Damaged Bone Tissue Research Articles

Ceramic/polymer nanocomposites with tunable drug delivery capability at specific disease sites

Pharmaceutical agents are often used to stimulate new bone formation for the treatment of bone injuries or diseases (such as osteoporosis). However, there are several problems associated with current orthopedic drug delivery methods. First, conventional systemic administration of pharmaceutical agents may not effectively reach targeted sites and, thus, they can cause nonspecific bone formation in areas not affected by injury or disease. Second, even if intentionally delivered or implanted locally to the damaged bone tissue, these agents tend to rapidly diffuse into adjacent tissues due to weak physical bonding to their drug carriers, which limits their potential to promote prolonged bone formation in targeted areas of bone disease. Therefore, in this study, biodegradable ceramic/polymer nanocomposites were explored as novel drug carriers for orthopedic applications to prolong local drug release and, thus, improve drug effectiveness at bone disease sites. Specifically, a bone morphogenetic protein (BMP-7) derived peptide (DIF-7c) was used as a model drug in this study and was first loaded onto nanocrystalline hydroxyapatite (nano-HA) by either covalent chemical attachment or physical adsorption. These drug-carrying nano-HA particles were then dispersed into a degradable polymer (poly-lactide-co-glycolide or PLGA) matrix to create an implantable system capable of long-term drug release. The aminophase silane covalent chemical immobilization process was utilized in this study. These nanocomposite-based drug delivery systems were then characterized for drug loading efficiency and in vitro drug release. Results demonstrated that DIF-7c was successfully immobilized onto nano-HA placed in PLGA. Moreover, a greater prolonged two-phase release profile (of more than 3 months) was achieved when using aminophase silane chemical immobilization to nano-HA particles. Since previous studies have demonstrated greater in vivo bone growth on nano- compared with micron-HA particles placed in rat calvaria, this study continued to demonstrate that ceramic/polymer nanocomposites are promising candidates as novel orthopedic materials to promote bone regeneration.

In vitro biofilms formation on polymer matrix composites

Aim of this work was a surface modification and characterisation of composite membrane materials destined for regeneration of damaged bone tissue. The materials consisted of stable, hydrophobic PTFE–PVDF–PP polymer and resorbable, hydrophilic biopolymer fibres made of sodium alginate (NaAlg). The fibres were washed-out with water to create open porosity in the membranes, and part of the dissolved sodium alginate deposited on the composite surface. Distribution of a biopolymer layer modifying the composite surface was investigated with FT-IR method. FT-IR reflection (ATR) and transmission techniques revealed that the surface modification had a domain-type character. The deposited sodium alginate modified physicochemical properties of the membrane i.e., lowered the wetting angle, and increased the surface free energy. Such surface characteristics may be advantageous for cells adhesion and proliferation process in in vitro and in vivo conditions.

Non-invasive monitoring of BMP-2 retention and bone formation in composites for bone tissue engineering using SPECT/CT and scintillation probes

Non-invasive imaging can provide essential information for the optimization of new drug delivery-based bone regeneration strategies to repair damaged or impaired bone tissue. This study investigates the applicability of nuclear medicine and radiological techniques to monitor growth factor retention profiles and subsequent effects on bone formation. Recombinant human bone morphogenetic protein-2 (BMP-2, 6.5 μg/scaffold) was incorporated into a sustained release vehicle consisting of poly(lactic-co-glycolic acid) microspheres embedded in a poly(propylene fumarate) scaffold surrounded by a gelatin hydrogel and implanted subcutaneously and in 5-mm segmental femoral defects in 9 rats for a period of 56 days. To determine the pharmacokinetic profile, BMP-2 was radiolabeled with 125I and the local retention of 125I-BMP-2 was measured by single photon emission computed tomography (SPECT), scintillation probes and ex vivo scintillation analysis. Bone formation was monitored by micro-computed tomography (µCT). The scaffolds released BMP-2 in a sustained fashion over the 56-day implantation period. A good correlation between the SPECT and scintillation probe measurements was found and there were no significant differences between the non-invasive and ex-vivo counting method after 8 weeks of follow up. SPECT analysis of the total body and thyroid counts showed a limited accumulation of 125I within the body. Ectopic bone formation was induced in the scaffolds and the femur defects healed completely. In vivo µCT imaging detected the first signs of bone formation at days 14 and 28 for the orthotopic and ectopic implants, respectively, and provided a detailed profile of the bone formation rate. Overall, this study clearly demonstrates the benefit of applying non-invasive techniques in drug delivery-based bone regeneration strategies by providing detailed and reliable profiles of the growth factor retention and bone formation at different implantation sites in a limited number of animals.

