An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro.

In the field of biomaterials for prosthetic reconstructive surgery, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials before in vivo tests. Despite the complex osteointegration process being difficult to recreate in vitro, this study proposes an advanced in vitro tissue culture model of osteointegration using human bone. Cubic samples of trabecular bone were harvested, as waste material, from hip arthroplasty; inner cylindrical defects were created and assigned to the following groups: (1) empty defects (CTRneg); (2) defects implanted with a cytotoxic copper pin (CTRpos); (3) defects implanted with standard titanium pins (Ti). Tissues were dynamically cultured in mini rotating bioreactors and assessed weekly for viability and sterility. After 8 weeks, immunoenzymatic, microtomographic, histological, and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, while Ti appears to have a trophic effect on bone. MicroCT and a histological analysis supported the results, with signs of matrix and bone deposition at the Ti implant site. Data suggest the reliability of the tested model in recreating the osteointegration process in vitro with the aim of reducing and refining in vivo preclinical models.

Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts.

Chondrocytes proliferate and mature into hypertrophic chondrocytes. Vascular invasion into the cartilage occurs in the terminal hypertrophic chondrocyte layer, and terminal hypertrophic chondrocytes die by apoptosis or transdifferentiate into osteoblasts. Runx2 is essential for osteoblast differentiation and chondrocyte maturation. Runx2-deficient mice are composed of cartilaginous skeletons and lack the vascular invasion into the cartilage. However, the requirement of Runx2 in the vascular invasion into the cartilage, mechanism of chondrocyte transdifferentiation to osteoblasts, and its significance in bone development remain to be elucidated. To investigate these points, we generated Runx2fl/flCre mice, in which Runx2 was deleted in hypertrophic chondrocytes using Col10a1 Cre. Vascular invasion into the cartilage was similarly observed in Runx2fl/fl and Runx2fl/flCre mice. Vegfa expression was reduced in the terminal hypertrophic chondrocytes in Runx2fl/flCre mice, but Vegfa was strongly expressed in osteoblasts in the bone collar, suggesting that Vegfa expression in bone collar osteoblasts is sufficient for vascular invasion into the cartilage. The apoptosis of terminal hypertrophic chondrocytes was increased and their transdifferentiation was interrupted in Runx2fl/flCre mice, leading to lack of primary spongiosa and osteoblasts in the region at E16.5. The osteoblasts appeared in this region at E17.5 in the absence of transdifferentiation, and the number of osteoblasts and the formation of primary spongiosa, but not secondary spongiosa, reached to levels similar those in Runx2fl/fl mice at birth. The bone structure and volume and all bone histomophometric parameters were similar between Runx2fl/fl and Runx2fl/flCre mice after 6 weeks of age. These findings indicate that Runx2 expression in terminal hypertrophic chondrocytes is not required for vascular invasion into the cartilage, but is for their survival and transdifferentiation into osteoblasts, and that the transdifferentiation is necessary for trabecular bone formation in embryonic and neonatal stages, but not for acquiring normal bone structure and volume in young and adult mice.

CCL2 is a critical mechano-responsive mediator in crosstalk between osteoblasts and bone mesenchymal stromal cells.

It has been known that moderate mechanical loading, like that caused by exercise, promotes bone formation. However, its underlying mechanisms remain elusive. Here we showed that moderate running dramatically improved trabecular bone in mice tibias with an increase in bone volume fraction and trabecular number and a decrease in trabecular pattern factor. Results of immunohistochemical and histochemical staining revealed that moderate running mainly increased the number of osteoblasts but had no effect on osteoclasts. In addition, we observed a dramatic increase in the number of colony forming unit-fibroblast in endosteal bone marrow and the percentage of CD45- Leptin receptor+ (CD45- LepR+ ) endosteal mesenchymal progenitors. Bioinformatics analysis of the transcriptional data from gene expression omnibus (GEO) database identified chemokine c-c-motif ligands (CCL2) as a critical candidate induced by mechanical loading. Interestingly, we found that CCL2 was up-regulated mainly in osteoblastic cells in the tibia of mice after moderate running. Further, we found that mechanical loading up-regulated the expression of CCL2 by activating ERK1/2 pathway, thereby stimulating migration of endosteal progenitors. Finally, neutralizing CCL2 abolished the recruitment of endosteal progenitors and the increased bone formation in mice after 4 weeks running. These results therefore uncover an unknown connection between osteoblasts and endosteal progenitors recruited in the increased bone formation induced by mechanical loading.

