Thyroid-Stimulating Hormone Favors Runx2-Mediated Matrix Mineralization in HOS and SaOS2 Cells: An In Vitro and In Silico Approach.

Osteoporosis is a skeletal disease that is both systemic and silent characterized by an unbalanced activity of bone remodeling leading to bone loss. Rising evidences demonstrate that thyroid stimulating hormone (TSH) has an important role in the regulation on the metabolism of bone. However, TSH regulation on human osteoblast essential transcriptional factors has not been identified. Current study examined the role of TSH on human osteoblastic Runx2 expression and their functional genes by in vitro and in slico analysis. Human osteoblast like (HOS and SaoS-2) cells were cultured with DMEM and treated with hTSH at the concentration of 0.01 ng/mL and 10 ng/mL. After treatment, osteoblastic Runx2 and IGF-1R beta expression were studied using RT-PCR and western blot analysis. TSH treatment induced osteoblastic essential transcriptional factor, Runx2 in HOS and SaOS2 cells on 48 h duration and elevated the expression of IGF-IR ß gene and Protein in SaoS-2 cells. TSH also promotes Runx2 responsive genes such as ALP, Collagen and osteocalcin in SaOS2 cells on day 2 to day 14 of 10 ng/mL of treatment and favors' matrix mineralization matrix in these cells. In addition, TSH facilitated human osteoblastic cells to mineralize their matrix confirmed by day 21 of alizarin red calcium staining. In silico study was performed to check CREB and ELK1 interaction with Runx2. Results of in silico analysis showed that TSH mediated signalling molecules such as CREB and ELK1 showed interaction with Runx2 which involve in osteobalstic gene expression and differentiation. Present findings confirm that TSH promotes Runx2 expression, osteoblastic responsive genes and bone matrix formation.

Clinical Importance of Bone Matrix Damage Mechanisms for Fracture Prevention.

PURPOSE OF REVIEW: Bone matrix exhibits great complexity in its composition, structure and mechanics. Here, we provide a review of recent research articles and appraise the evidence that bone matrix quality is clinically important and possibly targetable for fracture prevention. RECENT FINDINGS: Deformation of mineralised collagen fibrils determines bone fracture mechanics. Slipping and separation at the mineral-fibril and fibril-fibril interfaces, respectively, are the structural mechanisms for plastic deformation and microcrack nucleation. Existing technologies for assessing bone tissue in vivo cannot measure matrix structure or fracture mechanics but have shown limited use in clinical settings for identifying fragility or following treatment outcomes based on composition. Matrix is biomechanically and clinically important, but the knowledge has not translated into clinical practice. The structural mechanisms by which a load is transferred from mineralised collagen fibrils to the whole bone via microcracking have been proven too complex to measure in vivo. The mineral-fibril or fibril-fibril interfaces might be suitable targets for diagnosing fragility or delivering molecules that reduce fracture risk by strengthening the mineral bonds while maintaining flexibility in the fibrils.

In Vivo Biological Behavior of Polymer Scaffolds of Natural Origin in the Bone Repair Process.

Autologous bone grafts, used mainly in extensive bone loss, are considered the gold standard treatment in regenerative medicine, but still have limitations mainly in relation to the amount of bone available, donor area, morbidity and creation of additional surgical area. This fact encourages tissue engineering in relation to the need to develop new biomaterials, from sources other than the individual himself. Therefore, the present study aimed to investigate the effects of an elastin and collagen matrix on the bone repair process in critical size defects in rat calvaria. The animals (Wistar rats, n = 30) were submitted to a surgical procedure to create the bone defect and were divided into three groups: Control Group (CG, n = 10), defects filled with blood clot; E24/37 Group (E24/37, n = 10), defects filled with bovine elastin matrix hydrolyzed for 24 h at 37 °C and C24/25 Group (C24/25, n = 10), defects filled with porcine collagen matrix hydrolyzed for 24 h at 25 °C. Macroscopic and radiographic analyses demonstrated the absence of inflammatory signs and infection. Microtomographical 2D and 3D images showed centripetal bone growth and restricted margins of the bone defect. Histologically, the images confirmed the pattern of bone deposition at the margins of the remaining bone and without complete closure by bone tissue. In the morphometric analysis, the groups E24/37 and C24/25 (13.68 ± 1.44; 53.20 ± 4.47, respectively) showed statistically significant differences in relation to the CG (5.86 ± 2.87). It was concluded that the matrices used as scaffolds are biocompatible and increase the formation of new bone in a critical size defect, with greater formation in the polymer derived from the intestinal serous layer of porcine origin (C24/25).

