Remodeling of bone and bones: effects of altered mechanical stress on the regeneration of transplanted bones

We divided 116 rats weighing 50 gm into four groups with tails either left in situ or transplanted as follows: straight in situ: untreated controls; bent in situ: five caudal vertebrae (CV) in the loop; straight transplants: three CV skinned and transplanted autologously; and bent transplants: five CV skinned, bent to form a loop, and transplanted autologously. Tails were radiographed weekly up to 6 weeks and at 12 weeks, and microradiographic and histological studies were undertaken on selected specimens. At 12 weeks the bones in the apex of the loop of tails left in situ appeared bent with a straight-to-convex shaft on the outer side and a thicker, more concave one on the inner side. In the transplanted bent segments the bone shaft died and initially the reverse occurred: the outer shaft thickened and the inner resorbed completely. A new concave inner diaphysis then formed so that the bones in both instances were essentially similar in final shape. In the bent transplants the surviving osteogenic tissues regenerated and, adapting to the altered forces, formed a new bone shaft. This involved a change in the direction, amount, and nature of endochondral, periosteal, and regenerative growth and subsequent remodeling of bone. The results support previous observations that, within limits, the strain in the osteogenic envelope is an important factor in adaptation of bones to changing stress and that, where the envelope is deficient, the surviving tissues have the capacity to regenerate and repair defects in the bone so that it best resists the changing stresses applied to it.     

Fractal dimension and mechanical properties of human cortical bone

Fractal dimension (FD) can be used to characterize microstructure of porous media, particularly bone tissue. The porous microstructure of cortical bone is observable in micro-CT (μCT) images. Estimations of fractal dimensions of μCT images of coupons of human cortical bone are obtained. The same samples were tested on a tensile test machine and Young's modulus (YM) and Failure stress were obtained. When both types of measures were compared, a clear correlation was found (R = ?81%, P < 0.01). Young's modulus of each sample and the FD of its μCT images are correlated. From the assumption that cortical bone is approximately a fractal set, a non-linear constitutive relation involving FD is obtained for YM. Experimental results show good agreement with this constitutive relation. Additional parameters in the non-linear relation between YM and FD have been estimated from experimental results and related to physical parameters.     

大鼠皮质骨上机械性显微损伤的修复机制研究

目的研究大鼠皮质骨上植入物造成的机械性显微损伤的修复机制。方法 30只SD大鼠随机分为两组,一组行卵巢切除术,另一组行假手术。3个月后在右侧胫骨行钻孔术,分别于钻孔后1、2、4周后处死,处死前注射四环素和钙黄绿素进行标记。将含有钻孔的骨段(1 cm)应用大块碱性品红染色,甲基丙烯酸甲酯包埋后切成约50μm厚的切片。应用Bioquant图象分析系统对切片进行骨形态计量学分析。结果去卵巢大鼠和假手术大鼠都出现了与显微损伤有关的骨吸收腔,其中去卵巢组的孔隙率和骨吸收腔明显高于假手术组(P<0.05);随着术后时间的延长,吸收腔的数目逐渐增加,各时间点之间的差异有明显的统计学意义(P<0.05)。结论去卵巢大鼠的孔隙率和骨吸收腔数明显高于假手术大鼠,反映了在雌激素缺乏和较多裂纹形成两种因素刺激下,去卵巢大鼠皮质骨内的重建更为活跃,这会降低骨的强度从而增加骨折的危险性。     

The microcirculation of bone and marrow in the diaphysis of the rat hemopoietic long bones.

