique was useful when maxillary osteotomies were combined with alveolar bone grafting, because it provided structural bone grafts and cancellous bone. On the basis of these findings, the bone grinder is the preferred technique for harvesting alveolar bone grafts when no structural support is required. These authors no longer use the traditional bone window open harvesting technique.     

An immunocytochemical method for quantification of lung tissue fibronectin

A technique to quantify tissue fibronectin was developed, using peroxidase-antiperoxidase immunocytochemistry and automated scanning light microscopy. This technique was developed using isolated perfused rat lungs, some of which were subjected to acute oxidant lung injury. Both injured and control lungs were perfused with solutions containing heterologous fibronectin. The technique clearly demonstrated differences in the amount of tissue fibronectin in injured and noninjured lung as well as differences between lungs exposed to fibronectin and those not exposed. The described technique offers a reliable method for quantifying tissue fibronectin and is sensitive enough to detect differences in tissue fibronectin under experimental conditions of acute lung injury.     

Bone poroelasticity

Poroelasticity is a well-developed theory for the interaction of fluid and solid phases of a fluid-saturated porous medium. It is widely used in geomechanics and has been applied to bone by many authors in the last 30 years. The purpose of this work is, first, to review the literature related to the application of poroelasticity to the interstitial bone fluid and, second, to describe the specific physical and modeling considerations that establish poroelasticity as an effective and useful model for deformation-driven bone fluid movement in bone tissue. The application of poroelasticity to bone differs from its application to soft tissues in two important ways. First, the deformations of bone are small while those of soft tissues are generally large. Second, the bulk modulus of the mineralized bone matrix is about six times stiffer than that of the fluid in the pores while the bulk moduli of the soft tissue matrix and the pore water are almost the same. Poroelasticity and electrokinetics can be used to explain strain-generated potentials in wet bone. It is noted that strain-generated potentials can be used as an effective tool in the experimental study of local bone fluid flow, and that the knowledge of this technique will contribute to the answers of a number of questions concerning bone mineralization, osteocyte nutrition and the bone mechanosensory system.     

Fine Needle Tissue Aspiration for Accurate Localization of Histological Specimens from Complex Structures

A procedure is presented for exact, detailed comparison of light and electron microscopic analyses of tissues with complex architecture. Earlier techniques require one to make drawings of tissue pieces to be analyzed by electron microscopy to permit rough localization of the origin of the tissue pieces. Specifically, exact analysis of fetal cartilage and bone is hampered by the complicated arrangement of both tissue components, severely limiting the assessment of electron microscopic analyses. The advantage of the technique described here is that it allows precise localization of the tissue sample in the original tissue area. Punches 1 mm in diameter were obtained from femora and coxae with a syringe and embedded for light and electron microscopy. The remaining tissue with its exactly defined punctures is prepared for standard histology. Human fetal cartilage and bone tissue were used to demonstrate this technique, but this procedure may be used for other kinds of tissues.     

应用3D适形打印制备网状复合体对兔颅骨缺损的修复作用及安全性研究

目的:探讨采用3D适形打印技术制备的羟基磷灰石/聚乳酸网状复合体在兔颅骨缺损中的修复作用及安全性。方法:以24只新西兰兔为研究对象,以羟基磷灰石/聚乳酸为材料,采用3D适形打印技术制备网状复合体,于兔颅骨顶部制成两个颅骨全层缺损,分别为孔A(左)和孔B(右),孔A(阳性对照组)以自体颅骨为修复材料,孔B(实验组)以复合体为修复材料,观察缺损修复区域的形态学、影像学(X线及CT扫描)及组织学检查结果。结果:植入后24周时,形态学显示:阳性对照组可见致密的骨组织修复,与缺损边缘界限不清,实验组中支架孔隙内纤维组织由新生骨质取代,且新生骨成熟度较提高,材料表面有部分吸收。CT扫描观察显示:冠状面上,阳性对照组缺损修复区域与周围正常骨组织融合为一体,实验组修复材料与缺损边缘融合紧密,与周围正常骨组织结合良好,部分边缘结合不连贯。组织学观察显示:实验组材料部分降解,材料间隔可见新生骨小梁。研究中无实验动物死亡,皮肤切口处缝合良好,无皮下积液,无移植物脱出、红肿感染等情况出现。结论:以3D适形打印技术制备的羟基磷灰石/聚乳酸复合体对兔颅骨缺损有较好的修复作用,能促进缺损区域新骨的形成和生长,且安全性较高。     

The elastic properties of trabecular and cortical bone tissues are similar: results from two microscopic measurement techniques

Acoustic microscopy (30-60 microm resolution) and nanoindentation (1-5 microm resolution) are techniques that can be used to evaluate the elastic properties of human bone at a microstructural level. The goals of the current study were (1) to measure and compare the Young's moduli of trabecular and cortical bone tissues from a common human donor, and (2) to compare the Young's moduli of bone tissue measured using acoustic microscopy to those measured using nanoindentation. The Young's modulus of cortical bone in the longitudinal direction was about 40% greater than (p<0.01) the Young's modulus in the transverse direction. The Young's modulus of trabecular bone tissue was slightly higher than the transverse Young's modulus of cortical bone, but substantially lower than the longitudinal Young's modulus of cortical bone. These findings were consistent for both measurement methods and suggest that elasticity of trabecular tissue is within the range of that of cortical bone tissue. The calculation of Young's modulus using nanoindentation assumes that the material is elastically isotropic. The current results, i.e., the average anisotropy ratio (E(L)/E(T)) for cortical bone determined by nanoindentation was similar to that determined by the acoustic microscope, suggest that this assumption does not limit nanoindentation as a technique for measurement of Young's modulus in anisotropic bone.     

