Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Spatial and temporal variations of mechanical properties and mineral content of the external callus during bone healing

After bone fracture, various cellular activities lead to the formation of different tissue types, which form the basis for the process of secondary bone healing. Although these tissues have been quantified by histology, their material properties are not well understood. Thus, the aim of this study is to correlate the spatial and temporal variations in the mineral content and the nanoindentation modulus of the callus formed via intramembranous ossification over the course of bone healing. Midshaft tibial samples from a sheep osteotomy model at time points of 2, 3, 6 and 9 weeks were employed. PMMA embedded blocks were used for quantitative back scattered electron imaging and nanoindentation of the newly formed periosteal callus near the cortex. The resulting indentation modulus maps show the heterogeneity in the modulus in the selected regions of the callus. The indentation modulus of the embedded callus is about 6 GPa at the early stage. At later stages of mineralization, the average indentation modulus reaches 14 GPa. There is a slight decrease in average indentation modulus in regions distant to the cortex, probably due to remodelling of the peripheral callus. The spatial and temporal distribution of mineral content in the callus tissue also illustrates the ongoing remodelling process observed from histological analysis. Most interestingly the average indentation modulus, even at 9 weeks, remains as low as 13 GPa, which is roughly 60% of that for cortical sheep bone. The decreased indentation modulus in the callus compared to cortex is due to the lower average mineral content and may be perhaps also due to the properties of the organic matrix which might be different from normal bone.

Pressure, oxygen tension and temperature in the periosteal callus during bone healing - An in vivo study in sheep

Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply; hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm Hg [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, pressure and temperature.

Mechanical Behavior of Articular Cartilage After Osteochondral Autograft Transfer in an Ovine Model

BACKGROUND: Grafting of autologous hyaline cartilage and bone for articular cartilage repair is a well-accepted technique. Although encouraging midterm clinical results have been reported, no information on the mechanical competence of the transplanted joint surface is available. HYPOTHESIS: The mechanical competence of osteochondral autografts is maintained after transplantation. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral defects were filled with autografts (7.45 mm in diameter) in one femoral condyle in 12 mature sheep. The ipsilateral femoral condyle served as the donor site, and the resulting defect (8.3 mm in diameter) was left empty. The repair response was examined after 3 and 6 months with mechanical and histologic assessment and histomorphometric techniques. RESULTS: Good surface congruity and plug placement was achieved. The Young modulus of the grafted cartilage significantly dropped to 57.5% of healthy tissue after 3 months (P < .05) but then recovered to 82.2% after 6 months. The aggregate and dynamic moduli behaved similarly. The graft edges showed fibrillation and, in some cases (4 of 6), hypercellularity and chondrocyte clustering. Subchondral bone sclerosis was observed in 8 of 12 cases, and the amount of mineralized bone in the graft area increased from 40% to 61%. CONCLUSIONS: The mechanical quality of transplanted cartilage varies considerably over a short period of time, potentially reflecting both degenerative and regenerative processes, while histologically signs of both cartilage and bone degeneration occur. CLINICAL RELEVANCE: Both the mechanically degenerative and restorative processes illustrate the complex progression of regeneration after osteochondral transplantation. The histologic evidence raises doubts as to the long-term durability of the osteochondral repair.

Osteoclastic activity begins early and increases over the course of bone healing

Osteoclasts are specialised bone-resorbing cells. This particular ability makes osteoclasts irreplaceable for the continual physiological process of bone remodelling as well as for the repair process during bone healing. Whereas the effects of systemic diseases on osteoclasts have been described by many authors, the spatial and temporal distribution of osteoclasts during bone healing seems to be unclear so far. In the present study, healing of a tibial osteotomy under standardised external fixation was examined after 2, 3, 6 and 9 weeks (n = 8) in sheep. The osteoclastic number was counted, the area of mineralised bone tissue was measured histomorphometrically and density of osteoclasts per square millimetre mineralised tissue was calculated. The osteoclastic density in the endosteal region increased, whereas the density in the periosteal region remained relatively constant. The density of osteoclasts within the cortical bone increased slightly over the first 6 weeks, however, there was a more rapid increase between the sixth and ninth weeks. The findings of this study imply that remodelling and resorption take place already in the very early phase of bone healing. The most frequent remodelling process can be found in the periosteal callus, emphasising its role as the main stabiliser. The endosteal space undergoes resorption in order to recanalise the medullary cavity, a process also started in the very early phase of healing at a low level and increasing significantly during healing. The cortical bone adapts in its outward appearance to the surrounding callus structure. This paradoxic loosening is caused by the continually increasing number and density of osteoclasts in the cortical bone ends. This study clearly emphasises the osteoclastic role especially during early bone healing. These cells do not simply resorb bone but participate in a fine adjusted system with the bone-producing osteoblasts in order to maintain and improve the structural strength of bone tissue.