The nature of the microcirculation of the diaphyseal portion of long bones and the adjacent bone marrow is poorly understood. The purpose of this study was to describe the blood supply in the diaphyseal cortex and the relationship of the bone vascular circulation to that of the bone marrow in the growing rat. India ink-gelatin was infused in the arterial system of 3-month-old rats and the vascularization was determined from histological sections. In some studies the periosteal circulation was blocked but the nutrient and metaphyseal arteriole systems were left intact. In the growing rat, most of the vascular flow appears to be centripetally through the diaphyseal cortex and this appears to be the primary blood supply for the adjacent bone marrow. The India ink traversed the cortex and entered the marrow through osteal canals at the endocortical surface. At the marrow-endocortical bone surface interface, ink exiting from the osteal canals filled the adjacent marrow sinusoids in what appeared as "bush-like" structures. From the bone marrow the ink appeared to drain into the central vein. Some arterioles from the nutrient system were found to penetrate the inner two thirds of the cortical bone and then re-enter the bone marrow. The centripetal flow of blood and the importance of the cortical flow for perfusion of the hemopoietic tissue was further documented when periosteal flow was obstructed. In this situation, the cortical bone and adjacent bone marrow were not perfused while the nutrient system and central vein were filled with ink.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the mechanical and ultrastructural properties of heat-treated cortical bone for use as a bone substitute.

Heat-treated bovine cortical bone has been proposed as an alternative to bone grafts and synthetic bone substitutes because it may combine the advantages of allografts (high stiffness and strength) and synthetic materials (abundant supply, reduced risk of rejection and disease transfer). Its mechanical properties and ultrastructure, however, are not well characterized. To address this, we compared the compressive (n = 20, bovine bone) and tensile (n = 26, bovine bone) mechanical properties and the ultrastructure (n = 12, human bone) of intact versus 350 degrees C heat-treated cortical bone. The 350 degrees C heat-treated bone had a mean +/- SD elastic modulus similar to the intact bone for both compression (16.3 +/- 2.2 GPa, pooled; p = 0.68) and tension (16.3 +/- 3.7 GPa, pooled; p = 0.95). It also maintained 63% of the intact strength in compression but only 9% in tension (p < 0.001). Infrared scans and X-ray diffraction patterns showed no differences between the 350 degrees C heat-treated and intact bone but large differences between ashed (700 degrees C) and intact bone. Similarly, heat-treated bone previously has been shown to be biocompatible and osteoconductive. We conclude, therefore, that 350 degrees C heat-treated cortical bone may be an excellent load-bearing bone substitute provided that it is loaded in compression only in vivo and is shown by future work to have acceptable fatigue properties.     

Simultaneous injection of bone marrow cells and stromal cells into bone marrow accelerates hematopoiesis in vivo

We have previously demonstrated that stromal cells can support the proliferation and differentiation of hematopoietic cells in vitro and in vivo and that a major histocompatibility complex restriction exists between hematopoietic stem cells and stromal cells. We have also found that intra-bone marrow (IBM) injection of allogeneic bone marrow cells (BMCs) leads to more rapid reconstitution of hematopoietic cells than intravenous injection. In the present study, we examine the effect of simultaneous injection of stromal cells and BMCs into the same bone marrow on the recovery of donor hematopoietic cells and demonstrate that simultaneous IBM injection of BMCs plus stromal cells is more effective in reconstituting recipients with donor hematopoietic cells than intravenous injection of BMCs plus stromal cells or IBM injection of BMCs alone.     

Matrix metalloproteinase-9 and cell division in neuroblastoma cells and bone marrow macrophages

Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions in cell development, cancer, injury, and regeneration. In addition to its well recognized extracellular action, functional intracellular MMP activity under certain conditions is supported by increasing evidence. In this study, we observed higher gelatinase activity by in situ zymography and increased MMP-9 immunoreactivity in human neuroblastoma cells and in bone marrow macrophages undergoing mitosis compared with resting cells. We studied the pattern of immunoreactivity at the different stages of cell division by confocal microscopy. Immunostaining with different monoclonal antibodies against MMP-9 revealed a precise, dynamic, and well orchestrated localization of MMP-9 at the different stages of cell division. The cellular distribution of MMP-9 staining was studied in relation to that of microtubules. The spatial pattern of MMP-9 immunoreactivity suggested some participation in both the reorganization of the nuclear content and the process of chromatid segmentation. We then used several MMP-9 inhibitors to find out whether MMP-9 might be involved in the cell cycle. These drugs impaired the entry of cells into mitosis, as revealed by flow cytometry, and reduced cell culture growth. In addition, the silencing of MMP-9 expression with small interfering RNA also reduced cell growth. Taken together, these results suggest that intracellular MMP-9 is involved in the process of cell division in neuroblastoma cells and in primary cultures of macrophages.     

