Increased osteoblast and osteoclast activity in female senescence-accelerated, osteoporotic SAMP6 mice during fracture healing.

BACKGROUND: Previous studies have shown that fracture healing depends on gender and that in females, ovariectomy-induced osteoporosis impairs the healing process. There is no information, however, whether the alteration of fracture healing in osteoporosis also depends on gender. MATERIALS AND METHODS: Therefore, we herein studied fracture healing in female and male senescence-accelerated osteoporotic mice, strain P6 (SAMP6), including biomechanical, histomorphometric, and protein biochemical analysis. RESULTS: Bending stiffness was reduced in male and female SAMP6 mice compared with senescence-resistant strain 1 (SAMR1) controls. This was associated with elevated serum concentrations of tartrate-resistent acid phosphatase form 5b (TRAP) in both female and male SAMP6 mice. Callus size, however, was significantly larger in female SAMP6 mice compared with male SAMP6 mice and female SAMR1 controls. This indicates a delayed remodeling process in female SAMP6 mice. The delay of callus remodeling in female SAMP6 mice was associated with a significantly higher osteoprotegerin (OPG) callus tissue expression and increased serum concentrations of osteocalcin (OC) and deoxypyridinoline (DPD), indicating elevated osteoblast and osteoclast activities. CONCLUSION: The present study shows that remodeling during fracture healing in female, but not in male, SAMP6 mice is delayed, most probably due to an increased osteoblast and osteoclast activity.

Influence of trabecular microstructure and cortical index on the complexity of proximal humeral fractures.

OBJECTIVES: Poor bone quality increases the susceptibility to fractures of the proximal humerus. It is unclear whether local trabecular and cortical measures influence the severity of fracture patterns. The goal of this study was to assess parameters of trabecular and cortical bone properties and to compare these parameters with the severity of fractures and biomechanical testing. METHODS: Twenty patients with displaced proximal humeral fractures planned for osteosynthesis were included. Fractures were classified as either 2-part fractures or complex fractures. Bone after core drilling was harvested during surgery from the humeral head in each patient. Twenty bone cores obtained from nonpaired cadaver humeral heads served as nonfractured controls. Micro-CT (µCT) was performed and bone volume/total volume (BV/TV), connectivity density (CD), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and bone mineral density (BMD) were assessed. The cortical index (CI) was determined from AP plain films. Biomechanical testing was done after µCT scanning by axially loading until failure, and ultimate strength and E modulus were recorded. RESULTS: BV/TV, BMD and CD showed moderate to strong correlations with biomechanical testing (r = 0.45-0.76, all p < 0.05). No significant differences were detected between the 2-part and complex fracture groups and controls regarding µCT and biomechanical parameters. CI was not significantly different between the 2-part and complex fracture groups. CONCLUSIONS: In our study population local trabecular bone structure and cortical index could not predict the severity of proximal humeral fractures in the elderly. Complex fractures do not necessarily imply lower bone quality compared to simple fractures.

Cortex of the pedicle of the vertebral arch. Part I: Deformation characteristics during screw insertion.

OBJECT: Elastic deformation has been proposed as a mechanism by which vertebral pedicles can maintain pullout strength when conical screws are backed out from full insertion. The response to the insertion technique may influence both the extent of deformation and the risk of acute fracture during screw placement. The aim of this study was to determine the deformation characteristics of the lumbar pedicle cortex during screw placement. METHODS: Lumbar pedicles with linear strain gauges attached at the lateral and medial cortices were instrumented using 7.5-mm pedicle screws with or without preconditioning by insertion and removal of 6.5-mm screws. The strains and elastic recoveries of the medial and lateral cortices were determined. RESULTS: Mean medial wall strains tended to be lower than mean lateral wall strains when the 6.5-mm and 7.5-mm screw data were pooled (p = 0.07). After the screws had been removed, 71 to 79% of the deformation at the lateral cortex and 70 to 96% of the deformation at the medial cortex recovered. When inserted first, the 7.5-mm screw caused more plastic deformation at the cortex than it did when inserted after the 6.5-mm screw. Occasional idiosyncratic strain patterns were observed. No gross fracture was observed during screw placement. CONCLUSIONS: Screw insertion generated plastic deformation at the pedicle cortex even though the screw did not directly contact the cortex. The lateral and medial cortices responded differently to screw insertion. The technique of screw insertion affected the deformation behavior of the lumbar pedicles. With myriad options for screw selection and placement available, further study is needed before optimal placement parameters can be verified.