Bioactive glass sol‐gel foam scaffolds: Evolution of nanoporosity during processing and in situ monitoring of apatite layer formation using small‐ and wide‐angle X‐ray scattering

Recent work has highlighted the potential of sol-gel-derived calcium silicate glasses for the regeneration or replacement of damaged bone tissue. The work presented herein provides new insight into the processing of bioactive calcia-silica sol-gel foams, and the reaction mechanisms associated with them when immersed in vitro in a simulated body fluid (SBF). Small-angle X-ray scattering and wide-angle X-ray scattering (diffraction) have been used to study the stabilization of these foams via heat treatment, with analogous in situ time-resolved data being gathered for a foam immersed in SBF. During thermal processing, pore sizes have been identified in the range of 16.5-62.0 nm and are only present once foams have been heated to 400 degrees C and above. Calcium nitrate crystallites were present until foams were heated to 600 degrees C; the crystallite size varied from 75 to 145 nm and increased in size with heat treatment up to 300 degrees C, then decreased in size down to 95 nm at 400 degrees C. The in situ time-resolved data show that the average pore diameter decreases as a function of immersion time in SBF, as calcium phosphates grow on the glass surfaces. Over the same time, Bragg peaks indicative of tricalcium phosphate were evident after only 1-h immersion time, and later, hydroxycarbonate apatite was also seen. The hydroxycarbonate apatite appears to have preferred orientation in the (h,k,0) direction.

Loss of trabeculae by mechano-biological means may explain rapid bone loss in osteoporosis.

Osteoporosis is characterized by rapid and irreversible loss of trabecular bone tissue leading to increased bone fragility. In this study, we hypothesize two causes for rapid loss of bone trabeculae; firstly, the perforation of trabeculae is caused by osteoclasts resorbing a cavity so deep that it cannot be refilled and, secondly, the increases in bone tissue elastic modulus lead to increased propensity for trabecular perforation. These hypotheses were tested using an algorithm that was based on two premises: (i) bone remodelling is a turnover process that repairs damaged bone tissue by resorbing and returning it to a homeostatic strain level and (ii) osteoblast attachment is under biochemical control. It was found that a mechano-biological algorithm based on these premises can simulate the remodelling cycle in a trabecular strut where damaged bone is resorbed to form a pit that is subsequently refilled with new bone. Furthermore, the simulation predicts that there is a depth of resorption cavity deeper than which refilling of the resorption pits is impossible and perforation inevitably occurs. However, perforation does not occur by a single fracture event but by continual removal of microdamage after it forms beneath the resorption pit. The simulation also predicts that perforations would occur more easily in trabeculae that are more highly mineralized (stiffer). Since both increased osteoclast activation rates and increased mineralization have been measured in osteoporotic bone, either or both may contribute to the rapid loss of trabecular bone mass observed in osteoporotic patients.

Comparative Study of Ceramics Structure for Culturing Human Mesenchymal Stromal Cells

Hydroxyapatite (HA) ceramics together with various kinds of osteogenic cells have been used in bone tissue engineering. It is well known that the ceramics structure and composition affect cell proliferation / differentiation. In this study, three different types of HA ceramics were used to investigate initial cell attachment followed by osteoblastic differentiation of human mesenchymal stromal cells (MSCs). The results indicated that micro-pore affected the cell attachment and porosity (pore diameter and inter-pore connection) was the key to allow spacious distribution of the viable cells in the ceramics. This study also confirmed that surface pore areas of HA ceramics support the differentiation of human MSCs and thus the ceramics have the capability to regenerate damaged bone tissue.