Towards a Connectomic Description of the Osteocyte Lacunocanalicular Network in Bone.

PURPOSE OF REVIEW: Osteocytes are the most abundant bone cells. They are completely encased in mineralized tissue, sitting inside lacunae that are connected by a multitude of canaliculi. In recent years, the osteocyte network has been shown to fulfill endocrine functions and to communicate with a number of other organs. This review addresses emerging knowledge on the connectome of the lacunocanalicular network in different types of bone tissue. RECENT FINDINGS: Recent advances in three-dimensional imaging technology started to reveal parameters that are well known from general theory to characterize the function of networks, such as network density, degree of nodes, or shortest path length through the network. The connectome of the lacunocanalicular network differs in some aspects between lamellar and woven bone and seems to change with age. More research is needed to relate network structure to function, such as intercellular transport or communication and its role in mechanosensation, as well as to understand the effect of diseases.

Exosomes in Extracellular Matrix Bone Biology.

PURPOSE OF REVIEW: Exosomes are membrane vesicles that are released by most cell types into the extracellular environment. The purpose of this article is to discuss the main morphological features and contents of bone-derived exosomes, as well as their major isolation and physical characterization techniques. Furthermore, we present various scenarios and discuss potential clinical applications of bone-derived exosomes in bone repair and regeneration. RECENT FINDINGS: Exosomes were believed to be nanosized vesicles derived from the multivesicular body. Reports now suggest that nanovesicles could bud directly from the plasma membrane. However, the exosome cargo is cell-type specific and is derived from the parent cell. In the bone matrix, several intracellular proteins lacking a signal peptide are transported to the ECM by exosomes. Besides proteins, several mRNA, miRNA, and lipids are exported to the ECM by bone cells and bone marrow stromal cells. Recent evidence suggests that several of the functional components in the cargo could regulate processes of bone formation, inhibit osteoclast activity, and promote fracture repair. Exosomes are powerful cellular molecular machines produced without human intervention and packaged with physiological cargo that could be utilized for molecular therapy in several skeletal disorders such as osteoporosis, osteogenesis imperfecta, and fracture healing. Although much work has been done, there is a lot of information that is still unknown, as exosomes contain a multitude of molecules whose identity and function have yet to be identified.

In Vitro Impact of Conditioned Medium From Demineralized Freeze-Dried Bone on Human Umbilical Endothelial Cells.

Angiogenesis is essential for the consolidation of bone allografts. The underlying molecular mechanism, however, remains unclear. Soluble factors released from demineralized freeze-dried bone target mesenchymal cells; however, their effect on endothelial cells has not been investigated so far. The aim of the present study was therefore to examine the effect of conditioned medium from demineralized freeze-dried bone on human umbilical endothelial cells in vitro. Conditioned medium was first prepared from demineralized freeze-dried bone following 24 hours incubation at room temperature to produce demineralized bone conditioned media. Thereafter, conditioned medium was used to stimulate human umbilical vein endothelial cells in vitro by determining the cell response based on viability, proliferation, expression of apoptotic genes, a Boyden chamber to determine cell migration, and the formation of branches. The authors report here that conditioned medium decreased viability and proliferation of endothelial cells. Neither of the apoptotic marker genes was significantly altered when endothelial cells were exposed to conditioned medium. The Boyden chamber revealed that endothelial cells migrate toward conditioned medium. Moreover, conditioned medium moderately stimulated the formation of branches. These findings support the concept that conditioned medium from demineralized freeze-dried bone targets endothelial cells by decreasing their proliferation and enhancing their motility under these in vitro conditions.

Bone matrix mineralization is preserved during early perimenopausal stage in healthy women: a paired biopsy study.