Cortex of the pedicle of the vertebral arch. Part II: Microstructure.

OBJECT: Although the gross anatomy of the pedicle in the human spine has been investigated in great detail, knowledge of the microanatomy of trabecular and cortical structures of the pedicle is limited. An understanding of the mechanical properties and structure of the pedicle bone is essential for improving the quality of pedicle screw placement. To enhance this understanding, the authors examined human cadaveric lumbar vertebrae. METHODS: In this study, the authors obtained seven human cadaveric lumbar vertebrae. The lateral and medial cortices of these pedicle specimens were sectioned and embedded in polymethylmethacrylate. Cross-sectional slices of cortex were obtained from each specimen and imaged with the aid of a high-resolution light microscope. Assessments of osteonal orientation, determinations of relative dimensions, and histomorphometric studies were performed. RESULTS: The cortex of the pedicle in each human lumbar vertebra had an osteonal structure with haversian canals laid down mainly in the anteroposterior (longitudinal) direction. The organization of osteons across the transverse cross-section was not homogeneous. The layer of lamellar bone that typically envelops cortical bone structures (such as in long bones) was not observed, and the lateral cortex was significantly thinner than the medial cortex (p < 0.05). CONCLUSIONS: The cortical bone surrounding the pedicle differed from bone in other anatomical regions such as the anterior vertebral body and femur. The osteonal orientation and lack of a lamellar sheath may account for the unique deformation characteristics of the pedicle cortex seen during pedicle screw placement.

The osteocyte.

Osteocytes are the most numerous cells in mature bone and have the potential to live as long as the organism itself. However, study and subsequent understanding of osteocyte biology has been thwarted by the remote location of the cell in the mineralized matrix. This review is intended to synthesize current understanding of osteocyte biology and to suggest future paths that will promote understanding of this obscure cell and translation of knowledge to disease prophylaxis and management.

Trabecular bone adaptation to low-magnitude high-frequency loading in microgravity.

Exposure to microgravity causes loss of lower body bone mass in some astronauts. Low-magnitude high-frequency loading can stimulate bone formation on earth. Here we hypothesized that low-magnitude high-frequency loading will also stimulate bone formation under microgravity conditions. Two groups of six bovine cancellous bone explants were cultured at microgravity on a Russian Foton-M3 spacecraft and were either loaded dynamically using a sinusoidal curve or experienced only a static load. Comparable reference groups were investigated at normal gravity. Bone structure was assessed by histology, and mechanical competence was quantified using µCT and FE modelling; bone remodelling was assessed by fluorescent labelling and secreted bone turnover markers. Statistical analyses on morphometric parameters and apparent stiffness did not reveal significant differences between the treatment groups. The release of bone formation marker from the groups cultured at normal gravity increased significantly from the first to the second week of the experiment by 90.4% and 82.5% in response to static and dynamic loading, respectively. Bone resorption markers decreased significantly for the groups cultured at microgravity by 7.5% and 8.0% in response to static and dynamic loading, respectively. We found low strain magnitudes to drive bone turnover when applied at high frequency, and this to be valid at normal as well as at microgravity. In conclusion, we found the effect of mechanical loading on trabecular bone to be regulated mainly by an increase of bone formation at normal gravity and by a decrease in bone resorption at microgravity. Additional studies with extended experimental time and increased samples number appear necessary for a further understanding of the anabolic potential of dynamic loading on bone quality and mechanical competence.

Mechanical assessment of local bone quality to predict failure of locked plating in a proximal humerus fracture model.