Preparation of Fossil Bone Specimens for Scanning Electron Microscopy

A method is described for preparing fossil bone specimens for scanning electron microscopy. To obtain bone surfaces suitable for study, material was embedded in Epon 812 and selected faces exposed by grinding were subjected to controlled etching with a 4:1 mixture of 5% HNO₃ and 1% OsO₄, Surfaces thus prepared were further processed by the so-called clearing replicas technique. As a result of this procedure the bone surfaces revealed a network of anastomosing vascular canals the inner surface of whose walls could be examined in the scanning electron microscope. By etching extremely thin ground sections of bone stuck to plastic tape the contents of vascular canals as well as osteocytes can be isolated. This method ensures the good preservation of spatial relations between bone elements essential for studies of fossil bones, which an sometimes very brittle.     

An embedding method for histochemical studies of undecalcified skeletal growth plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tisssues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

An Embedding Method for Histochemical Studies of Undecalcified Skeletal Growth Plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tissues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

Proteinases involved in matrix turnover during cartilage and bone breakdown

The joint is a discrete unit that consists of cartilage, bone, tendon and ligaments. These tissues are all composed of an extracellular matrix made of collagens, proteoglycans and specialised glycoproteins that are actively synthesised, precisely assembled and subsequently degraded by the resident connective tissue cells. A balance is maintained between matrix synthesis and degradation in healthy adult tissues. Different classes of proteinases play a part in connective tissue turnover in which active proteinases can cleave matrix protein during resorption, although the proteinase that predominates varies between different tissues and diseases. The metalloproteinases are potent enzymes that, once activated, degrade connective tissue and are inhibited by tissue inhibitors of metalloproteinases (TIMPs); the balance between active matrix metalloproteinases and TIMPs determines, in many tissues, the extent of extracellular matrix degradation. The serine proteinases are involved in the initiation of activation cascades and some, such as elastase, can directly degrade the matrix. Cysteine proteinases are responsible for the breakdown of collagen in bone following the removal of the osteoid layer and the attachment of osteoclasts to the exposed bone surface. Various growth factors increase the synthesis of matrix and proteinase inhibitors, whereas cytokines (alone or in combination) can inhibit matrix synthesis and stimulate proteinase production and matrix destruction.     

PIXE technique for calcium analysis of human bone

The determination of calcium content in human bone tissue is very useful in metabolic diseases of bone, such as renal osteodystrophy, osteoporosis, hyperparathyroidism, and osteomalacia of diverse etiology. The PIXE technique allows calcium to be directly determined in bioptic tissue sections properly, sampled for histological optical and/or electron microscopy examination. Bone semithin sections (3 μm thick, 4×4 mm² dimensions), cut by ultramicrotome and deposited onto polyvinyl acetate films, underwent PIXE analysis using the CISE set-up. Histomorphometric (after standard staining), evaluation of calcified bone volume (CBV) in absolute value allows calcium density to be determined. A total of nine bone biopsies were analyzed (three sections each) obtaining values ranging between 352 and 482, with an average value of 421.5±15.3 (M±SE) μg/μL, in good agreement with literature data (obtained by AAS technique on dissected bone samples). The aim of this paper is to emphasize the usefulness, of combined PIXE and histomorphometric techniques for the study of calcium content in bone tissue in both healthy and diseased bones.     

Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds

In this work, we investigated whether osteoinductive constructs can be generated by isolation and expansion of sheep bone marrow stromal cells (BMSC) directly within three-dimensional (3D) ceramic scaffolds, bypassing the typical phase of monolayer (2D) expansion prior to scaffold loading. Nucleated cells from sheep bone marrow aspirate were seeded into 3D ceramic scaffolds either by static loading or under perfusion flow and maintained in culture for up to 14 days. The resulting constructs were exposed to enzymatic treatment to assess the number and lineage of extracted cells, or implanted subcutaneously in nude mice to test their capacity to induce bone formation. As a control, BMSC expanded in monolayer for 14 days were also seeded into the scaffolds and implanted. BMSC could be isolated and expanded directly in the 3D ceramic scaffolds, although they proliferated slower than in 2D. Upon ectopic implantation, the resulting constructs formed a higher amount of bone tissue than constructs loaded with the same number of 2D-expanded cells. Constructs cultivated for 14 days generated significantly more bone tissue than those cultured for 3 days. No differences in bone formation were found between samples seeded by static loading or under perfusion. In conclusion, the culture of bone marrow nucleated cells directly on 3D ceramic scaffolds represents a promising approach to expand BMSC and streamline the engineering of osteoinductive grafts.     

RESPONSE OF THE PREOSTEOBLAST AND STEM CELL OF RAT BONE MARROW TO A LETHAL DOSE OF X-IRRADIATION OR CYCLOPHOSPHAMIDE

Following syngeneic or autotransplantation of hemopoietic tissue to a heterotopic location, bone formation has been observed to occur in the implanted tissue. the characteristics of the cell residing in hemopoietic tissue with bone forming potential (preosteoblast) are unknown. to define some properties of this cell, its response to X-irradiation and cyclophosphamide (CTX) was compared to the response of the hemopoietic stem cell. Adult, male rats were exposed to 900 R whole body X-irradiation or 220 mg/kg of intraperitoneal CTX. With either treatment the dose was sufficient to kill the animals by bone marrow failure. At intervals following the X-irradiation or CTX, hemopoietic tissue was examined for the presence of viable hemopoietic stem cells and preosteoblasts. Following X-irradiation, viable hemopoietic stem cells and preosteoblasts could not be detected. Following CTX these cells could be detected. It is suggested that in the rat CTX at 220 mg/kg, although causing death by bone marrow failure, does not reduce the population of the preosteoblast or hemopoietic stem cell as effectively as 900 R X-irradiation.