Comparison of Nanocrystalline Hydroxyapatite Bone Graft with Empty Defects in Long Bone Fractures: A Retrospective Case-Control Study.

BACKGROUND The regeneration of bone defects is indicated to restore lost tissue mass and functionality. Ostim®, an absorbable nanocrystalline hydroxyapatite (NCHA) paste, is indicated to enhance bone regeneration in bone defects due to trauma or surgery. This retrospective study of 110 patients with long-bone fracture defects presenting at a single trauma center between 2010 and 2012 aimed to compare outcomes with and without the use of Ostim® absorbable nanocrystalline hydroxyapatite paste. MATERIAL AND METHODS The study encompassed fractures in 110 patients - 55 patients received any defect augmentation (ED) and 55 patients were treated with NCHA augmentation. Fractures were located at the distal radius (66.4%, n=73), proximal humerus (5.5%, n=6), and proximal tibia (28.2%, n=31). Evaluating the clinical follow-up, the study encompassed post-surgery complications (eg, non-unions, infection). Bone healing was evaluated by conventional radiographs. RESULTS Postoperative complications occurred in 45.5% of patients regardless of the treatment (P=1.0). The non-union rate in both groups was 5.5% (n=8, P=1.0), and the risk for infection was lower in the NCHA group (3.6%, ED: n=3, NCHA: n=1, p=0.62). Patients suffered open fractures were treated in the NCHA group (100%, n=7, P=0.003). Radiological assessment demonstrated comparable healing of the fracture border, fracture gap, and articular surface (P>0.05). CONCLUSIONS The findings from this retrospective study support previous studies that have shown Ostim® absorbable nanocrystalline hydroxyapatite paste enhances outcomes and reduces the risk of complications when used to repair bone defects in long-bone fractures in trauma patients. NCHA paste augmentation is suitable for use in traumatic long-bone fractures.

Landscape of Well-Coordinated Fracture Healing in a Mouse Model Using Molecular and Cellular Analysis.

The success of fracture healing relies on overlapping but coordinated cellular and molecular events. Characterizing an outline of differential gene regulation throughout successful healing is essential for identifying crucial phase-specific markers and may serve as the basis for engineering these in challenging healing situations. This study analyzed the healing progression of a standard closed femoral fracture model in C57BL/6N (age = 8 weeks) wild-type male mice. The fracture callus was assessed across various days post fracture (D = days 0, 3, 7, 10, 14, 21, and 28) by microarray, with D0 serving as a control. Histological analyses were carried out on samples from D7 until D28 to support the molecular findings. Microarray analysis revealed a differential regulation of immune response, angiogenesis, ossification, extracellular matrix regulation, mitochondrial and ribosomal genes during healing. In-depth analysis showed differential regulation of mitochondrial and ribosomal genes during the initial phase of healing. Furthermore, the differential gene expression showed an essential role of Serpin Family F Member 1 over the well-known Vascular Endothelial Growth Factor in angiogenesis, especially during the inflammatory phase. The significant upregulation of matrix metalloproteinase 13 and bone sialoprotein from D3 until D21 asserts their importance in bone mineralization. The study also shows type I collagen around osteocytes located in the ossified region at the periosteal surface during the first week of healing. Histological analysis of matrix extracellular phosphoglycoprotein and extracellular signal-regulated kinase stressed their roles in bone homeostasis and the physiological bone-healing process. This study reveals previously unknown and novel candidates, that could serve as a target for specific time points in healing and to remedy cases of impaired healing.

Retrospective Analysis of Bone Substitute Material for Traumatic Long Bone Fractures: Sex-Specific Outcomes.

Male patients often experience increased bone and muscle loss after traumatic fractures. This study aims to compare the treatment outcomes of male and female patients with large bone defects. A total of 345 trauma patients underwent surgery, with participants divided into two groups: one receiving bone substitute material (BSM) for augmented defects (n = 192) and the other without augmentation (empty defects = ED, n = 153). Outcome parameters were assessed among female (n = 184) and male (n = 161) patients. Descriptive statistics revealed no significant differences between male and female patients. Approximately one-half of the fractures resulted from high-energy trauma (n = 187). The BSM group experienced fewer complications (p = 0.004), including pseudarthrosis (BSM: n = 1, ED: n = 7; p = 0.02). Among female patients over 65, the incidence of pseudarthrosis was lower in the BSM group (p = 0.01), while younger females showed no significant differences (p = 0.4). Radiologically, we observed premature bone healing with subsequent harmonization. Post hoc power analysis demonstrated a power of 0.99. Augmenting bone defects, especially with bone substitute material, may reduce complications, including pseudarthrosis, in female patients. Additionally, this material accelerates bone healing. Further prospective studies are necessary for confirmation.