The importance of osteoporosis in proximal humerus fractures is well recognized. However, the local distribution of bone quality in the humeral head may also have a significant effect because it remains unclear in what quality of bone screws of standard implants purchase. The goal of this study was to investigate whether the failure of proximal humerus locked plating can be predicted by the DensiProbe (ARI, Davos, Switzerland). A 2-part fracture with metaphyseal impaction was simulated in 12 fresh-frozen human cadaveric humeri. Using the DensiProbe, local bone quality was determined in the humeral head in the course of 6 proximal screws of a standard locking plate (Philos; Synthes GmbH, Solothurn, Switzerland). Cyclic mechanical testing with increasing axial loading until failure was performed. Bone mineral density (BMD) significantly correlated with cycles until failure. Head migration significantly increased between 1000 and 2000 loading cycles and significantly correlated with BMD after 3000 cycles. DensiProbe peak torque in all screw positions and their respective mean torque correlated significantly with the BMD values. In 3 positions, the peak torque significantly correlated with cycles to failure; here BMD significantly influenced mechanical stability. The validity of the DensiProbe was proven by the correlation between its peak torque measurements and BMD. The correlation between the peak torque and cycles to failure revealed the potential of the DensiProbe to predict the failure of locked plating in vitro. This method provides information about local bone quality, potentially making it suitable for intraoperative use by allowing the surgeon to take measures to improve stability.

Where do locking screws purchase in the humeral head?

INTRODUCTION: One of the limiting factors in finding the best osteosynthesis approach in proximal humerus fractures is the current lack of information on the properties of the cancellous bone regions engaged by the implants fixing the epiphysis. The aim of this study is to assess the densitometric and mechanical characteristics of these regions when using a proximal humerus locking plate (PHLP). MATERIALS AND METHODS: Nineteen PHLPs were mounted on cadaveric humeri using only their three most distal screws. Subsequently, the plates were removed and the bones were scanned using high-resolution peripheral quantitative computed tomography. Bone mineral density (BMD) was determined in the intact proximal epiphysis and in the exact locations where the six proximal screws would have been positioned concluding the instrumentation. Each plate was then repositioned on its bone and a minimally destructive local torque measurement was performed in the same six locations. A statistical analysis was performed to detect significant differences in the investigated parameters between screw positions, and to test the ability of local torque values to discriminate the bone mineral density of the entire humeral head (BMD(TOT)). RESULTS: Novel data about the cancellous bone engaged by the screws of a PHLP are provided. Different epiphyseal locations showed statistically significant different properties. A local torque measurement was a good predictor of the BMD(TOT). CONCLUSION: Position and direction of the epiphyseal screws on a locking implant are determinant to engage bone regions with significantly better bone quality. A breakaway torque measurement in a given screw position can distinguish between humeral heads with different densitometric properties.

Prediction of bone strength at the distal tibia by HR-pQCT and DXA.

BACKGROUND: Areal bone mineral density (aBMD) at the distal tibia, measured at the epiphysis (T-EPI) and diaphysis (T-DIA), is predictive for fracture risk. Structural bone parameters evaluated at the distal tibia by high resolution peripheral quantitative computed tomography (HR-pQCT) displayed differences between healthy and fracture patients. With its simple geometry, T-DIA may allow investigating the correlation between bone structural parameter and bone strength. METHODS: Anatomical tibiae were examined ex vivo by DXA (aBMD) and HR-pQCT (volumetric BMD (vBMD) and bone microstructural parameters). Cortical thickness (CTh) and polar moment of inertia (pMOI) were derived from DXA measurements. Finally, an index combining material (BMD) and mechanical property (polar moment of inertia, pMOI) was defined and analyzed for correlation with torque at failure and stiffness values obtained by biomechanical testing. RESULTS: Areal BMD predicted the vBMD at T-EPI and T-DIA. A high correlation was found between aBMD and microstructural parameters at T-EPIas well as between aBMD and CTh at T-DIA. Finally, at T-DIA both indexes combining BMD and pMOI were strongly and comparably correlated with torque at failure and bone stiffness. CONCLUSION: Ex vivo, at the distal tibial diaphysis, a novel index combining BMD and pMOI, which can be calculated directly from a single DXA measurement, predicted bone strength and stiffness better than either parameter alone and with an order of magnitude comparable to that of HR-pQCT. Whether this index is suitable for better prediction of fracture risk in vivo deserves further investigation.