UNLABELLED: Bone matrix mineralization based on quantitative backscatter electron imaging remained unchanged during the first year of menopause in paired transiliac biopsy samples from healthy women. This suggests that the reported early perimenopausal reductions in bone mineral density are caused by factors other than decreases in the degree of mineralization. INTRODUCTION: It is unknown whether perimenopausal loss of bone mass is associated with a drop in bone matrix mineralization. METHODS: For this purpose, we measured the bone mineralization density distribution (BMDD) by quantitative backscatter electron imaging (qBEI) in n = 17 paired transiliac bone biopsy samples at premenopausal baseline and 12 months after last menses (obtained at average ages of 49 ± 2 and 55 ± 2 years, respectively) in healthy women. For interpretation of BMDD outcomes, previously measured bone mineral density (BMD) and biochemical and histomorphometric markers of bone turnover were revisited for the present biopsy cohort. RESULTS: Menopause significantly decreased BMD at the lumbar spine (-4.5 %) and femoral neck (-3.8 %), increased the fasting urinary hydroxyproline/creatinine ratio (+60 %, all p < 0.01) and histomorphometric bone formation rate (+25 %, p < 0.05), but affected neither cancellous nor cortical BMDD variables (paired comparison p > 0.05). Mean calcium concentrations of cancellous (Cn.CaMean) and cortical bone (Ct.CaMean) were within normal range (p > 0.05 compared to established reference data). Ct.CaMean was significantly correlated with Cn.CaMean before (R = 0.81, p < 0.001) and after menopause (R = 0.80, p < 0.001) and to cortical porosity of mineralized tissue (Ct.Po.) after menopause (R = -0.57, p = 0.02). CONCLUSIONS: Surprisingly, the BMDD was found not affected by the changes in bone turnover rates in this cohort. This suggests that the substantial increase in bone formation rates took place shortly before the second biopsy, and the bone mineralization changes lag behind. We conclude that during the first year after the last menses, the degree of bone matrix mineralization is preserved and does not contribute to the observed reductions in BMD.

Bone Formation is Affected by Matrix Advanced Glycation End Products (AGEs) In Vivo.

Advanced glycation end products (AGEs) accumulate in bone extracellular matrix as people age. Although previous evidence shows that the accumulation of AGEs in bone matrix may impose significant effects on bone cells, the effect of matrix AGEs on bone formation in vivo is still poorly understood. To address this issue, this study used a unique rat model with autograft implant to investigate the in vivo response of bone formation to matrix AGEs. Fluorochrome biomarkers were sequentially injected into rats to label the dynamic bone formation in the presence of elevated levels of matrix AGEs. After sacrificing animals, dynamic histomorphometry was performed to determine mineral apposition rate (MAR), mineralized surface per bone surface (MS/BS), and bone formation rate (BFR). Finally, nanoindentation tests were performed to assess mechanical properties of newly formed bone tissues. The results showed that MAR, MS/BS, and BFR were significantly reduced in the vicinity of implant cores with high concentration of matrix AGEs, suggesting that bone formation activities by osteoblasts were suppressed in the presence of elevated matrix AGEs. In addition, MAR and BFR were found to be dependent on the surrounding environment of implant cores (i.e., cortical or trabecular tissues). Moreover, MS/BS and BFR were also dependent on how far the implant cores were away from the growth plate. These observations suggest that the effect of matrix AGEs on bone formation is dependent on the biological milieu around the implants. Finally, nanoindentation test results indicated that the indentation modulus and hardness of newly formed bone tissues were not affected by the presence of elevated matrix AGEs. In summary, high concentration of matrix AGEs may slow down the bone formation process in vivo, while imposing little effects on bone mineralization.

Osteogenic potential of in vitro osteo-induced adipose-derived mesenchymal stem cells combined with platelet-rich plasma in an ectopic model.