Complication and Infection Risk Using Bone Substitute Materials to Treat Long Bone Defects in Geriatric Patients: An Observational Study.

Background and Objectives: he treatment of large bone defects in geriatric patients often presents a major surgical challenge because of age-related bone loss. In such patients, the scarcity of healthy makes autologous grafting techniques hard to perform. On the one hand, clinicians' fear of possible infections limits using bone substitute materials (BSM). On the other hand, BSM is limitless and spares patients another surgery to harvest autologous material. Materials and Methods: To address the aptness of BSM in geriatric patients, we performed a retrospective analysis of all patients over the age of 64 years who visited our clinic between the years 2011-2018. The study assessed postoperative complications clinically and healing results radiologically. The study included 83 patients with bone defects at the distal radius, proximal humerus, and proximal tibia. The defect zones were filled with BSM based on either nanocrystalline hydroxyapatite (NHA) or calcium phosphate (CP). For comparison, a reference group (empty defect, ED) without the void filling with a BSM was also included. Results: 106 patients sustained traumatic fractures of the distal radius (71.7%), proximal humerus (5.7%), and proximal tibia (22.6%). No difference was found between the BSM groups in infection occurrence (p = 1.0). Although not statistically significant, the BSM groups showed a lower rate of pseudarthrosis (p = 0.09) compared with the ED group. Relative risk (RR) of complications was 32.64% less in the BSM groups compared with the ED group. The additional beneficial outcome of BSM was demonstrated by calculating the number needed to treat (NNT). The calculation showed that with every six patients treated, at least one complication could be avoided. Radiological assessment of bone healing showed significant improvement in the bridging of the defect zone (p < 0.001) when BSM was used. Conclusions: In contrast to previous studies, the study showed that BSM could support bone healing and does not present an infection risk in geriatric patients. The NNT calculation indicates a wider potential benefit of BSM.

Characterisation of Sr2+ mobility in osteoporotic rat bone marrow by cryo-ToF-SIMS and cryo-OrbiSIMS.

Strontium (Sr2+) ions are an effective therapeutic agent for the healing of osteoporotic bone fractures and are therefore used, for example, in form of strontium-modified bone cements. In order to reduce animal testing in further implant materials development in the future, a simulation of the Sr2+ release and transport in bone would be helpful. For such a simulation, knowledge of the experimental parameters for Sr2+ mobility in different compartments of bone (mineralised bone, bone marrow) is essential. In a previous study, we developed an experimental protocol for transport studies in bovine bone marrow by time-of-flight secondary ion mass spectrometry (ToF-SIMS). In the current proof-of-concept study, we investigated Sr2+ diffusion for the first time in bone marrow of rat bone sections. Additionally, orbitrap secondary ion mass spectrometry (OrbiSIMS) was applied for unambiguous signal identification of lipids and fatty acid species in rat bone marrow. Detailed 2D and 3D mass spectrometric imaging analyses, depth profiling as well as OrbiSIMS spectrometric analysis revealed faster Sr2+ diffusion in rat bone marrow areas with low intensity of lipid and fatty acid signals than in areas with higher lipid/fatty acid content. These results could be confirmed by histological staining and additional analysis of Sr2+ diffusion into pure fat sections.

Structural Analysis of Mitochondrial Dynamics-From Cardiomyocytes to Osteoblasts: A Critical Review.

Mitochondria play a crucial role in cell physiology and pathophysiology. In this context, mitochondrial dynamics and, subsequently, mitochondrial ultrastructure have increasingly become hot topics in modern research, with a focus on mitochondrial fission and fusion. Thus, the dynamics of mitochondria in several diseases have been intensively investigated, especially with a view to developing new promising treatment options. However, the majority of recent studies are performed in highly energy-dependent tissues, such as cardiac, hepatic, and neuronal tissues. In contrast, publications on mitochondrial dynamics from the orthopedic or trauma fields are quite rare, even if there are common cellular mechanisms in cardiovascular and bone tissue, especially regarding bone infection. The present report summarizes the spectrum of mitochondrial alterations in the cardiovascular system and compares it to the state of knowledge in the musculoskeletal system. The present paper summarizes recent knowledge regarding mitochondrial dynamics and gives a short, but not exhaustive, overview of its regulation via fission and fusion. Furthermore, the article highlights hypoxia and its accompanying increased mitochondrial fission as a possible link between cardiac ischemia and inflammatory diseases of the bone, such as osteomyelitis. This opens new innovative perspectives not only for the understanding of cellular pathomechanisms in osteomyelitis but also for potential new treatment options.