Injectable rhBMP-2-loaded chitosan hydrogel composite: osteoinduction at ectopic site and in segmental long bone defect.

Carriers for bone morphogenetic protein-2 (BMP-2) used in clinical practice still suffer from limitations such as insufficient protein retention. In addition, there is a clinical need for injectable carriers. The main objective of this study was to assess bone forming ability of rhBMP-2 combined either with chitosan hydrogel (rhBMP-2/CH) or chitosan hydrogel containing ß-tricalcium phosphate (ß-TCP) (rhBMP-2/CH/TCP). Formulations were first compared in a rat ectopic intramuscular bone formation model, and the optimal formulation was further evaluated in healing of 15-mm critical size defect in the radius of a rabbit. Three weeks after injection ectopically formed bone was analyzed by microcomputerized tomography (micro-CT) and histology. Significantly higher (4.7-fold) mineralized bone formation was observed in the rhBMP-2/CH/TCP group compared to rhBMP-2/CH group. In a pilot study, defect in a rabbit radius treated with rhBMP-2/CH/TCP showed incomplete regeneration at 8 weeks with composite leakage from the defect, indicating the need for formulation refinement when segmental defect repair is foreseen.

Influence of defective bone marrow osteogenesis on fracture repair in an experimental model of senile osteoporosis.

Bone marrow osteogenesis in senile osteoporotic bone is impaired and, as such, may have significant implications on the successful outcome of fracture repair. Here we utilize a well-established murine model of senile osteoporosis, the P6 strain of senescence-accelerated mice (SAMP6), to investigate fracture healing in aged osteoporotic bone. A femoral osteotomy was created in SAMP6 and in non-osteoporotic age-matched control R1 senescence-resistant mice (SAMR1). The course of fracture healing was evaluated over a period of 42 days using quantitative microCT and histological analysis. The differentiation capabilities of bone mesenchymal progenitor cells derived from SAMP6 and SAMR1 mice was examined, and their osteogenic potential determined. Although preliminary in vitro analysis confirmed that bone marrow-derived stem cells (BMSC) isolated from SAMP6 mice had a reduced osteogenic capacity, no significant deficit in fracture repair as determined by quantitative microCT could be detected. This was supported by histology assessment, where complete bridging of the fracture gap was evident by day 28 and was fully healed day 42 in both SAMP6 and SAMR1 mice. Further in vitro studies revealed that periosteal-derived progenitor cells (PDPC) isolated from SAMP6 mice had an osteogenic potential comparable to that observed in SAMR1 mice. In conclusion, fracture healing in SAMP6 mice is not detrimentally affected by impairment of BMSC osteogenesis, suggesting that bone marrow-mediated repair processes are dispensable for normal bone healing in this senile osteoporotic fracture model. Furthermore, the influence of PDPC in the repair process may partly explain the absence of any detectable deficits in fracture repair in SAMP6 mice.

Endothelial progenitor cells and mesenchymal stem cells seeded onto beta-TCP granules enhance early vascularization and bone healing in a critical-sized bone defect in rats.