TLR4 knockout ameliorates streptozotocin-induced osteoporosis in a mouse model of diabetes.

Type 1 diabetes mellitus (T1DM) is characterized by hyperglycemia manifesting as insufficient insulin. Toll-like receptor-4 (TLR4) has been implicated in diabetic osteoporosis. We established streptozotocin (STZ)-induced diabetic mouse model and examined the relevant osteoporosis factors in different experimental groups, the WT-CON group, WT-STZ group, KO-CON group and KO-STZ group, respectively. No obvious protection of TLR4 deletion was shown in mice with diabetes. There was no obvious difference in the body weight or blood glucose concentration between WT-STZ group and KO-STZ group. However, TLR4 deletion reduced the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. Furthermore, TLR4 knockout attenuated STZ-induced diabetic osteoporosis via inhibiting osteoblasts and pre-inflammation factors mediated by the NF-κB pathway. TLR4 deletion ameliorated STZ-induced diabetic osteoporosis in mice, and TLR4 may be used as a potential therapeutic target for the treatment of diabetic osteoporosis.

Collagenated Porcine Heterologous Bone Grafts: Histomorphometric Evaluation of Bone Formation Using Different Physical Forms in a Rabbit Cancellous Bone Model.

Collagenated porcine-derived bone graft materials exhibit osteoconductive properties and the development of different formulations intends to enhance bone regeneration. This study aims to evaluate bone healing in a rabbit cancellous bone defect in response to grafting with different physicochemical forms of heterologous porcine bone. Twenty-six adult male New Zealand White rabbits received two critical size femoral bone defects per animal (n = 52), each randomly assigned to one of the five tested materials (Apatos, Gen-Os, mp3, Putty, and Gel 40). Animals were sacrificed at 15- and 30-days post-surgery. Qualitative and quantitative (new bone, particle and connective tissue percentages) histological analyses were performed. Histomorphometry showed statistically significant differences in all evaluated parameters between mp3 and both Putty and Gel 40 groups, regardless of the timepoint (p < 0.05). Moreover, statistical differences were observed between Apatos and both Putty (p = 0.014) and Gel 40 (p = 0.007) groups, at 30 days, in regard to particle percentage. Within each group, regarding new bone formation, mp3 showed significant differences (p = 0.028) between 15 (40.93 ± 3.49%) and 30 (52.49 ± 11.04%) days. Additionally, intragroup analysis concerning the percentage of particles revealed a significant reduction in particle occupied area from 15 to 30 days in mp3 and Gen-Os groups (p = 0.009). All mp3, Gen-Os and Apatos exhibited promising results in terms of new bone formation, thus presenting suitable alternatives to be used in bone regeneration.

Comparative proteomic analysis of osteogenic differentiated human adipose tissue and bone marrow-derived stromal cells.

Mesenchymal stromal cells are promising candidates for regenerative applications upon treatment of bone defects. Bone marrow-derived stromal cells (BMSCs) are limited by yield and donor morbidity but show superior osteogenic capacity compared to adipose-derived stromal cells (ASCs), which are highly abundant and easy to harvest. The underlying reasons for this difference on a proteomic level have not been studied yet. Human ASCs and BMSCs were characterized by FACS analysis and tri-lineage differentiation, followed by an intraindividual comparative proteomic analysis upon osteogenic differentiation. Results of the proteomic analysis were followed by functional pathway analysis. 29 patients were included with a total of 58 specimen analysed. In these, out of 5148 identified proteins 2095 could be quantified in >80% of samples of both cell types, 427 in >80% of ASCs only and 102 in >80% of BMSCs only. 281 proteins were differentially regulated with a fold change of >1.5 of which 204 were higher abundant in BMSCs and 77 in ASCs. Integrin cell surface interactions were the most overrepresented pathway with 5 integrins being among the proteins with highest fold change. Integrin 11a, a known key protein for osteogenesis, could be identified as strongly up-regulated in BMSC confirmed by Western blotting. The integrin expression profile is one of the key distinctive features of osteogenic differentiated BMSCs and ASCs. Thus, they represent a promising target for modifications of ASCs aiming to improve their osteogenic capacity and approximate them to that of BMSCs.