Awareness of predatory journals and open access publishing among orthopaedic and trauma surgeons - results from an online survey in Germany.

BACKGROUND: Along with emerging open access journals (OAJ) predatory journals increasingly appear. As they harm accurate and good scientific research, we aimed to examine the awareness of predatory journals and open access publishing among orthopaedic and trauma surgeons. METHODS: In an online survey between August and December 2019 the knowledge on predatory journals and OAJ was tested with a hyperlink made available to the participants via the German Society for Orthopaedics and Trauma Surgery (DGOU) email distributor. RESULTS: Three hundred fifty orthopaedic and trauma surgeons participated, of which 291 complete responses (231 males (79.4%), 54 females (18.6%) and 5 N/A (2.0%)) were obtained. 39.9% were aware of predatory journals. However, 21.0% knew about the "Directory of Open Access Journals" (DOAJ) as a register for non-predatory open access journals. The level of profession (e.g. clinic director, consultant) (p = 0.018) influenced the awareness of predatory journals. Interestingly, participants aware of predatory journals had more often been listed as corresponding authors (p < 0.001) and were well published as first or last author (p < 0.001). Awareness of OAJ was masked when journal selection options did not to provide any information on the editorial board, the peer review process or the publication costs. CONCLUSION: The impending hazard of predatory journals is unknown to many orthopaedic and trauma surgeons. Early stage clinical researchers must be trained to differentiate between predatory and scientifically accurate journals.

Osteocytes Influence on Bone Matrix Integrity Affects Biomechanical Competence at Bone-Implant Interface of Bioactive-Coated Titanium Implants in Rat Tibiae.

Osseointegration is a prerequisite for the long-term success of implants. Titanium implants are preferred for their biocompatibility and mechanical properties. Nonetheless, the need for early and immediate loading requires enhancing these properties by adding bioactive coatings. In this preclinical study, extracellular matrix properties and cellular balance at the implant/bone interface was examined. Polyelectrolyte multilayers of chitosan and gelatin or with chitosan and Hyaluronic acid fabricated on titanium alloy using a layer-by-layer self-assembly process were compared with native titanium alloy. The study aimed to histologically evaluate bone parameters that correlate to the biomechanical anchorage enhancement resulted from bioactive coatings of titanium implants in a rat animal model. Superior collagen fiber arrangements and an increased number of active osteocytes reflected a significant improvement of bone matrix quality at the bone interface of the chitosan/gelatin-coated titan implants over chitosan/hyaluronic acid-coated and native implants. Furthermore, the numbers and localization of osteoblasts and osteoclasts in the reparative and remodeling phases suggested a better cellular balance in the chitosan/Gel-coated group over the other two groups. Investigating the micro-mechanical properties of bone tissue at the interface can elucidate detailed discrepancies between different promising bioactive coatings of titanium alloys to maximize their benefit in future medical applications.

Bone Status in a Mouse Model of Experimental Autoimmune-Orchitis.

Investigations in male patients with fertility disorders revealed a greater risk of osteoporosis. The rodent model of experimental autoimmune-orchitis (EAO) was established to analyze the underlying mechanisms of male infertility and causes of reduced testosterone concentration. Hence, we investigated the impact of testicular dysfunction in EAO on bone status. Male mice were immunized with testicular homogenate in adjuvant to induce EAO (n = 5). Age-matched mice were treated with adjuvant alone (adjuvant, n = 6) or remained untreated (control, n = 7). Fifty days after the first immunization specimens were harvested. Real-time reverse transcription-PCR indicated decreased bone metabolism by alkaline phosphatase and Cathepsin K as well as remodeling of cell-contacts by Connexin-43. Micro computed tomography demonstrated a loss of bone mass and mineralization. These findings were supported by histomorphometric results. Additionally, biomechanical properties of femora in a three-point bending test were significantly altered. In summary, the present study illustrates the induction of osteoporosis in the investigated mouse model. However, results suggest that the major effects on bone status were mainly caused by the complete Freund's adjuvant rather than the autoimmune-orchitis itself. Therefore, the benefit of the EAO model to transfer laboratory findings regarding bone metabolism in context with orchitis into a clinical application is limited.

Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds.

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen 'in air' and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of 'ready-to-use' TECs.

Gelatin-Modified Calcium/Strontium Hydrogen Phosphates Stimulate Bone Regeneration in Osteoblast/Osteoclast Co-Culture and in Osteoporotic Rat Femur Defects-In Vitro to In Vivo Translation.

The development and characterization of biomaterials for bone replacement in case of large defects in preconditioned bone (e.g., osteoporosis) require close cooperation of various disciplines. Of particular interest are effects observed in vitro at the cellular level and their in vivo representation in animal experiments. In the present case, the material-based alteration of the ratio of osteoblasts to osteoclasts in vitro in the context of their co-cultivation was examined and showed equivalence to the material-based stimulation of bone regeneration in a bone defect of osteoporotic rats. Gelatin-modified calcium/strontium phosphates with a Ca:Sr ratio in their precipitation solutions of 5:5 and 3:7 caused a pro-osteogenic reaction on both levels in vitro and in vivo. Stimulation of osteoblasts and inhibition of osteoclast activity were proven during culture on materials with higher strontium content. The same material caused a decrease in osteoclast activity in vitro. In vivo, a positive effect of the material with increased strontium content was observed by immunohistochemistry, e.g., by significantly increased bone volume to tissue volume ratio, increased bone morphogenetic protein-2 (BMP2) expression, and significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio. In addition, material degradation and bone regeneration were examined after 6 weeks using stage scans with ToF-SIMS and µ-CT imaging. The remaining material in the defects and strontium signals, which originate from areas exceeding the defect area, indicate the incorporation of strontium ions into the surrounding mineralized tissue. Thus, the material inherent properties (release of biologically active ions, solubility and degradability, mechanical strength) directly influenced the cellular reaction in vitro and also bone regeneration in vivo. Based on this, in the future, materials might be synthesized and specifically adapted to patient-specific needs and their bone status.

Computed tomography for managing periprosthetic femoral fractures. A retrospective analysis.

BACKGROUND: Periprosthetic fractures (PPF) present a common cause for revision surgery after arthroplasty. The choice of performing either an osteosynthesis or revision arthroplasty depends on the orthopedic implant anchored and loosening. Standard diagnostics include x-ray imaging. CT is usually performed to confirm implant loosening in case of ambiguous diagnosis on standard x-ray imaging. This study aimed to examine the role of CT as a diagnostic modality and its implications for treatment planning and outcome. METHODS: Patients treated for PPF from January 2010 to February 2018 were included. X-ray and CT reports were analyzed to assess implant loosening. The planning for surgery and the final surgical treatment were evaluated. In addition, patient characteristics were analyzed and compared between patients with and without additional CT as a preoperative diagnostic procedure. RESULTS: Seventy-five patients were eligible for the study. X-ray imaging was performed in 90.7% of cases. CT was performed in 60% of the cases as part of the preoperative diagnostic. A clear statement on implant stability or loosening could not be made in 69.1% after X-ray imaging and in 84.4% following CT imaging. Revision arthroplasty for loosened femoral prosthesis components was necessary in 40% of cases. No difference could be determined comparing patients with X-ray imaging to those with X-ray and additional CT. In both groups, operative treatment did not deviate from the preoperative planning. DISCUSSION: In two thirds of the conventional radiographic findings, no reliable evaluation of implant loosening was possible in femoral PPFs. Intriguingly, additional CT did not improve the evaluation of implant loosening. Nonetheless, CT scans are often performed if loosening assessment is unclear on regular radiographs. This fact can explain the bias CT results in comparison to regular radiography. However, software-supported CT diagnosis could help to adequately answer the question of loosened implants in PPF in the near future. Since the diagnosis of fracture and their morphology assessment is currently adequately performed using X-rays, CT shall not be considered as the gold standard.

Predatory journals: a major threat in orthopaedic research.