No effects of in vivo micro‐CT radiation on structural parameters and bone marrow cells in proximal tibia of wistar rats detected after eight weekly scans

Recently developed in vivo animal high-resolution micro-CT scanners offer the possibility to monitor longitudinal changes in bone microstructure of small rodents, but may impose high radiation doses that could damage bone tissue. The goal of this study was to determine the effects on the bone of 8 weeks of in vivo scanning of the proximal tibia in female Wistar rats. Eight weekly CT scans were made of the right proximal tibia of nine female, 30-week-old, retired-breeder, Wistar rats. Two weeks after the last weekly scan, a final scan was made. The left leg was only scanned during first and final measurements and served as a control. A two-way ANOVA with repeated measures was performed on the first and last measurements of left and right tibiae for six bone structural parameters. Bone marrow cells were flushed out and tested for cell viability. No significant difference was found between left and right for any of the bone structural parameters (p > 0.05). Structure model index and trabecular separation significantly changed as a result of aging, while none of the other parameters did. No significant difference was found between left and right in absolute and percentage number of cell viability. We did not find any indication that the applied scanning regime, in combination with the particular settings used, would affect the results of in vivo bone structural measurements in long-term studies using aged, female Wistar rats. However, careful consideration should be made when determining the number of scans, particularly when a different experimental design is used.

Bioactive composite ceramics in the hydroxyapatite-tricalcium phosphate system

Synthetic materials based on hydroxyapatite (HA), an analogue of the mineral component of the bone tissue, are used in medicine for the repair of damaged bone tissue [1‐3]. A new repair technology is based on implanting porous ceramic scaffolds containing cultivated cells and proteins into the bone tissue [4]. The formation of new bone tissue is a complex process that includes protein adsorption as an important stage [5, 6]. Scaffolds should be resorbable with time in the human body with gradual replacement by newly formed bone tissue and should ensure a high protein-adsorption capacity. Hydroxyapatite is stable against dissolution by body fluids, whereas tricalcium phosphate (TCP) has a much higher resorption rate compared to that of HA. By varying the component ratio in an HA/TCP composite, one can control the resorption rate. The problems of design of such composite materials have been considered in reviews [7, 8]. Two-phase composites (TCMs) are produced by thermal decomposition of calcium-deficient HA followed by sintering [8]. Using this method, it is difficult to control the component ratio in the material. Some aspects of the influence of the phase composition on the biological behavior of TCMs remain obscure. This study is devoted to the production of TCMs with specified composition and porosity and elucidation of the question of which factor, porosity or phase composition, is more important for protein adsorption.

SEM and 3D synchrotron radiation micro‐tomography in the study of bioceramic scaffolds for tissue‐engineering applications

Different biomaterials have been proposed as scaffolds for the delivery of cells and/or biological molecules to repair or regenerate damaged or diseased bone tissues. Particular attention is being given to porous bioceramics that mimic trabecular bone chemistry and structure. Chemical composition, density, pore shape, pore size, and pore interconnection are elements that have to be considered to improve the efficiency of these biomaterials. Commonly, two-dimensional (2D) systems of analysis such as scanning electron microscope (SEM) are used for the characterization and comparison of the scaffolds. Unfortunately, these systems do not allow a complete investigation of the three-dimensional (3D) spatial structure of the scaffold. In this study, we have considered two different techniques, that is, SEM and 3D synchrotron radiation (SR) micro-CT to extract information on the geometry of two hydroxyapatite (HA) bioceramics with identical chemical composition but different micro-porosity, pore size distribution, and pore interconnection pathway. The two scaffolds were obtained with two different procedures: (a) sponge matrix embedding (scaffold FB), and (b) foaming (scaffold EP). Both scaffolds showed structures suitable for tissue-engineering applications, but scaffold EP appeared superior with regard to interconnection of pores, surface on which the new bone could be deposited, and percentage of volume available to bone deposition.