Skeletal Stem Cells for Bone Development and Repair: Diversity Matters.

PURPOSE OF REVIEW: Skeletal stem cells (SSCs) are considered to play important roles in bone development and repair. These cells have been historically defined by their in vitro potential for self-renewal and differentiation into "trilineage" cells; however, little is known about their in vivo identity. Here, we discuss recent progress on SSCs and how they potentially contribute to bone development and repair. RECENT FINDINGS: Bone is composed of diverse tissues, which include cartilage and its perichondrium, cortical bone and its periosteum, and bone marrow and its trabecular bone and stromal compartment. We are now at the initial stage of understanding the precise identity of SSCs in each bone tissue. The emerging concept is that functionally dedicated SSCs are encased by their own unique cellular and extracellular matrix microenvironment, and locally support its own compartment. Diverse groups of SSCs are likely to work in concert to achieve development and repair of the highly functional skeletal organ.

Bif-1/Endophilin B1/SH3GLB1 regulates bone homeostasis.

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 -/- ) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 -/- mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 -/- mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 -/- mice. Treatment with ß-glycerophosphate (ß-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 -/- mice. Finally, bone marrow cells from Bif-1 -/- mice showed a significantly higher colony-forming efficacy by the treatment with or without ß-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 -/- mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).

Melatonin prevents bone destruction in mice with retinoic acid-induced osteoporosis.

BACKGROUND: The protective effect of melatonin against bone metabolism imbalance in osteoporosis (OP) induced by drugs such as retinoic acid (RA) is unclear. The aim of this study was to explore the role of melatonin in bone destruction based on a mouse model. METHODS: RA-induced OP model mice were established. To assess the effect of melatonin on these mice, micro-CT was used to characterize the trabecular structure of normal mice and those treated with RA (model), RA + low-dose melatonin (Mlt-L), RA + high-dose melatonin (Mlt-H), and RA + alendronate sodium (positive control). The shape of the trabecular bone, the length and diameter of the femoral head and the height and diameter of vertebra(L1) of each group were also measured and the number of osteoclasts was determined by Tartrate-resistant acid phosphatase (TRACP) staining. Meanwhile, the expression of alkaline phosphatase (ALP) was evaluated by immunohistochemistry assays. The differences between groups in terms of liver and kidney oxidation-related indexes and serum and urinary indicators related to bone metabolism were also analyzed. Furthermore, qRT-PCR and western blotting were used to evaluate the effect of melatonin on osteogenic and osteoclastic differentiation in MC3T3-E1 and RAW264.7 cells, respectively. RESULTS: RA induction led to a decrease in the amount and density of trabecular bone, a decrease in the length and diameter of the femur and height, diameter of the vertebra (L1), a decrease in bone mass and density and the expression of ALP, and an increase in the number of osteoclasts. Melatonin treatment alleviated these effects induced by RA, increasing the amount of trabecular bone in OP mice, improving the microstructure of the femur and vertebra(L1) and increasing bone mass bone density and the expression of ALP, as well as decreasing the number of osteoclasts. Additionally, blood and urinary bone metabolism-related indicators showed that melatonin promoted bone formation and inhibited bone resorption. Determination of oxidant and antioxidant biomarkers in the livers and kidneys of the mice revealed that melatonin promoted the antioxidant level and suppressed the level of oxidant molecules in these organs. In vitro, RA promoted osteoclasts and inhibit osteogenesis by increasing oxidative stress levels in the RAW264.7 and MC3T3-E1 cells, but melatonin reversed this effect. Melatonin may, therefore, play a role in the ERK/SMAD and NF-κB pathways. CONCLUSIONS: Melatonin can alleviate bone loss in RA-induced OP model mice, repair the trabecular microstructure, and promote bone formation. These effects may be related to reducing oxidation levels in vivo and vitro through the ERK/SMAD and NF-κB pathways.