UNLABELLED: QUESTION/AIM: Lack of vessels indicates an insufficient nutritional supply of a bone graft and may limit the recruitment of bone-forming cells. Our aim was to evaluate the influence of endothelial progenitor cells (EPCs) alone or in combination with mesenchymal stem cells (MSCs) on early vascularization and bone healing in critical-sized defect (CSD) in vivo. METHODS: MSCs from human bone marrow and EPCs from buffy coat were used. A femoral CSD in adult athymic rats was created and stabilized by an external fixateur. The remaining defects were filled with fibronectin-coated beta-tricalcium phosphate (beta-TCP) granules, EPCs seeded on beta-TCP, MSCs seeded on beta-TCP, coculture of EPCs/MSCs seeded on beta-TCP, or autologous bone. Vascularization and bone formation were determined by immunohistology, microCT analysis, and biomechanical testing after 1, 4, and 8 weeks. RESULTS: Early vascularization was significantly improved in EPC/MSC group or EPC group, respectively. At 4 weeks bone formation increased significantly when the CSD was treated with coculture of MSCs/EPCs. Eight weeks after transplantation CSD showed significantly more bony bridgings and significantly increased ultimate load in the EPC/MSC group compared to the other groups. DISCUSSION: This cell approach suggests that there is a synergistic effect and that the initial stage of neovascularization by EPCs is considered to be crucial for complete bone regeneration in the late phase.

Assessment of bone quality within the tuberosities of the osteoporotic humeral head: relevance for anchor positioning in rotator cuff repair.

BACKGROUND: Tears of the rotator cuff are highly prevalent in patients older than 60 years, thereby presenting a population also suffering from osteopenia or osteoporosis. Suture fixation in the bone depends on the holding strength of the anchoring technique, whether a bone tunnel or suture anchor is selected. Because of osteopenic or osteoporotic bone changes, suture anchors in the older patient might pull out, resulting in failure of repair. HYPOTHESIS: The aim of our study was to analyze the bone quality within the tuberosities of the osteoporotic humeral head using high-resolution quantitative computed tomography (HR-pQCT). STUDY DESIGN: Descriptive laboratory study. METHODS: Thirty-six human cadaveric shoulders were analyzed using HR-pQCT. The mean bone volume to total volume (BV/TV) as well as trabecular bone mineral densities (trabBMDs) of the greater tuberosity (GT) and the lesser tuberosity (LT) were determined. Within the GT, 6 volumes of interest (VOIs) within the LT, and 2 VOIs and 1 control volume within the subchondral area beyond the articular surface were set. RESULTS: Comparing BV/TV of the medial and the lateral row, significantly higher values were found medially (P < .001). The highest BV/TV, 0.030% + or - 0.027%, was found in the posteromedial portion of the GT (P < .05). Regarding the analysis of the LT, no difference was found comparing the superior (BV/TV: 0.024% + or - 0.022%) and the inferior (BV/TV: 0.019% + or - 0.016%) portion. Analyzing trabBMD, equal proportions were found. An inverse correlation with a correlation coefficient of -0.68 was found regarding BV/TV of the posterior portion of the GT and age (P < .05). CONCLUSION: Significant regional differences of trabecular microarchitecture were found in our HR-pQCT study. The volume of highest bone quality resulted for the posteromedial aspect of the GT. Moreover, a significant correlation of bone quality within the GT and age was found, while the bone quality within the LT seems to be independent from it. CLINICAL RELEVANCE: The shape of the rotator cuff tear largely determines the bony site of tendon reattachment, although the surgeon has distinct options to modify anchor positioning. According to our results, placement of suture anchors in a medialized way at the border to the articular surface might guarantee a better structural bone stock.

Fixation compliance in a mouse osteotomy model induces two different processes of bone healing but does not lead to delayed union.

Delayed unions are a problematic complication of fracture healing whose pathophysiology is not well understood. Advanced molecular biology methods available with mice would be advantageous for investigation. In humans, decreased fixation rigidity and poor reduction are generally associated with delayed unions. In this study, these two factors were combined to observe their effect on bone healing in mice. Two plates with locking screws, one with 14 the bending stiffness of the other, were used to stabilize a 0.45mm gap osteotomy. muCT, radiographs, 4pt-bending tests and histological analysis demonstrated that the different plate types led to two different healing pathways. The less flexible bridging plate induced only intramembranous ossification whereas the more flexible bridging plate induced a mixture of endochondral and intramembranous ossification. However, the different plates led to a delay in healing of only 3-5 days in the period between 14 and 21 post-operative days. In mice, considerable fixation flexibility is necessary to induce secondary bone healing similar to that which occurs in humans, but this was not sufficient to induce a substantial delay in bone healing as would be expected in humans.