The course of bone healing is influenced by the initial shear fixation stability

Fracture healing is influenced by fixation stability and experimental evidence suggests that the initial mechanical conditions may determine the healing outcome. We hypothesised that mechanical conditions influence not only the healing outcome, but also the early phase of fracture healing. Additionally, it was hypothesised that decreased fixation stability characterised by an increased shear interfragmentary movement results in a delay in healing. Sixty-four sheep underwent a mid-shaft tibial osteotomy which 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 callus was analysed using radiological, biomechanical and histological techniques. The tibia treated with semi-rigid fixation showed inferior callus stiffness and quality after 6 weeks. At 9 weeks, the calluses were no longer distinguishable in their mechanical competence. The calluses at 9 weeks produced under rigid fixation were smaller and consisted of a reduced fibrous tissue component. These results demonstrate that the callus formation over the course of healing differed both morphologically and in the rate of development. In this study, we provide evidence that the course of healing is influenced by the initial fixation stability. The semi-rigid fixator did not result in delayed healing, but a less optimal healing path was taken. An upper limit of stability required for successful healing remains unknown, however a limit by which healing is less optimal has been determined.

Subchondral bone osteoblasts can induce chondrocyte mineralization during osteoarthritis and this process is relevant to cartilage degradation

Pathological mineralization of articular cartilage is a characteristic feature of osteoarthritis (OA); however, the underlying mechanisms, and their relevance to cartilage degeneration, are not clear. The involvement of subchondral bone changes in OA have been reported previously with the characterization of abnormal subchondral bone mineral density (BMD), osteiod volume, altered bone mechanical parameters and an increase in bone turnover markers. A number of osteoarthritic animal models have demonstrated that subchondral bone changes often precede cartilage degeneration. In this study site specific localization of mineralization markers were detected in the OA cartilage. Chondrocytes and osteoblasts derived from OA cartilage and subchondral bone showed a significant increase in the mRNA expressions of mineralization markers. Interestingly, osteoblasts from OA subchondral bone could significantly decrease cartilage matrix expression; whereas, increase mineralization of chondrocytes (Figure 1). Osteogenic factors, such as CBFA1, ALP, and type X collagen (Col-X), were detected in chondrocytes under mineralization conditions (Figure 2). Furthermore, chondrocyte mineralization was followed by increased mRNA and protein levels of MMP-2, MMP-9 and MMP-13, all of which are detrimental to cartilage integrity in vivo. The data reported here suggests that the upregulation of subchondral bone-mineralization, typical of OA progression, causes cartilage mineralization, and that the mineralization of chondrocytes induce increased MMP levels with a subsequent degradation of the articular cartilage.

Altered cell interactions of subchondral bone osteoblasts and articular chondrocytes in osteoarthritis is through the mediation of ERK1/2 phosphorylation

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo a typical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. However, the mechanism(s) by which these changes occur during the OA development are not completely understood. Materials and Methods: ACCs and subchondral bone osteoblasts (SBOs) were harvested from OA and healthy patients for the cross-talk studies between normal and OA ACCs and SBOs. The involvement of mitogen activated protein kinase (MAPK) signalling pathway during the cell-cell interactions was analysed by zymography, ELISA and western blotting methods. Results: The direct and in-direct co-culture studies showed that OA (ACCs and SBOs) cells induced osteoarthritic changes of normal (ACC and SBOs) cells. This altered cell interaction induced by OA cells significantly aggravated the proteolytic activity, which resulted cartilage degeneration. The altered cell interaction appeared to significantly activate ERK 1/2 phosphorylation and inhibition of MAPK-ERK 1/2 pathway reversed the osteoarthrtitic phenotypic changes. Discussion and Conclusion: Our study has demonstrated that the altered bi-directional communication of SBOs and ACCs are critical for initiation and progression of OA related changes and that this process is mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA related disorders.

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40 ng/ml in the culture medium, but decreased at 80 ng/ml. Under CoCl2- induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or antiangiogenic activities of BMSCs.

Can the contra-lateral limb be used as a control with respect to analyses of bone remodelling?

Bone loss may result from remodelling initiated by implant stress protection. Quantifying remodelling requires bone density distributions which can be obtained from computed tomography scans. Pre-operative scans of large animals however are rarely possible. This study aimed to determine if the contra-lateral bone is a suitable control for the purpose of quantifying bone remodelling. CT scans of 8 pairs of ovine tibia were used to determine the likeness of left and right bones. The deviation between the outer surfaces of the bone pairs was used to quantify geometric similarity. The density differences were determined by dividing the bones into discrete volumes along the shaft of the tibia. Density differences were also determined for fractured and contra-lateral bone pairs to determine the magnitude of implant related remodelling. Left and right ovine tibiae were found to have a high degree of similarity with differences of less than 1.0 mm in the outer surface deviation and density difference of less than 5% in over 90% of the shaft region. The density differences (10–40%) as a result of implant related bone remodelling were greater than left-right differences. Therefore, for the purpose of quantifying bone remodelling in sheep, the contra-lateral tibia may be considered an alternative to a pre-operative control.