P.3.c.049 Aggression and psychopathology in an acute psychiatric sample:clinical and therapeutic considerations

Obesity and type 2 diabetes (T2D) significantly increase the risk of developing an arthritic condition.We performed a review of literature on the pathophysiological mechanisms that underpin the relationships between obesity, T2D and osteoarthritis (OA).The pathophysiology of the link between obesity and OA is related to both the direct effect of excess mechanical loads being placed on the cartilage and to an adipose tissue effect. Adipocytes produce and release adipokines (e.g. leptin). They are also the seat of a local inflammatory reaction when the adipose tissue is ectopic (visceral vs. subcutaneous adipose tissue), and then systemic effects that add even more to a micro-inflammatory mechanism. In diabetics, insulin resistance can add to these mechanisms, which can damage cartilage, bone and synovial tissue. These all act together to reduce mobility in obese subjects and contribute to a vicious cycle centered on OA, especially when the obesity is predominantly abdominal and/or associated with T2D.Prevention of obesity-related OA must be the focus in high-risk subjects, such as those who are obese with metabolic syndrome > “metabolically healthy” obese, have T2D, and normal weight subjects with abdominal obesity (defined as waist circumference > 102 cm for men and 88 cm for women). The primary component of this prevention effort is weight loss combined with a balanced diet and regular physical activity.

The structure of a bioactive calcia–silica sol–gel glass

We have used neutron diffraction with isotopic substitution to gain new insights into the nature of the atomic scale calcium environment in bioactive sol–gel glasses, and also used high energy X-ray total diffraction to probe the nature of the processes initiated when bioactive glass is immersed in vitro in simulated body fluid (SBF). Recent work has highlighted the potential of sol–gel derived calcium silicate glasses for the regeneration or replacement of damaged bone tissue. The mechanism of bioactivity and the requirements for optimisation of the properties of these materials are as yet only partially understood but have been strongly linked to calcium dissolution from the glass matrix. The data obtained point to a complex calcium environment in which calcium is loosely bound within the glass network and may therefore be regarded as facile. Complex multi-stage dissolution and mineral growth phases were observed as a function of reaction time between 1 min and 30 days, leading eventually to the formation of a disordered hydroxyapatite (HA) layer on the glass surface, which is similar to the polycrystalline bone mineral hydroxyapatite. This methodology provides insight into the structure of key sites in these materials and key stages involved in their reactions, and thereby more generally into the behaviour of bone-regenerative materials that may facilitate improvements in tissue engineering applications.

Microcracking and porosity in calcium phosphates and the implications for bone tissue engineering

Calcium phosphates including hydroxyapatite (HA) have been widely studied as bone scaffold materials. However their mechanical properties are highly variable and may be a function of thermal expansion anisotropy (TEA) induced stresses and microcracking. There is confusion concerning the mechanisms and microscopic identification of microcracks in HA. This study presents clear evidence of microcracking from micrographs of as-sintered HA surfaces which avoids the complications of identifying TEA-induced microcracks on fracture surfaces. Additionally, the existing literature of TEA-induced microcracking is briefly reviewed and pertinent papers involving likely microcracking in HA are analyzed. The recent realization in the biomedical literature notes that microcracks are of critical importance in remodeling and repair of damaged bone tissue. Hence, the similarities between microcracking in HA used for scaffolds in bone tissue engineering and that in the normal bone repair process is of importance in designing HA scaffolds with improved mechanical properties and biocompatibility.

骨と結合する人工骨における傾斜構造

Artificial bone, i.e. implant material, is widely used for reconstruction of damaged bone tissue. Implant material is required to show high biological affinity to the surrounding living tissue. Artificial materials are generally encapsulated by fibrous collagen tissues to be isolated from surrounding bone, when implanted in bony defects. However, some special types of ceramics are known to show ability of direct bone-bonding, i.e. bioactivity. Previous studies reported that the essential requirement for materials to exhibit the bioactivity is formation of bone-like apatite on their surfaces in body environment. The same type of apatite formation can be observed in a simulated body fluid (SBF) that mimics ion concentrations of human extracellular fluid. The apatite formation is triggered by specific functional groups such as Si-OH, Ti-OH, Zr-OH, Ta-OH, which can induce heterogeneous nucleation on the surface of materials. These findings bring us an idea that even bioinert metals show bioactivity, if such functional groups are formed on their surfaces with gradient structure. As a novel kind of surface treatment, the alkali treatment has been developed to induce bioactivity on various kinds of metal such as titanium, tantalum, zirconium and molybdenum. Bioactive surface layer composed of metal hydroxide is formed by the treatment and it takes a graded structure toward the interior of the substrates. Bioactive materials with graded structure are useful as artificial bones with ability of tight bone-bonding.

