Instability prolongs the chondral phase during bone healing in sheep

In this sheep study, we investigated the influence of fixation stability on the temporal and spatial distribution of tissues in the fracture callus. As the initial mechanical conditions have been cited as being especially important for the healing outcome, it was hypothesized that differences in the path of healing would be seen as early as the initial phase of healing. ----- ----- Sixty-four sheep underwent a mid-shaft tibial osteotomy that was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture calluses were analyzed using radiological, biomechanical and histological techniques. Statistical comparison between the groups was performed using the Mann–Whitney U test for unpaired non-parametric data. ----- ----- In the callus of the tibia treated with semi-rigid fixation, remnants of the fracture haematoma remained present for longer, although new periosteal bone formation during early healing was similar in both groups. The mechanical competence of the healing callus at 6 weeks was inferior compared to tibiae treated with rigid fixation. Semi-rigid fixation resulted in a larger cartilage component of the callus, which persisted longer. Remodeling processes were initiated earlier in the rigid group, while new bone formation continued throughout the entire investigated period in the semi-rigid group. ----- ----- In this study, evidence is provided that less rigid fixation increased the time required for healing. The process of intramembranous ossification appeared during the initial stages of healing to be independent of mechanical stability. However, the delay in healing was related to a prolonged chondral phase.

Effect of alendronate on endochondral ossification in mandibular condyles of growing rats

The replacement of the calcified cartilage by bone tissue during the endochondral ossification of the mandibular condyle is dependent of the resorbing activity of osteoclats. After partial resorption, calcified cartilage septa are covered by a primary bone matrix secreted by osteoblasts. Osteoadherin (OSAD) is a small proteoglycan present in bone matrix but absent in cartilage during the endochondral ossification. The aim of this study was to analyze the effect of alendronate, a drug known to inhibit bone resorption by osteoclasts, on the endochondral ossification of the mandibular condyle of young rats, by evaluating the distribution of osteoclasts and the presence of OSAD in the bone matrix deposited. Wistar newborn rats (n = 45) received daily injections of alendronate (n = 27) or sterile saline solution as control (n = 18) from the day of birth until the ages of 4, 14 and 30 days. At the days mentioned, the mandibular condyles were collected and processed for transmission electron microscopy analysis. Specimens were also submitted to tartrate resistant acid phosphatase (TRAP) histochemistry and ultrastructural immunodetection of OSAD. Alendronate treatment did not impede the recruitment and fusion of osteoclasts at the ossification zone during condyle growth, but they presented inactivated phenotype. The trabeculae at the ossification area consisted of cartilage matrix covered by a layer of primary bone matrix that was immunopositive to OSAD at all time points studied. Apparently, alendronate impeded the removal of calcified cartilage and maturation of bone trabeculae in the mandibular ramus, while in controls they occurred normally. These findings highlight for giving attention to the potential side-effects of bisphosphonates administered to young patients once it may represent a risk of disturbing maxillofacial development.

Endochondral ossification in vitro is influenced by mechanical bending

Bone development is influenced by the local mechanical environment. Experimental evidence suggests that altered loading can change cell proliferation and differentiation in chondro- and osteogenesis during endochondral ossification. This study investigated the effects of three-point bending of murine fetal metatarsal bone anlagen in vitro on cartilage differentiation, matrix mineralization and bone collar formation. This is of special interest because endochondral ossification is also an important process in bone healing and regeneration. Metatarsal preparations of 15 mouse fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days. After 3 days in culture to allow adherence they were stimulated 4 days for 20 min twice daily by a controlled bending of approximately 1000-1500 microstrain at 1 Hz. The paraffin-embedded bone sections were analyzed using histological and histomorphometrical techniques. The stimulated group showed an elongated periosteal bone collar while the total bone length was not different from controls. The region of interest (ROI), comprising the two hypertrophic zones and the intermediate calcifying diaphyseal zone, was greater in the stimulated group. The mineralized fraction of the ROI was smaller in the stimulated group, while the absolute amount of mineralized area was not different. These results demonstrate that a new device developed to apply three-point bending to a mouse metatarsal bone culture model caused an elongation of the periosteal bone collar, but did not lead to a modification in cartilage differentiation and matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more pronounced effects on the process of endochondral ossification and may provide further insights into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration.

Influences of age and mechanical stability on volume, microstructure, and mineralization of the fracture callus during bone healing : Is osteoclast activity the key to age-related impaired healing?