Predatory publishing is a major threat to contemporary publishing, as it offers 'to unaware scientist's', a quick open-access publication against fees without peer-review procedures.. Lack of peer-review leads to unethical practices, as plagiarism, publication of unscientific falsified data, and even unsafe clinical practices. As these journals threaten the credibility of academic publishing, significant work has been done from many scientific teams, in the last years, in establishing discriminating criteria between predatory and legitimate publishing. In the present review, we include mechanisms used by predatory editors to convince eager researchers to submit to their journals. We also provide useful links giving information about potential predatory journals and publishers, as well as scholarly writing. Joining the efforts of different scientific disciplines which compiled "green" lists with journals in their field, we conducted a "green" list with genuine orthopaedic research journals based on the directory of open-access journals (DOAJ) and Thomson Reuters journal citation reports. Ninety-six legitimate orthopaedic journals were identified based on the Thomson Reuters journal citation reports. One hundred thirty hits were found on the DOAJ site using the keywords "orthopaedics, orthopedics, sports medicine, musculoskeletal, trauma, traumatology, osteoarthritis, osteoporosis, cartilage, bone, hand, shoulder, knee, hip, foot, wound." Twenty-one journals on the DOAJ site occurred overlapping with keywords. Researchers and clinicians in the field of orthopaedics are advised to use all available tools in order to recognize predatory practices and avoid publishing in predatory journals.

Osteocyte Regulation of Receptor Activator of NF-κB Ligand/Osteoprotegerin in a Sheep Model of Osteoporosis.

Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.

Computational segmentation of collagen fibers in bone matrix indicates bone quality in ovariectomized rat spine.

Bone loss varies according to disease and age and these variations affect bone cells and extracellular matrix. Osteoporosis rat models are widely investigated to assess mechanical and structural properties of bone; however, bone matrix proteins and their discrepant regulation of diseased and aged bone are often overlooked. The current study considered the spine matrix properties of ovariectomized rats (OVX) against control rats (Sham) at 16 months of age. Diseased bone showed less compact structure with inhomogeneous distribution of type 1 collagen (Col1) and changes in osteocyte morphology. Intriguingly, demineralization patches were noticed in the vicinity of blood vessels in the OVX spine. The organic matrix structure was investigated using computational segmentation of collagen fibril properties. In contrast to the aged bone, diseased bone showed longer fibrils and smaller orientation angles. The study shows the potential of quantifying transmission electron microscopy images to predict the mechanical properties of bone tissue.

Application of donepezil increased collagen 1 expression in mesenchymal stroma cells of an ovine osteoporosis model.

OBJECTIVES: Donepezil inhibits the acetylcholine degradation molecule acetylcholinesterase (AChE). Clinical studies reported that Alzheimer's disease (AD) patients with hip fractures had improved bone quality and better fracture healing if they were treated with AD medication donepezil. We asked whether mesenchymal stroma cells (MSC) from an osteoporosis sheep model treated with donepezil increased their proliferation rate and mRNA expression. METHODS: Sheep were divided into 4 groups: a) untreated control group, b) sheep with bilateral ovariectomy (OVX), c) sheep with OVX and malnutrition, and d) sheep with OVX, malnutrition, and application of corticosteroid. After 8 months MSC were isolated of iliac crest biopsy, treated with donepezil, and AChE activity, proliferation rate, and mRNA expression were analyzed. RESULTS: Application of donepezil resulted in a significant decrease of AChE activity. Inhibition of AChE did not lead to a significant increase in proliferation. Expression of the osteogenic marker osteocalcin was not regulated by donepezil while the mRNA concentration of collagen was increased. CONCLUSION: AChE inhibition via donepezil resulted in an increased synthesis of osteoid which consists mainly of collagen. Thus, we suppose that increased acetylcholine levels through AChE inhibition do not support MSC proliferation but osteogenic activity probably combined with osteogenic differentiation.

A tissue-based approach to selection of reference genes for quantitative real-time PCR in a sheep osteoporosis model.

BACKGROUND: In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability. METHODS: Twelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes. RESULTS: We identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples. CONCLUSION: This study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs.

Neovascularization of osteoporotic metaphyseal bone defects: A morphometric micro-CT study.