PURPOSE: The osteogenic potential of in vitro osteo-induced adipose-derived mesenchymal stem cells (ADSCs) combined with platelet-rich plasma (PRP) and implanted on bone mineral matrix (BMM) carrier was examined in a subcutaneous model in Balb/c mice. METHODS: In vitro osteogenic differentiation of ADSCs was monitored by relative bone-related gene expression and osteocalcin expression at the third, seventh and 15th day. Test implants consisting of in vitro osteo-induced ADSCs, PRP and BMM (OPC implants) and control implants consisting of PRP and BMM (PC implants) were examined. The relative expression of the bone-related genes encoding osterix, osteocalcin, collagen type I α1 and alkaline phosphatase was examined in implants extracted at one, two, four and eight weeks. Histochemical, immunohistochemical and histomorphometric analyses of implants extracted at two and eight weeks were performed. RESULTS: The highest relative expression of bone-related genes and osteocalcin expression was found at the 15th day of in vitro osteogenic induction of the ADSCs. Permanent and continuous increased expression of bone-related genes was noticed in OPC implants at eight weeks. Expression peaks of bone-related genes in PC implants were at two and four weeks, but they significantly decreased at eight weeks. The signs of resorption, formation of callus-like tissue positive for osteocalcin and increased presence of bone cells were found in OPC implants compared with PC implants. A higher percentage of infiltrated tissue and vascularisation was found in OPC than in PC implants. CONCLUSIONS: The combination of in vitro osteo-induced ADSCs and PRP on BMM carrier represents a promising approach for bone regeneration.

Evaluation of Osteogenic Potentials of Avian Demineralized Bone Matrix in the Healing of Osseous Defects in Pigeons.

OBJECTIVES: To evaluate avian allogeneic demineralized bone matrix (DBM) in the healing of long bone defects as a function of geometry and time in a pigeon model. STUDY DESIGN: Experimental. ANIMALS: Adult rock pigeons (n = 60). METHODS: Midshaft ulnar osseous defects were grafted with 2 geometric forms of DBM (tubular vs. chipped) and stabilized with a hybrid fixator. Autologous chips of sternal keel were used in a third group as control. Outcomes were evaluated by radiography and histology/histomorphometry at 4, 8, 12, and 24 weeks postoperatively. RESULTS: Despite an early rapid healing response, autografts plateaued (histologic score and new bone area) by 8 weeks with no significant improvement afterwards. Conversely, allogeneic DBM implants demonstrated continuous temporal improvement in bone healing, and tubular DBM finally outpaced autograft implants after week 12 with values for metrics achieving statistical significance by week 24. Chip DBM was inferior to tubular DBM and autograft. CONCLUSIONS: Avian DBM is osteogenic, biocompatible, and safe in orthotopic sites with potential usefulness in avian bone grafting. Implant geometry (shape and size) affects such osteogenic potentials.

The use of demineralized bone matrix for anterior cruciate ligament reconstruction: a radiographic, histologic, and immunohistochemical study in rabbits.

Tendon-bone healing is crucial in success of anterior cruciate ligament (ACL) reconstruction surgery. Demineralized bone matrix (DBM) is a physiological component that has the inherent potential of bone regeneration. We hypothesized that the alternative bone substitute can affect the structural properties of tendon graft in tibial tunnel healing. Five 12-week-old New Zealand white rabbits in study group underwent unilateral ACL reconstructions plus the application of 0.5 cc DBM in the tibial tunnel. The assessment included radiological assessment and histologic and immunohistochemical analyses. Radiological examination revealed that DBM group had the least displacement of tendon in tibial tunnel (0.4 ± 0.12; P = 0.03). Histologic examination showed significantly better integration between tendon and bone in DBM group (77.62 ± 2.08; P = 0.001). On immunohistochemical analysis, the DBM group showed significantly higher expressions of bone morphogenetic protein-2 and vascular endothelial growth factor than control group (51.98 ± 3.02, 84.06 ± 1.86; P = 0.001, P < 0.001). DBM enhances the tendon-bone healing in ACL reconstruction. DBM has the potential use in ACL surgery.

Preparation and characterization of an advanced medical device for bone regeneration.