Influence of cancellous bone microstructure on ultrasonic attenuation: a theoretical prediction.

BACKGROUND: Quantitative ultrasound has been used for the assessment of cancellous bone status. The attenuation mechanisms of cancellous bone, however, have not been well understood, because the microstructure of cancellous bone is significantly inhomogeneous and the interaction between ultrasound and the microstructure of cancellous bone is complex. In this study, a theoretical approach was applied to investigate the influence of the microstructure of cancellous bone on ultrasonic attenuation. RESULTS: The scattering from a trabecular cylinder was significantly angle dependent. The dependencies of the ultrasonic attenuation on frequency, scatterer size, and porosity were explored from the theoretical calculation. Prediction results showed that the ultrasonic attenuation increased with the increase of frequency and decreased linearly with the increase in porosity, and the broadband ultrasound attenuation decreased with the increase in porosity. All these predicted trends were consistent with published experimental data. In addition, our model successfully explained the principle of broadband ultrasound attenuation measurement (i.e., the attenuation over the frequency range 0.3-0.65 MHz was approximately linearly proportional to frequency) by considering the contributions of scattering and absorption to attenuation. CONCLUSION: The proposed theoretical model may be a potentially valuable tool for understanding the interaction of ultrasound with cancellous bone.

The in vitro behaviour of canine osteoblasts derived from different bone types.

BACKGROUND: Our understanding of the biology of osteoblasts is important as they underpin bone remodelling, fracture healing and processes such as osseointegration. Osteoblasts isolated from human humeral samples display distinctive biological activity in vitro, which relates to the samples' bone types (subchondral (S), trabecular (T), cortical (C)). Our aim was to isolate primary osteoblast cultures from different bone types from the proximal femur of a clinical population of dogs presented for total hip replacement and compare the behaviour of the osteoblasts derived from different bone types, to identify a preferred bone type for isolation. RESULTS: No differences were found for osteoblast doubling time (median for S = 2.9, T = 3.1 and C = 2.71 days, respectively; p = 0.33), final cell number (median for S = 54,849, T = 49,733, C = 61,390 cells/cm2; p = 0.34) or basal tissue non-specific alkaline phosphatase (TNAP) activity (median for S = 0.02, T = 0.02, C = 0.03 U/min/mg protein; p = 0.81) between bone types after 6 days of culture in basal media. There were no differences in mineralizing TNAP activity (S = 0.02, T = 0.02, C = 0.03 U/min/mg protein, p = 0.84) or in mineralized area (S = 0.05, T = 0.04, C = 0.04%, p = 0.92) among cells from different bone types. CONCLUSIONS: There is no significant difference in mean doubling time, basal or mineralizing TNAP activity or mineralized area in osteoblasts derived from subchondral, cortical, or trabecular bone types from the canine femoral head. However, there appears to be a high level of inter-animal variability in the studied parameters, which was independent of age, body mass, and sex. Trabecular isolate osteoblasts have the least variation of the bone types studied, and therefore should be considered a preferred source for primary osteoblast cultures. The work here provides baselines for canine osteoblast function, which has utility for future comparative studies.

NOX4 Deletion in Male Mice Exacerbates the Effect of Ethanol on Trabecular Bone and Osteoblastogenesis.