In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for BMP-7 gene delivery

Introduction and aims: For a scaffold material to be considered effective and efficient for tissue engineering it must be biocompatible as well as bioinductive. Silk fiber is a natural biocompatible material suitable for scaffold fabrication; however, silk is tissue-conductive and lacks tissue-inductive properties. One proposed method to make the scaffold tissue-inductive is to introduce plasmids or viruses encoding a specific growth factor into the scaffold. In this study, we constructed adenoviruses encoding bone morphogenetic protein-7 (BMP-7) and incorporated these into silk scaffolds. The osteo-inductive and new bone formation properties of these constructs were assessed in vivo in a critical-sized skull defect animal model. Materials and methods: Silk fibroin scaffolds containing adenovirus particles coding BMP-7 were prepared. The release of the adenovirus particles from the scaffolds was quantified by tissue-culture infective dose (TCID50) and the bioactivity of the released viruses was evaluated on human bone marrow mesenchymal stromal cells (BMSCs). To demonstrate the in vivo bone forming ability of the virus-carrying silk fibroin scaffold, the scaffold constructs were implanted into calvarial defects in SCID mice. Results: In vitro studies demonstrated that the virus-carrying silk fibroin scaffold released virus particles over a 3 week period while preserving their bioactivity. In vivo test of the scaffold constructs in critical-sized skull defect areas revealed that silk scaffolds were capable of delivering the adenovirus encoding BMP-7, resulting significantly enhanced new bone formation. Conclusions: Silk scaffolds carrying BMP-7 encoding adenoviruses can effectively transfect cells and enhance both in vitro and in vivo osteogenesis. The findings of this study indicate silk fibroin is a promising biomaterial for gene delivery to repair critical-sized bone defects.

Hylaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues : in vitro and in vivo evaluation in a PLDLLA-TCP-PCL-composite system

Smart matrices are required in bone tissueengineered grafts that provide an optimal environment for cells and retain osteo-inductive factors for sustained biological activity. We hypothesized that a slow-degrading heparin-incorporated hyaluronan (HA) hydrogel can preserve BMP-2; while an arterio–venous (A–V) loop can support axial vascularization to provide nutrition for a bioartificial bone graft. HA was evaluated for osteoblast growth and BMP-2 release. Porous PLDLLA–TCP–PCL scaffolds were produced by rapid prototyping technology and applied in vivo along with HA-hydrogel, loaded with either primary osteoblasts or BMP-2. A microsurgically created A–V loop was placed around the scaffold, encased in an isolation chamber in Lewis rats. HA-hydrogel supported growth of osteoblasts over 8 weeks and allowed sustained release of BMP-2 over 35 days. The A–V loop provided an angiogenic stimulus with the formation of vascularized tissue in the scaffolds. Bone-specific genes were detected by real time RT-PCR after 8 weeks. However, no significant amount of bone was observed histologically. The heterotopic isolation chamber in combination with absent biomechanical stimulation might explain the insufficient bone formation despite adequate expression of bone-related genes. Optimization of the interplay of osteogenic cells and osteo-inductive factors might eventually generate sufficient amounts of axially vascularized bone grafts for reconstructive surgery.

The effect of friction on indenter force and pile-up in numerical simulations of bone nanoindentation

Nanoindentation is a useful technique for probing the mechanical properties of bone, and finite element (FE) modeling of the indentation allows inverse determination of elasto-plastic constitutive properties. However, all but one FE study to date have assumed frictionless contact between indenter and bone. The aim of this study was to explore the effect of friction in simulations of bone nanoindentation. Two dimensional axisymmetric FE simulations were performed using a spheroconical indenter of tip radius 0.6 m and angle 90°. The coefficient of friction between indenter and bone was varied between 0.0 (frictionless) and 0.3. Isotropic linear elasticity was used in all simulations, with bone elastic modulus E=13.56GPa and Poisson‟s ratio f 0.3. Plasticity was incorporated using both Drucker-Prager and von Mises yield surfaces. Friction had a modest effect on the predicted force-indentation curve for both von Mises and Drucker-Prager plasticity, reducing maximum indenter displacement by 10% and 20% respectively as friction coefficient was increased from zero to 0.3 (at a maximum indenter force of 5mN). However, friction has a much greater effect on predicted pile-up after indentation, reducing predicted pile-up from 0.27 to 0.11 m with a von Mises model, and from 0.09 to 0.02 m with Drucker-Prager plasticity. We conclude that it is potentially important to include friction in nanoindentation simulations of bone if pile-up is used to compare simulation results with experiment.

Mesoporous bioactive glasses as drug delivery and bone tissue regeneration platforms

The use of mesoporous bioactive glasses (MBG) for drug delivery and bone tissue regeneration has grown significantly over the past 5 years. In this review, we highlight the recent advances made in the preparation of MBG particles, spheres, fibers and scaffolds. The advantages of MBG for drug delivery and bone scaffold applications are related to this material’s well-ordered mesopore channel structure, superior bioactivity, and the application for the delivery of both hydrophilic and hydrophobic drugs. A brief forward-looking perspective on the potential clinical applications of MBG in regenerative medicine is also discussed.