Tridimensional scaffolds can promote bone regeneration as a framework supporting the migration of cells from the surrounding tissue into the damaged tissue and as delivery systems for the controlled or prolonged release of cells, genes, and growth factors. The goal of the work was to obtain an advanced medical device for bone regeneration through coating a decellularized and deproteinized bone matrix of bovine origin with a biodegradable, biocompatible polymer, to improve the cell engraftment on the bone graft. The coating protocol was studied and set up to obtain a continuous and homogeneous polylactide-co-glycolide (PLGA) coating on the deproteinized bone matrix Orthoss® block without occluding pores and decreasing the scaffold porosity. The PLGA-coated scaffolds were characterized for their morphology and porosity. The effects of PLGA polymer coating on cell viability were assessed with the 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium assay. The polymer solution concentration and the number of polymeric layers were the main variables affecting coating efficiency and porosity of the original decellularized bone matrix. The designed polymer coating protocol did not affect the trabecular structure of the original decellularized bone matrix. The PLGA-coated decellularized bone matrix maintained the structural features, and it improved the ability in stimulating fibroblasts attachment and proliferation.

Cavities in the compact bone in tetrapods and fish and their effect on mechanical properties.

Bone includes cavities in various length scales, from nanoporosities occurring between the collagen fibrils and the mineral crystals all the way to macrocavities like the medullary cavity. In particular, bone is permeated by a vast number of channels (the lacunar-canalicular system), that reduce the stiffness and, more importantly, the strength of the bone that they permeate. These consequences are presumably a price worth paying for the ability of the lacunar-canalicular system to detect changes in the strain environment within the bone material and, when deleterious, to trigger processes like modeling or remodeling which 'rectify' it. Here we review the size and density of the various types of cavities in bone, and discuss their effect on the mechanical properties of cortical bone. In this respect the bones of advanced teleost fish species (probably the majority of all vertebrate species) are an unsolved conundrum because they lack bone cells (and therefore lacunae and canaliculi) in their skeleton. Yet, despite being acellular, some of these fish can undergo considerable remodeling in at least some parts of their skeleton. We address, but do not solve this mystery.

Of mice, rats and men: trabecular bone architecture in mammals scales to body mass with negative allometry.

Body mass (BM) in mammal species spans over six orders of magnitude. Although trabecular bone contributes to the mechanical properties of bones, we know much less about how trabecular bone scales with BM than about how cortical bone scales with BM. We therefore conducted a meta-analysis of the existing literature to test in rodents, humans and other mammals, predicted scaling properties between BM and several trabecular parameters: bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density (ConnD) and degree of anisotropy (DA). Our results show that BV/TV and DA are independent of BM and that Tb.N, Tb.Th and Tb.Sp scale with negative allometry relative to BM. Rodents appear to have relatively thicker and fewer trabeculae than humans, and we propose it that is due to a minimum thickness threshold "imposed" on mechanically functional trabeculae. Consequently, rodents (mice and rats) and humans demonstrate two distinct mechanisms to achieve variations in BV/TV. Although Tb.Th variation is the main contributing factor for differences in BV/TV in humans, Tb.N variation is the main contributing factor for differences in BV/TV in rodents. Our results also demonstrate no correlation between Tb.N and Tb.Th within each taxon (mice, rats and humans). Since rodents are a common animal model for research on bone biomechanics, the evidence that trabecular bone parameters scale and correlate differently in rodents than in humans suggests that care should be applied when extrapolating bone biomechanical results from small animals to large-bodied humans.

Evolution of the vertebrate bone matrix: an expression analysis of the network forming collagen paralogues in amphibian osteoblasts.

The emergence of vertebrates is closely associated to the evolution of mineralized bone tissue. However, the molecular basis underlying the origin and subsequent diversification of the skeletal mineralized matrix is still poorly understood. One efficient way to tackle this issue is to compare the expression, between vertebrate species, of osteoblastic genes coding for bone matrix proteins. In this work, we have focused on the evolution of the network forming collagen family which contains the Col8a1, Col8a2, and Col10a1 genes. Both phylogeny and synteny reveal that these three paralogues are vertebrate-specific and derive from two independent duplications in the vertebrate lineage. To shed light on the evolution of this family, we have analyzed the osteoblastic expression of the network forming collagens in endochondral and intramembraneous skeletal elements of the amphibian Xenopus tropicalis. Remarkably, we find that amphibian osteoblasts express Col10a1, a gene strongly expressed in osteoblasts in actinopterygians but not in amniotes. In addition, while Col8a1 is known to be robustly expressed in mammalian osteoblasts, the expression levels of its amphibian orthologue are dramatically reduced. Our work reveals that while a skeletal expression of network forming collagen members is widespread throughout vertebrates, osteoblasts from divergent vertebrate lineages express different combinations of network forming collagen paralogues.