Remodeling of fracture callus in mice is consistent with mechanical loading and bone remodeling theory.

During the remodeling phase of fracture healing in mice, the callus gradually transforms into a double cortex, which thereafter merges into one cortex. In large animals, a double cortex normally does not form. We investigated whether these patterns of remodeling of the fracture callus in mice can be explained by mechanical loading. Morphologies of fractures after 21, 28, and 42 days of healing were determined from an in vivo mid-diaphyseal femoral osteotomy healing experiment in mice. Bone density distributions from microCT at 21 days were converted into adaptive finite element models. To assess the effect of loading mode on bone remodeling, a well-established remodeling algorithm was used to examine the effect of axial force or bending moment on bone structure. All simulations predicted that under axial loading, the callus remodeled to form a single cortex. When a bending moment was applied, dual concentric cortices developed in all simulations, corresponding well to the progression of remodeling observed experimentally and resulting in quantitatively comparable callus areas of woven and lamellar bone. Effects of biological differences between species or other reasons cannot be excluded, but this study demonstrates how a difference in loading mode could explain the differences between the remodeling phase in small rodents and larger mammals.

Hydroxyapatite particles maintain peri-implant bone mantle during osseointegration in osteoporotic bone.

In osteoporotic bones, resorption exceeds formation during the remodelling phase of bone turnover. As a consequence, decreased bone volume and bone contact result in the peri-implant region. This may subsequently lead to loss of fixation. In this study we investigated whether the presence of nonresorbable, osteoconductive hydroxyapatite (HA) particles could help maintain a denser and more functional peri-implant bone structure. Titanium screws were implanted into the proximal tibial metaphysis of four months old, ovariectomized Wistar rats (n=60). In the right tibia, the drill hole was first filled with HA particles, while the left tibia served as a control without HA particles. Histological analysis demonstrated that during the remodelling phase the amount of newly formed bone was significantly higher on the HA over the control side. Micro-CT analysis corroborated the significant changes over time as well as differences in peri-implant bone volume density between treatment and control group. Mechanical tests demonstrated that the pull-out force was greater with HA particles. These results indicate that HA particles are able to induce and maintain for a longer time a denser peri-implant bone mantle in osteoporotic bone, which may have important implications in the prevention of implant migration and cut-outs.

Hyperhomocysteinemia induces a tissue specific accumulation of homocysteine in bone by collagen binding and adversely affects bone.

BACKGROUND: Recently, hyperhomocysteinemia (HHCY) has been suggested to have adverse effects on bone. This study investigated if an experimental HHCY in rats induces an accumulation of homocysteine (HCY) in bone tissue that is accompanied by bone loss and reduced bone strength. MATERIAL AND METHODS: HHCY was induced in healthy rats by either a methionine (Meth)- or a homocystine (Homo)-enriched diet and compared with controls. Homocystine is the product of two disulfide linked HCY molecules. Tissue and plasma concentrations of HCY, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were measured. Bones were assessed by biomechanical testing, histomorphometry, microCT and the measurement of biochemical bone turnover markers in plasma. RESULTS: Meth and Homo animals developed a significant HHCY that was accompanied by a tissue specific accumulation of HCY (1300 to 2000% vs. controls). 65% of HCY in bone was bound to collagen of the extracellular matrix. The SAH / SAM-ratio in bone and plasma of Meth and Homo animals exhibited a tissue specific increase indicating a reduced methylation capacity. Accumulation of HCY in bone was characterized by a distinct reduction of cancellous bone (proximal femur: -25 to -35%; distal femur -56 to -58%, proximal tibia: -28 to -43%). Accordingly, bone strength was significantly reduced (-9 to -12%). CONCLUSION: A tissue specific accumulation of HCY in bone may be a promising mechanism explaining adverse effects of HHCY on bone. A reduced methylation capacity of bone cells might be another relevant pathomechanism.