Chronic alcohol consumption increases bone resorption and decreases bone formation. A major component of ethanol (EtOH) pathology in bone is the generation of excess reactive oxygen species (ROS). The ROS-generating NADPH oxidase-4 (NOX4) is proposed to drive much of the EtOH-induced suppression of bone formation. Here, 13-week-old male wild-type (WT) and NOX4-/- mice were pair fed (PF) a high-fat (35%), Lieber-DeCarli liquid diet with or without EtOH at 30% of their total calories for 12 weeks. Micro-computed tomography analysis demonstrated significant decreases in trabecular bone volume/total volume (BV/TV) percentage and cortical thickness in WT, EtOH-fed mice compared with PF controls. EtOH-fed NOX4-/- mice also displayed decreased trabecular BV/TV and trabecular number compared with PF (P < 0.05). However, NOX4-/- mice were protected against EtOH-induced decreases in cortical thickness (P < 0.05) and decreases in collagen1 and osteocalcin mRNA expression in cortical bone (P < 0.05). In WT and NOX4-/- vertebral bone, EtOH suppressed expression of Wnt signaling components that promote osteoblast maturation. A role for NOX4 in EtOH inhibition of osteoblast differentiation was further demonstrated by protection against EtOH inhibition of osteoblastogenesis in ex vivo bone marrow cultures from NOX4-/-, but not p47phox-/- mice lacking active NADPH oxidase-2. However, bone marrow cultures from NOX4-/- mice formed fewer osteoblastic colonies compared with WT cultures (P < 0.05), suggesting a role for NOX4 in the maintenance of mesenchymal progenitor cell populations. These data suggest that NOX4 deletion is partially protective against EtOH effects on osteoblast differentiation, but may predispose bone to osteogenic impairments.

EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling.

In this study, we examined the functional importance of EZH2 during skeletal development and homeostasis using the conditional deletion of Ezh2 (Ezh2fl/fl ) in early mesenchyme with the use of a Prrx-1-cre driver mouse (Ezh2+/+). Heterozygous (Ezh2+/-) newborn and 4-wk-old mice exhibited increased skeletal size, growth plate size, and weight when compared to the wild-type control (Ezh2+/+), whereas homozygous deletion of Ezh2 (Ezh2-/-) resulted in skeletal deformities and reduced skeletal size, growth plate size, and weight in newborn and 4-wk-old mice. Ezh2-/- mice exhibited enhanced trabecular patterning. Osteogenic cortical and trabecular bone formation was enhanced in Ezh2+/- and Ezh2-/- animals. Ezh2+/- and Ezh2-/- mice displayed thinner cortical bone and decreased mechanical strength compared to the wild-type control. Differences in cortical bone thickness were attributed to an increased number of osteoclasts, corresponding with elevated levels of the bone turnover markers cross-linked C-telopeptide-1 and tartrate-resistant acid phosphatase, detected within serum. Moreover, Ezh2+/- mice displayed increased osteoclastogenic potential coinciding with an upregulation of Rankl and M-csf expression by mesenchymal stem cells (MSCs). MSCs isolated from Ezh2+/- mice also exhibited increased trilineage potential compared with wild-type bone marrow stromal/stem cells (BMSCs). Gene expression studies confirmed the upregulation of known Ezh2 target genes in Ezh2-/- bone tissue, many of which are involved in Wnt/BMP signaling as promoters of osteogenesis and inhibitors of adipogenesis. In summary, EZH2 appears to be an important orchestrator of skeletal development, postnatal bone remodelling and BMSC fate determination in vitro and in vivo-Hemming, S., Cakouros, D., Codrington, J., Vandyke, K., Arthur, A., Zannettino, A., Gronthos, S. EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling.

The Effect of Autologous Platelet-Rich Plasma on Bone Regeneration by Autologous Mesenchymal Stem Cells Loaded onto Allogeneic Cancellous Bone Granules.