Earlier studies have shown that the influence of fixation stability on bone healing diminishes with advanced age. The goal of this study was to unravel the relationship between mechanical stimulus and age on callus competence at a tissue level. Using 3D in vitro micro-computed tomography derived metrics, 2D in vivo radiography, and histology, we investigated the influences of age and varying fixation stability on callus size, geometry, microstructure, composition, remodeling, and vascularity. Compared were four groups with a 1.5-mm osteotomy gap in the femora of Sprague–Dawley rats: Young rigid (YR), Young semirigid (YSR), Old rigid (OR), Old semirigid (OSR). Hypothesis was that calcified callus microstructure and composition is impaired due to the influence of advanced age, and these individuals would show a reduced response to fixation stabilities. Semirigid fixations resulted in a larger ΔCSA (Callus cross-sectional area) compared to rigid groups. In vitro μCT analysis at 6 weeks postmortem showed callus bridging scores in younger animals to be superior than their older counterparts (pb0.01). Younger animals showed (i) larger callus strut thickness (pb0.001), (ii) lower perforation in struts (pb0.01), and (iii) higher mineralization of callus struts (pb0.001). Callus mineralization was reduced in young animals with semirigid fracture fixation but remained unaffected in the aged group. While stability had an influence, age showed none on callus size and geometry of callus. With no differences observed in relative osteoid areas in the callus ROI, old as well as semirigid fixated animals showed a higher osteoclast count (pb0.05). Blood vessel density was reduced in animals with semirigid fixation (pb0.05). In conclusion, in vivo monitoring indicated delayed callus maturation in aged individuals. Callus bridging and callus competence (microstructure and mineralization) were impaired in individuals with an advanced age. This matched with increased bone resorption due to higher osteoclast numbers. Varying fixator configurations in older individuals did not alter the dominant effect of advanced age on callus tissue mineralization, unlike in their younger counterparts. Age-associated influences appeared independent from stability. This study illustrates the dominating role of osteoclastic activity in age-related impaired healing, while demonstrating the optimization of fixation parameters such as stiffness appeared to be less effective in influencing healing in aged individuals.

Nano- and macro-scale characterisation of the mechanical properties of bovine bone

In the present study, nano- and macro-scale characterisations on the mechanical properties of bovine cortical bones have been performed by using nanoindentation and conventional compressive tests. Nanoindentation results showed that the elastic modulus for the osteons and the interstitial lamellae in the longitude direction were 24.7 ± 2.5 GPa and 30.1 ± 2.4 GPa. As it’s difficult to distinguish osteons from interstitial lamellae in the transverse direction, the average elastic modulus for cortical bovine bone in the transverse direction was 19.8 ± 1.6 GPa. Significant differences were found in the modulus values between different microstructures of bone tissue and in different testing direction. It was found that the elastic modulus of bone bovine material in nano-level was higher than that in macro-level. The elastic modulus and
ultimate stress of large bone samples were 12.5 ± 1.9 GPa and 195 ± 19 MPa respectively from the compression test.

Uptake of bone-related matrix proteins into implanted deproteinized bovine bone mineral

Deproteinized bovine bone mineral (DBBM) (Bio-Oss®, Geistlich-Pharma, Wohlhusen, Switzerland) is widely used as a bone substitute for the preservation or augmentation of bone volume. After implantation near native bone, new bone may form around the DBBM particles. Since DBBM is very resistant to resorption, it will hardly ever be replaced by bone and, therefore, the mechanical stability largely depends on the extent of bridging between the newly formed bone and the DBBM particles. The molecular factors responsible for the deposition of new bone to the DBBM particles have not been determined. The aim of this study was, therefore, to test the hypothesis that DBBM implanted near bone take up bone-related matrix proteins that are involved in cell-matrix interactions. Cylindrical biopsies harvested from tooth extraction sites filled with DBBM particles were fixed in aldehydes, decalcified, and embedded in LR White resin. Thin sections were incubated with antibodies against bone sialoprotein (BSP) and osteopontin (OPN), two bone proteins involved in cell attachment, signaling, and mineralization. High-resolution immunogold labeling was used to examine protein distribution. BSP and OPN were immunodetected in all DBBM particles and yielded an identical distribution pattern. Most gold particles were found over the peripheral DBBM matrix, although some peripheral regions lacked immunolabeling. The bulk of the interior DBBM portion was mainly free of labeling with the exception of the peripheral matrix of some osteocyte lacunae and canaliculi. It is concluded that DBBM selectively takes up at least BSP and OPN after its implantation at a bone site. BSP and OPN or other molecules accommodating in DBBM may modulate events associated with cell attachment and differentiation.