Tensile damage and its effects on cortical bone

Plexiform bovine bone samples are repeatedly loaded in tension along their longitudinal axis. In order to induce damage in the bone tissue, bone samples are loaded past their yield point. Half of the bone samples from the damaged group were stored in saline to allow for viscoelastic recovery while the others were decalcified. Tensile tests were conducted on these samples to characterize the effects of damage on the mechanical behavior of the organic matrix (decalcified samples) as well as on bone tissue (stored in saline). The ultimate strain of the damaged decalcified bone is 29% higher compared to that of non-damaged decalcified (control) bone. The ultimate stresses as well as the elastic moduli are similar in both decalcified groups. This phenomenon is also observed in other collagenous tissue (tendon and ligament). This may suggest that damage in bone is caused by shear failure of the organic matrix; transverse separation of the collagen molecules or microfibrils from each other. In contrast, there is a trend towards lowered ultimate strains in damaged bone, which is soaked in saline, with respect to control bone samples (not damaged). The damaged bone tissue exhibits a bi-linear behavior in contrast to the mechanical behavior of non-damaged bone. The initial elastic modulus (below 55 MPa) and ultimate strength of damaged bone are similar to that in non-damaged bone.

Strain distribution analysis of braided composite bone plates

Bone plates made of braided composites have low elastic modulus compared to conventional metal bone plates. This low elastic modulus of ensures only a part of load is transferred through bone plate and the remaining load is borne by the bone itself, thus creating a small movement at fracture. The movement induced by external loading gives stimulus to the damaged bone tissue and encourages callus formation. It is necessary to calculate magnitudes of strains induced in bone plate system subjected to loading. The results from stress–strain curves of the composite indicate that at 1% strain, there is no danger of fibre and matrix separation. The suitability of a braided carbon–epoxy composite for bone plate application is studied.

Damaged-bone adaptation under steady homogeneous stress.

In this work an extension of the adaptive-elasticity theory is proposed in order to include the contribution of bone microdamage as a stimulus. Some aspects of damaged-bone tissue adaptation, brought about by a change of the daily loading history, are investigated. In particular, under the assumption of a small strain approximation and isothermal conditions, the solution of the remodeling rate equation for steady homogeneous stress is discussed and the damage effect upon the remodeling time constant is shown. The result is both theoretical and numerical, based on a recent theory of internal damaged-bone remodeling (Ramtani, S., and Zidi, M., 1999, "Damaged-Bone Remodeling Theory: Thermodynamical Approach, " Mechanics Research Communications, Vol. 26, pp. 701-708. Ramtani, S., and Zidi, M., 2001, "A Theoretical Model of the Effect of Continum Damage on a Bone Adaption Model," Journal of Biomechanics, Vol. 34, pp. 471-479) and motivated by the works of Cowin, S. C., and Hegedus, D. M., 1976, "Bone Remodeling I: Theory and Adaptive Elasticity," Journal of Elasticity, Vol. 6, pp. 471-479 and Hegedus, D. H., and Cowin, S. C., 1976, "Bone Remodeling II: Small Strain Adaptive Elasticity," Journal of Elasticity, Vol. 6, pp. 337-352.

Mechanical strain, induced noninvasively in the high-frequency domain, is anabolic to cancellous bone, but not cortical bone