To develop a clinically effective bone regeneration strategy, we compared bone regeneration using allogeneic cancellous bone granule scaffolds loaded with autologous bone marrow-derived mesenchymal stem cells (BM-MSC) with or without autologous platelet-rich plasma (PRP). Critical-sized segmental bone defects were made at the mid-shaft of both radiuses in 41 New Zealand White rabbits. Small-sized allogeneic cancellous bone granules (300-700 µm in diameter) loaded with BM-MSC were implanted on one side, and PRP was added. On the other side, autologous BM-MSC loaded onto allogeneic cancellous granules were grafted as a control. Bone regeneration was assessed by radiographic evaluation at 4, 8, and 16 weeks postimplantation and by micro-computed tomography (micro-CT) and histological evaluation of the retrieved specimens at 8 and 16 weeks. The experimental group did not show significantly higher bone quantity indices than the control group at any time point. Micro-CT analysis revealed that both groups had similar mean total volumes, surface areas, and other parameters at 8 and 16 weeks. Histological evaluation of 8- and 16-week specimens also showed a similar progression of new bone formation and maturation. In this experiment using a contralateral control group in the same individual, an initial single addition of PRP in allogeneic cancellous bone granules loaded with BM-MSC for critical-sized bone defects in the weight-bearing area did not induce a consequent difference in bone healing. Further research into the optimal preparation and application of PRP is necessary. Furthermore, studies involving a greater number of subjects and larger experimental animals could determine the clinical relevance of PRP treatment.

Wnt10b and Dkk-1 gene therapy differentially influenced trabecular bone architecture, soft tissue integrity, and osteophytosis in a skeletally mature rat model of osteoarthritis.

AIMS: Our goals in the current experiments were to determine if (a) upregulation of Wnt signaling would induce osteoarthritis changes in stable stifle joints and (b) if downregulation of Wnt signaling in destabilized joints would influence the progression of OA. METHODS: At 37 weeks of age, rats were injected in the stifle joint with a recombinant adeno-associated viral vector containing the Wnt-inhibitor Dkk-1 or a Wnt10b transgene. At 40 weeks of age, rats underwent surgical destabilization of the joint. At 50 weeks of age, stifle joints were submitted for micro-computed tomography and histopathological analysis. RESULTS: Injection of either Wnt10b or Dkk-1 transgenes in stable joints improved bone architectural parameters, but worsened soft tissue integrity. Osteophytosis was decreased by Dkk-1, but unchanged by Wnt10b. Destabilization negatively influenced bone architecture, increased osteophytosis, and decreased soft tissue integrity. Dkk-1 exacerbated the negative effects of destabilization, whereas Wnt10b had little effect on these parameters. Osteophytosis was improved, whereas soft tissue integrity was worsened by both transgenes in destabilized joints. CONCLUSIONS: The Wnt-inhibitor Dkk-1 does not appear to completely inhibit the effects of Wnt signaling on bone remodeling. In vivo upregulation of Wnt10b and its inhibitor, Dkk-1, can produce both parallel or contrasting phenotypic responses depending on the specific parameter measured and the fidelity of the examined joint. These observations elucidate different roles for Wnt signaling in stable versus destabilized joints and may help to explain the conflicting results previously reported for the role of Dkk-1 in joint disease.

Chronic alcohol abuse in men alters bone mechanical properties by affecting both tissue mechanical properties and microarchitectural parameters.

OBJECTIVE OF THE STUDY: Alcohol-induced secondary osteoporosis in men has been characterized by higher fracture prevalence and a modification of bone microarchitecture. Chronic alcohol consumption impairs bone cell activity and results in an increased fragility. A few studies highlighted effects of heavy alcohol consumption on some microarchitectural parameters of trabecular bone. But to date and to our knowledge, micro- and macro-mechanical properties of bone of alcoholic subjects have not been investigated. PATIENTS: In the present study, mechanical properties and microarchitecture of trabecular bone samples from the iliac crest of alcoholic male patients (n=15) were analyzed and compared to a control group (n=8). MATERIALS AND METHODS: Nanoindentation tests were performed to determine the tissue's micromechanical properties, micro-computed tomography was used to measure microarchitectural parameters, and numerical simulations provided the apparent mechanical properties of the samples. RESULTS: Compared to controls, bone tissue from alcoholic patients exhibited an increase of micromechanical properties at tissue scale, a significant decrease of apparent mechanical properties at sample scale, and significant changes in several microarchitectural parameters. In particular, a crucial role of structure model index (SMI) on mechanical properties was identified. CONCLUSIONS: 3D microarchitectural parameters are at least as important as bone volume fraction to predict bone fracture risk in the case of alcoholic patients.