Influence of Particle Size of Deproteinized Bovine Bone Mineral on New Bone Formation and Implant Stability after Simultaneous Sinus Floor Elevation: A Histomorphometric Study in Minipigs

BACKGROUND Deproteinized bovine bone mineral (DBBM) is one of the best-documented bone substitute materials for sinus floor elevation (SFE). PURPOSE DBBM is available in two particle sizes. Large particles are believed to facilitate improved neoangiogenesis compared with small ones. However, their impact on the rate of new bone formation, osteoconduction, and DBBM degradation has never been reported. In addition, the implant stability quotient (ISQ) has never been correlated to bone-to-implant contact (BIC) after SFE with simultaneous implant placement. MATERIALS AND METHODS Bilateral SFE with simultaneous implant placement was performed in 10 Göttingen minipigs. The two sides were randomized to receive large or small particle size DBBM. Two groups of 5 minipigs healed for 6 and 12 weeks, respectively. ISQ was recorded immediately after implant placement and at sacrifice. Qualitative histological differences were described and bone formation, DBBM degradation, BIC and bone-to-DBBM contact (osteoconduction) were quantified histomorphometrically. RESULTS DBBM particle size had no qualitative or quantitative impact on the amount of newly formed bone, DBBM degradation, or BIC for either of the healing periods (p > 0.05). Small-size DBBM showed higher osteoconduction after 6 weeks than large-size DBBM (p < 0.001). After 12 weeks this difference was compensated. There was no significant correlation between BIC and ISQ. CONCLUSION Small and large particle sizes were equally predictable when DBBM was used for SFE with simultaneous implant placement.

Mesenchymal Stem Cells and Bovine Bone Mineral in Sinus Lift Procedures-An Experimental Study in Sheep

Introduction: New reconstructive and less invasive methods have been searched to optimize bone formation and osseointegration of dental implants in maxillary sinus augmentation. Purpose: The aim of the presented ovine split-mouth study was to compare bovine bone mineral (BBM) alone and in combination with mesenchymal stem cells (MSCs) regarding their potential in sinus augmentation. Material and Methods: Bilateral sinus floor augmentations were performed in six adult sheep. BBM and MSCs were placed into the test side and only BBM in the contra-lateral control side of each sheep. Animals were sacrificed after 8 and 16 weeks. Augmentation sites were analyzed by computed tomography, histology, and histomorphometry. Results: The initial volumes of both sides were similar and did not change significantly with time. A tight connection between the particles of BBM and the new bone was observed histologically. Bone formation was significantly (p = 0.027) faster by 49% in the test sides. Conclusion: The combination of BBM and MSCs accelerated new bone formation in this model of maxillary sinus augmentation. This could allow early placement of implants.

Bone healing around nanocrystalline hydroxyapatite, deproteinized bovine bone mineral, biphasic calcium phosphate, and autogenous bone in mandibular bone defects.

The individual healing profile of a given bone substitute with respect to osteogenic potential and substitution rate must be considered when selecting adjunctive grafting materials for bone regeneration procedures. In this study, standardized mandibular defects in minipigs were filled with nanocrystalline hydroxyapatite (HA-SiO), deproteinized bovine bone mineral (DBBM), biphasic calcium phosphate (BCP) with a 60/40% HA/β-TCP (BCP 60/40) ratio, or particulate autogenous bone (A) for histological and histomorphometric analysis. At 2 weeks, percent filler amongst the test groups (DBBM (35.65%), HA-SiO (34.47%), followed by BCP 60/40 (23.64%)) was significantly higher than the more rapidly substituted autogenous bone (17.1%). Autogenous bone yielded significantly more new bone (21.81%) over all test groups (4.91%-7.74%) and significantly more osteoid (5.53%) than BCP 60/40 (3%) and DBBM (2.25%). At 8 weeks, percent filler amongst the test groups (DBBM (31.6%), HA-SiO (31.23%), followed by BCP 60/40 (23.65%)) demonstrated a similar pattern and was again significantly higher as compared to autogenous bone (9.29%). Autogenous bone again exhibited statistically significantly greater new bone (55.13%) over HA-SiO (40.62%), BCP 60/40 (40.21%), and DBBM (36.35%). These results suggest that the osteogenic potential of HA-SiO and BCP is inferior when compared to autogenous bone. However, in instances where a low substitution rate is desired to maintain the volume stability of augmented sites, particularly in the esthetic zone, HA-SiO and DBBM may be favored. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 1478-1487, 2015.

F-actin crosslinker : a key player for the mechanical stability of filopodial protrusion

Filopodial protrusion initiates cell migration, which decides the fate of cells in biological environments. In order to understand the structural stability of ultra-slender filopodial protrusion, we have developed an explicit modeling strategy that can study both static and dynamic characteristics of microfilament bundles. Our study reveals that the stability of filopodial protrusions is dependent on the density of F-actin crosslinkers. This cross-linkage strategy is a requirement for the optimization of cell structures, resulting in the provision and maintenance of adequate bending stiffness and buckling resistance while mediating the vibration. This cross-linkage strategy explains the mechanical stability of filopodial protrusion and helps understand the mechanisms of mechanically induced cellular activities.

Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early endochondral ossification

The distribution, phenotype, and requirement of macrophages for fracture-associated inflammation and/or early anabolic progression during endochondral callus formation were investigated. A murine femoral fracture model [internally fixed using a flexible plate (MouseFix)] was used to facilitate reproducible fracture reduction. IHC demonstrated that inflammatory macrophages (F4/80+Mac-2+) were localized with initiating chondrification centers and persisted within granulation tissue at the expanding soft callus front. They were also associated with key events during soft-to-hard callus transition. Resident macrophages (F4/80+Mac-2neg), including osteal macrophages, predominated in the maturing hard callus. Macrophage Fas-induced apoptosis transgenic mice were used to induce macrophage depletion in vivo in the femoral fracture model. Callus formation was completely abolished when macrophage depletion was initiated at the time of surgery and was significantly reduced when depletion was delayed to coincide with initiation of early anabolic phase. Treatment initiating 5 days after fracture with the pro-macrophage cytokine colony stimulating factor-1 significantly enhanced soft callus formation. The data support that inflammatory macrophages were required for initiation of fracture repair, whereas both inflammatory and resident macrophages promoted anabolic mechanisms during endochondral callus formation. Overall, macrophages make substantive and prolonged contributions to fracture healing and can be targeted as a therapeutic approach for enhancing repair mechanisms. Thus, macrophages represent a viable target for the development of pro-anabolic fracture treatments with a potentially broad therapeutic window...

Thermo-mechanical stability of a cellular assembly of carbon nanotubes in air

Carbon nanotubes (CNT) in bulk form offer outstanding structural and functional properties, and are shown to remain viscoelastic over a wide temperature range (77-1273 K) under inert conditions. We examine the quasi-static and dynamic compressive mechanical response of these cellular CNT materials in ambient air up to a temperature of 773 K. In uniaxial quasi-static compression, several displacement bursts are noted at large strains. These are results of the slippage and zipping of the CNT, and lead to significant mechanical energy absorption. Results of the dynamic mechanical analysis experiments show no degradation in storage modulus and loss coefficient for up to 20 h at 673 K. Hence, these stable cellular CNT structures can be utilized up to a maximum temperature of 673 K in air, which is much higher than the best polymers. (C) 2012 Elsevier Ltd. All rights reserved.

Chemical and mechanical stability of sodium sulfate activated slag after exposure to elevated temperature

The chemical and mechanical stability of slag activated with two different concentrations of sodium sulfate (Na₂SO₄) after exposure to elevated temperatures ranging from 200 to 800 °C with an increment of 200 °C has been examined. Compressive strengths and pH of the hardened pastes before and after the exposure were determined. The various decomposition phases formed were identified using X-ray diffraction, thermogravimetric analysis and scanning electron microscopy. The results indicated that Na₂SO₄ activated slag has a better resistance to the degradation caused by exposure to elevated temperature up to 600 °C than Portland cement system as its relative strengths are superior. The finer slag and higher Na₂SO₄ concentration gave better temperature resistance. Whilst the pH of the hardened pastes decreased with an increase in temperature, it still maintained a sufficiently high pH for the protection of reinforcing bar against corrosion.

Mesenchymal stem cells and inorganic bovine bone mineral in sinus augmentation: comparison with augmentation by autologous bone in adult sheep

Our aim was to compare the osteogenic potential of mononuclear cells harvested from the iliac crest combined with bovine bone mineral (BBM) (experimental group) with that of autogenous cancellous bone alone (control group). We studied bilateral augmentations of the sinus floor in 6 adult sheep. BBM and mononuclear cells (MNC) were mixed and placed into one side and autogenous bone in the other side. Animals were killed after 8 and 16 weeks. Sites of augmentation were analysed radiographically and histologically. The mean (SD) augmentation volume was 3.0 (1.0) cm(3) and 2.7 (0.3) cm(3) after 8 and 16 weeks in the test group, and 2.8 (0.3) cm(3) (8 weeks) and 2.8 (1.2) cm(3) (16 weeks) in the control group, respectively. After 8 weeks, histomorphometric analysis showed 24 (3)% BBM, and 19 (11)% of newly formed bone in the test group. The control group had 20 (13%) of newly formed bone. Specimens after 16 weeks showed 29 (12%) of newly formed bone and 19 (3%) BBM in the test group. The amount of newly formed bone in the control group was 16 (6%). The results show that mononuclear cells, including mesenchymal stem cells, in combination with BBM as the biomaterial, have the potential to form bone. (C) 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.