Departing from the premise that it is the large-amplitude signals inherent to intense functional activity that define bone morphology, we propose that it is the far lower magnitude, high-frequency mechanical signals that continually barrage the skeleton during longer term activities such as standing, which regulate skeletal architecture. To examine this hypothesis, we proposed that brief exposure to slight elevations in these endogenous mechanical signals would suffice to increase bone mass in those bones subject to the stimulus. This was tested by exposing the hind limbs of adult female sheep (n = 9) to 20 min/day of low-level (0.3g), high-frequency (30 Hz) mechanical signals, sufficient to induce a peak of approximately 5 microstrain (μϵ) in the tibia. Following euthanasia, peripheral quantitative computed tomography (pQCT) was used to segregate the cortical shell from the trabecular envelope of the proximal femur, revealing a 34.2% increase in bone density in the experimental animals as compared with controls (p = 0.01). Histomorphometric examination of the femur supported these density measurements, with bone volume per total volume increasing by 32% (p = 0.04). This density increase was achieved by two separate strategies: trabecular spacing decreased by 36.1% (p = 0.02), whereas trabecular number increased by 45.6% (p = 0.01), indicating the formation of cancellous bone de novo. There were no significant differences in the radii of animals subject to the stimulus, indicating that the adaptive response was local rather than systemic. The anabolic potential of the signal was evident only in trabecular bone, and there were no differences, as measured by any assay, in the cortical bone. These data suggest that subtle mechanical signals generated during predominant activities such as posture may be potent determinants of skeletal morphology. Given that these strain levels are three orders of magnitude below strains that can damage bone tissue, we believe that a noninvasive stimulus based on this sensitivity has potential for treating skeletal complications such as osteoporosis.

Anabolism. Low mechanical signals strengthen long bones.

Although the skeleton's adaptability to load-bearing has been recognized for over a century, the specific mechanical components responsible for strengthening it have not been identified. Here we show that after mechanically stimulating the hindlimbs of adult sheep on a daily basis for a year with 20-minute bursts of very-low-magnitude, high-frequency vibration, the density of the spongy (trabecular) bone in the proximal femur is significantly increased (by 34.2%) compared to controls. As the strain levels generated by this treatment are three orders of magnitude below those that damage bone tissue, this anabolic, non-invasive stimulus may have potential for treating skeletal conditions such as osteoporosis.

Observation of the bone matrix structure of intact and regenerative zones of tibias by atomic force microscopy

Atomic force microscopy (AFM) was used to comparatively study the structure of the bone matrix of rat tibia from an intact region with that from regions submitted to surgical injury. We used young male adult rats (Wistar), with corporal masses between 250 and 300 g. Each injury was provoked by drilling a 1.5-mm-diam hole in one cortical tibia surface. The healing course was monitored at 8 and 15 days after the injury. Atomic force microscopy images, at different magnifications, allowed the identification of the time dependence of the osteoblast activity, measured by the increase in the area of neoformed primary bone and in the organization of the collagen fibers of the bone matrix. Characterization of the natural recovery of the damaged bone tissue by AFM is potentially of great importance because it allows the comparison of natural recovery processes with those induced by medicines and other therapeutic procedures.

Effect of aging time of sol on structure and in vitro calcium phosphate formation of sol-gel-derived titania films.

Titanium and its alloys have been used successfully in the manufacture of orthopedic and dental implants to replace damaged bone tissue. In this study, different sol-gel-derived TiO(2) coatings were produced on titanium substrates using different aging times (5, 10, 24, or 48 h) of the sol before dipping the coatings and varying numbers (one, three, or five) of coating layers. The influence of the aging time of the sol on the structure of the titania coatings with respect to in vitro bioactivity was investigated. The in vitro bioactivity tests were done in a simulated body fluid (SBF). The sol properties were monitored using a capillary viscometer and dynamic light scattering to determine the viscosity and particle size, respectively. The topography of the films was characterized using atomic force microscopy. The various sol aging times and numbers of layers produced differences in the topography of the titania films. For the coatings with one and three layers, the aging of the sols had an influence on the height of the peaks (lower with longer aging times) although the peak distance was about the same. The number of coating layers had a stronger influence. The distribution of the peak distances became narrower with an increasing number of coating layers. The coating with three layers (top coating prepared after 24 h of sol aging) and the coatings with five layers had a similar distribution of peak distances (15-50 nm), which was favorable for calcium phosphate formation. On these substrates, calcium phosphate formation started within 3-6 days of immersion in SBF. The aging time of the titania sol and the number of coating layers were found to have a strong influence on the surface topography in the nanometer scale of the titania films. The results indicate that the topography of the outermost surface is of importance for in vitro bioactivity.