Opposing effects of Sca-1(+) cell-based systemic FGF2 gene transfer strategy on lumbar versus caudal vertebrae in the mouse.

Our previous work showed that a Sca-1(+) cell-based FGF2 therapy was capable of promoting robust increases in trabecular bone formation and connectivity on the endosteum of long bones. Past work reported that administration of FGF2 protein promoted bone formation in red marrow but not in yellow marrow. The issue as to whether the Sca-1(+) cell-based FGF2 therapy is effective in yellow marrow is highly relevant to its clinical potential for osteoporosis, as most red marrows in a person of an advanced age are converted to yellow marrows. Accordingly, this study sought to compare the osteogenic effects of this stem cell-based FGF2 therapy on red marrow-filled lumbar vertebrae with those on yellow marrow-filled caudal vertebrae of young adult W(41)/W(41) mice. The Sca-1(+) cell-based FGF2 therapy drastically increased trabecular bone formation in lumbar vertebrae, but the therapy not only did not promote bone formation but instead caused substantial loss of trabecular bone in caudal vertebrae. The lack of an osteogenic response was not due to insufficient engraftment of FGF2-expressing Sca-1(+) cells or inadequate FGF2 expression in caudal vertebrae. Previous studies have demonstrated that recipient mice of this stem cell-based FGF2 therapy developed secondary hyperparathyroidism and increased bone resorption. Thus, the loss of bone mass in caudal vertebrae might in part be due to an increase in resorption without a corresponding increase in bone formation. In conclusion, the Sca-1(+) cell-based FGF2 therapy is osteogenic in red marrow but not in yellow marrow.

Isolation of Murine and Human Osteocytes.

Osteocytes are thought to be the mechanosensors of bone by sensing mechanical loads imposed upon the bone and transmitting these signals to the other bone cells to initiate bone modeling and remodeling. The location of osteocytes deep within bone is ideal for their function. However, this location makes the study of osteocytes in vivo technically difficult. There are several methods for obtaining and culturing primary osteocytes for in vitro experiments and ex vivo observation. In this chapter, several proven methods are discussed including the isolation of avian osteocytes from chicks and osteocytes from calvaria and long bones of young mice. A detailed protocol for the isolation of osteocytes from hypermineralized bone of mature and aged animals is provided. In addition, a modified version of this protocol that can be used to isolate osteocytes from human trabecular bone is described.

Improved osteogenesis in rat femur segmental defects treated with human allograft and zinc adjuvants.

Bone allograft is widely used to treat large bone defects or complex fractures. However, processing methods can significantly compromise allograft osteogenic activity. Adjuvants that can restore the osteogenic activity of processed allograft should improve clinical outcomes. In this study, zinc was tested as an adjuvant to increase the osteogenic activity of human allograft in a Rag2 null rat femoral defect model. Femoral defects were treated with human demineralized bone matrix (DBM) mixed with carboxy methyl cellulose containing ZnCl2 (0, 75, 150, 300 µg) or Zn stearate (347 µg). Rat femur defects treated with DBM-ZnCl2 (75 µg) and DBM-Zn stearate (347 µg) showed increased calcified tissue in the defect site compared to DBM alone. Radiograph scoring and µCT (microcomputed tomography) analysis showed an increased amount of bone formation at the defects treated with DBM-Zn stearate. Use of zinc as an adjuvant was also tested using human cancellous bone chips. The bone chips were soaked in ZnCl2 solutions before being added to defect sites. Zn adsorbed onto the chips in a time- and concentration-dependent manner. Rat femur defects treated with Zn-bound bone chips had more new bone in the defects based on µCT and histomorphometric analyses. The results indicate that zinc supplementation of human bone allograft improves allograft osteogenic activity in the rat femur defect model.