Histovariability in human clavicular cortical bone microstructure and its mechanical implications

The human clavicle (i.e. collarbone) is an unusual long bone due to its signature S‐shaped curve and variability in macrostructure observed between individuals. Because of the complex nature of how the upper limb moves, as well as due to its complex musculoskeletal arrangement, the biomechanics, in particular the mechanical loadings, of the clavicle are not fully understood. Given that bone remodeling can be influenced by bone stress, the histologic organization of Haversian bone offers a hypothesis of responses to force distributions experienced across a bone. Furthermore, circularly polarized light microscopy can be used to determine the orientation of collagen fibers, providing additional information on how bone matrix might organize to adapt to direction of external loads. We examined Haversian density and collagen fiber orientation, along with cross‐sectional geometry, to test whether the clavicle midshaft shows unique adaptation to atypical load‐bearing when compared with the sternal (medial) and acromial (lateral) shaft regions. Because fractures are most common at the midshaft, we predicted that the cortical bone structure would show both disparities in Haversian remodeling and nonrandomly oriented collagen fibers in the midshaft compared with the sternal and acromial regions. Human clavicles (n = 16) were sampled via thin‐sections at the sternal, middle, and acromial ends of the shaft, and paired sample t‐tests were employed to evaluate within‐individual differences in microstructural or geometric properties. We found that Haversian remodeling is slightly but significantly reduced in the middle of the bone. Analysis of collagen fiber orientation indicated nonrandom fiber orientations that are overbuilt for tensile loads or torsion but are poorly optimized for compressive loads throughout the clavicle. Geometric properties of percent bone area, polar second moment of area, and shape (I_max/I_min) confirmed the conclusions drawn by existing research on clavicle macrostructure. Our results highlight that mediolateral shape changes might be accompanied by slight changes in Haversian density, but bone matrix organization is predominantly adapted to resisting tensile strains or torsion throughout and may be a major factor in the risk of fracture when experiencing atypical compression.     

Regional variation in the mechanical properties of cortical bone from the porcine femur

Despite the widespread use of porcine bone as a substitute for human bone in the development of surgical technique and the use of fixation devices, relatively few studies have reported on the mechanical behaviour of porcine long bones. Regional variation in the mechanical properties of cortical bone from porcine femora was investigated using three-point bending and cutting tests. Results were related to measurements of bone architecture and composition and Rutherford backscattering spectrometry (RBS) was used to calculate the calcium to phosphorus ratio. There was significant, but limited, regional variation in the strength of the femur with bone from the distal, posterior quadrant (241.4 ± 10.43 MPa) being significantly stronger than that of the lateral quadrant (162.3 ± 17.96 MPa). Cortical bone was also anisotropic; samples cut transverse to the bone's axis were around six times tougher than those cut parallel to the axis (p<0.05). This corresponded with a significant negative correlation between the Young's modulus and toughness when cut along the longitudinal axis. RBS analysis of cortical bone samples gave a Ca:P ratio of 1.37 ± 0.035, somewhat lower than that reported for cortical bone of adult human femora. These results indicate that the mechanical properties of cortical bone show significant, but limited, variation around the porcine femur and that this should be taken into consideration when sampling and choosing an appropriate animal model for orthopaedic biomechanics research.     