PURPOSE: Neovascularization is essential for bone regeneration in fractures. This study aimed to investigate the microvascular morphology and distribution in the non-injured femur and the neovascularization of the metaphyseal critical size defect in a small animal model of osteoporosis. MATERIALS AND METHODS: Female rats (n=7) were ovariectomized (OVX) and received a multideficiency diet. Three months after OVX, a 5mm wedge shaped critical size defect was cut at the distal femoral metaphysis and stabilized with a T-shaped mini-plate. After six weeks, the animals were euthanized, and femora were removed and decalcified for micro-CT measurement of fracture neovascularization. RESULTS: No fracture healing was observed along the critical size defects. In the non-injured bone, micro-vessel distribution showed a specific pattern, thereby enabling a differentiation between epi-, meta- and diaphysis. Micro-CT based morphometry revealed a significant reduction of the vascular volume fraction as well as the vascular thickness (p<0.001) in the critical size defect compared to the intact contralateral femur. Blood volume related vascular surface (vascular surface/volume) increased significantly (p<0.001). Connectivity density and tissue volume related vascular surface (vascular surface density) did not change significantly. CONCLUSIONS: Micro-CT based vascular morphometry demonstrated differences between epi-, meta- and diaphysis in the non-injured bone as well as differences between the critical size defect and the non-injured metaphysis. As angiogenesis is a crucial prerequisite that precedes osteogenesis, our results may influence further evaluation of osteoconductive or osteogenic biomaterials in this small animal model of osteoporosis.

Impaired extracellular matrix structure resulting from malnutrition in ovariectomized mature rats.

Bone loss is a symptom related to disease and age, which reflects on bone cells and ECM. Discrepant regulation affects cell proliferation and ECM localization. Rat model of osteoporosis (OVX) was investigated against control rats (Sham) at young and old ages. Biophysical, histological and molecular techniques were implemented to examine the underlying cellular and extracellular matrix changes and to assess the mechanisms contributing to bone loss in the context of aging and the widely used osteoporotic models in rats. Bone loss exhibited a compromised function of bone cells and infiltration of adipocytes into bone marrow. However, the expression of genes regulating collagen catabolic process and adipogenesis was chronologically shifted in diseased bone in comparison with aged bone. The data showed the involvement of Wnt signaling inhibition in adipogenesis and bone loss due to over-expression of SOST in both diseased and aged bone. Further, in the OVX animals, an integrin-mediated ERK activation indicated the role of MAPK in osteoblastogenesis and adipogenesis. The increased PTH levels due to calcium and estrogen deficiency activated osteoblastogenesis. Thusly, RANKL-mediated osteoclastogenesis was initiated. Interestingly, the data show the role of MEPE regulating osteoclast-mediated resorption at late stages in osteoporotic bone. The interplay between ECM and bone cells change tissue microstructure and properties. The involvement of Wnt and MAPK pathways in activating cell proliferation has intriguing similarities to oncogenesis and myeloma. The study indicates the importance of targeting both pathways simultaneously to remedy metabolic bone diseases and age-related bone loss.

Bone matrix, cellularity, and structural changes in a rat model with high-turnover osteoporosis induced by combined ovariectomy and a multiple-deficient diet.

In estrogen-deficient, postmenopausal women, vitamin D and calcium deficiency increase osteoporotic fracture risk. Therefore, a new rat model of combined ovariectomy and multiple-deficient diet was established to mimic human postmenopausal osteoporotic conditions under nutrient deficiency. Sprague-Dawley rats were untreated (control), laparatomized (sham), or ovariectomized and received a deficient diet (OVX-Diet). Multiple analyses involving structure (micro-computed tomography and biomechanics), cellularity (osteoblasts and osteoclasts), bone matrix (mRNA expression and IHC), and mineralization were investigated for a detailed characterization of osteoporosis. The study involved long-term observation up to 14 months (M14) after laparotomy or after OVX-Diet, with intermediate time points at M3 and M12. OVX-Diet rats showed enhanced osteoblastogenesis and osteoclastogenesis. Bone matrix markers (biglycan, COL1A1, tenascin C, and fibronectin) and low-density lipoprotein-5 (bone mass marker) were down-regulated at M12 in OVX-Diet rats. However, up-regulation of matrix markers and existence of unmineralized osteoid were seen at M3 and M14. Osteoclast markers (matrix metallopeptidase 9 and cathepsin K) were up-regulated at M14. Micro-computed tomography and biomechanics confirmed bone fragility of OVX-Diet rats, and quantitative RT-PCR revealed a higher turnover rate in the humerus than in lumbar vertebrae, suggesting enhanced bone formation and resorption in OVX-Diet rats. Such bone remodeling caused disturbed bone mineralization and severe bone loss, as reported in patients with high-turnover, postmenopausal osteoporosis. Therefore, this rat model may serve as a suitable tool to evaluate osteoporotic drugs and new biomaterials or fracture implants.