Unique biomaterial compositions direct bone marrow stem cells into specific chondrocytic phenotypes corresponding to the various zones of articular cartilage

Numerous studies have reported generation of cartilage-like tissue from chondrocytes and stem cells, using pellet cultures, bioreactors and various biomaterials, especially hydrogels. However, one of the primary unsolved challenges in the field has been the inability to produce tissue that mimics the highly organized zonal architecture of articular cartilage; specifically its spatially varying mechanical properties and extra-cellular matrix (ECM) composition. Here we show that different combinations of synthetic and natural biopolymers create unique niches that can "direct" a single marrow stem cell (MSC) population to differentiate into the superficial, transitional, or deep zones of articular cartilage. Specifically, incorporating chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) into PEG hydrogels (PEG:CS:MMP-pep) induced high levels of collagen II and low levels of proteoglycan expression resulting in a low compressive modulus, similar to the superficial zone. PEG:CS hydrogels produced intermediate-levels of both collagen II and proteoglycans, like the transitional zone, while PEG:hyaluronic acid (HA) hydrogels induced high proteoglycan and low collagen II levels leading to high compressive modulus, similar to the deep zone. Additionally, the compressive moduli of these zone-specific matrices following cartilage generation showed similar trend as the corresponding zones of articular cartilage, with PEG:CS:MMP-pep having the lowest compressive modulus, followed by PEG:CS while PEG:HA had the highest modulus. These results underscore the potential for composite scaffold structures incorporating these biomaterial compositions such that a single stem-progenitor cell population can give rise to zonally-organized, functional articular cartilage-like tissue.     

A critical damping approach for assessing the role of marrow fat on the mechanical strength of trabecular bone

Several clinical findings revealed that post-menopausal osteoporosis and age-related osteopenia are accompanied by trabecular bone marrow fat (BMF) increase. To help understand this phenomenon, a vibrating string model is proposed, based on the hypothesis that, when bone marrow properties change, the trabecular bone structure remodels itself to preserve its critical damping state. It is found that an inverse relationship holds between trabecular average length and marrow damping coefficient. Such a result leads us to hypothesize the following bone-weakening mechanism. Since fat-rich bone marrow is a worse damper, a BMF increment causes an increase of trabecular average length, which is accomplished by the absorption of horizontal trabeculae (structurally less important than vertical trabeculae). The resulting bone patterns are in excellent agreement with clinical observations of osteoporotic bone. A definitive confirmation of the proposed mechanism will support a therapeutical approach to widespread osteopenic diseases aimed at avoiding, or limiting, BMF increase.     

Investigation into the material properties of beech wood and cortical bone

When testing medical implants it is very important to be able to test the implant using a suitable material. In the case of orthopaedic implants the optimum material is bone. Beech wood is considered a suitable substitute for bone as it has a similar Young's modulus in tension. Although it is widely used, no actual comparison of the two materials has been undertaken. The aim of this study was to compare the material properties of beech wood and cortical bone using conventional compression tests. Cortical bone samples 4 mm in diameter and 20 mm in length, were prepared from the tibia of an amputated leg. Beech wood samples were prepared to the same specifications. In compression, the Young's modulus for cortical bone was found to be 27+/-9.9 GPa (mean +/- standard deviation) and for beech wood 2.6+/-1.7 GPa. The failure load for cortical bone was 911+/-207 N and 732+/-62 N for beech wood. Although beech wood has been used as a substitute for bone in some studies, this study has shown that there are significant differences in the properties of the two materials when they are subjected to compression.     

Circadian variation in cell division of the mouse alimentary tract,bone marrow and corneal epithelium

Circadian rhythms in DNA synthesis are described for the tongue epithelium, five different regions of the alimentary canal (gut) –esophagus, stomach, duodenum, jejunum and rectum– and bone marrow in a group of BDF¹ male mice. A circadian rhythm is also described for the mitotic index in the corneal epithelium in the same mice. The data document for the first time in the same animals the dramatic variation in cell division encountered from one region of the gut to another. This variation is seen in the amplitudes of the rhythms as well as in the over-all 24-hour means. On the contrary, the phasings of the rhythms in the different regions of the gut are remarkably similar. In this study, where the mice were standardized to 12 hours of light (0600-1800) alternating with 12 hours of darkness, the peak of the DNA-synthesis rhythm occurred around the time of transition from dark to light, and the trough around the time of transition from light to dark. The implications of these findings, and those of others, to the study of cell kinetics and to cancer chemotherapy are discussed.