Osteoclasts on bone and dentin in vitro: mechanism of trail formation and comparison of resorption behavior.

The main function of osteoclasts in vivo is the resorption of bone matrix, leaving behind typical resorption traces consisting of pits and trails. The mechanism of pit formation is well described, but less is known about trail formation. Pit-forming osteoclasts possess round actin rings. In this study we show that trail-forming osteoclasts have crescent-shaped actin rings and provide a model that describes the detailed mechanism. To generate a trail, the actin ring of the resorption organelle attaches with one side outside the existing trail margin. The other side of the ring attaches to the wall inside the trail, thus sealing that narrow part to be resorbed next (3­21 lm). This 3D configuration allows vertical resorption layer-by-layer from the surface to a depth in combination with horizontal cell movement. Thus, trails are not just traces of a horizontal translation of osteoclasts during resorption. Additionally, we compared osteoclastic resorption on bone and dentin since the latter is the most frequently used in vitro model and data are extrapolated to bone. Histomorphometric analyses revealed a material-dependent effect reflected by an 11-fold higher resorption area and a sevenfold higher number of pits per square centimeter on dentin compared to bone. An important material-independent aspect was reflected by comparable mean pit area (µm²) and podosome patterns. Hence, dentin promotes the generation of resorbing osteoclasts, but once resorption has started, it proceeds independently of material properties. Thus, dentin is a suitable model substrate for data acquisition as long as osteoclast generation is not part of the analyses.

Calcium regulates key components of vascular smooth muscle cell-derived matrix vesicles to enhance mineralization.

RATIONALE: Matrix vesicles (MVs) are specialized structures that initiate mineral nucleation during physiological skeletogenesis. Similar vesicular structures are deposited at sites of pathological vascular calcification, and studies in vitro have shown that elevated levels of extracellular calcium (Ca) can induce mineralization of vascular smooth muscle cell (VSMC)-derived MVs. OBJECTIVES: To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress. METHODS AND RESULTS: Transmission electron microscopy showed that both nonmineralized and mineralized MVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites. CONCLUSIONS: In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes.

Histological remodelling of demineralised bone matrix allograft in posterolateral fusion of the spine--an ex vivo study.

BACKGROUND: Demineralised bone matrix (DBM) has shown to be effective in enhancing posterior fusion of the spine. Several animal studies and clinical investigations in humans showed its successful remodelling. The use of allogenic matrix may decrease the need of autologous bone graft and therefore helps prevent corresponding donor site morbidity. Since DBM products are very expensive, the question arises, whether it is completely remodelled into new bone, and therefore truly is comparable to autologous cancellous bone graft. To our knowledge there is no report of a consecutive series of patients where ex vivo histological analysis after postero-lateral fusion of the spine was performed. METHODS: Osseous biopsies of nine consecutive patients who underwent postero-lateral fusion of the spine for trauma were obtained at the time of elective removal of the hardware. Histological samples were then analyzed on ground and thin sections stained with toluidine blue and von Kossa stainings. RESULTS: Time span between index operation and removal of the metal ranged between 6 and 18 month. Histological analysis showed good incorporation and overall remodelling of DBM into new bone in all patients. No foreign body reaction was visible and new bone formation progressed time dependently with DBM in situ. Four out of nine patients showed more than 50% new bone formation after one year. CONCLUSION: DBM shows good overall remodelling properties in histological analysis and therefore seems to be an effective adjunct in postero-lateral fusion of the spine. Furthermore, DBM substitution increases over time.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral.

OBJECTIVES: The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS: Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS: EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION: The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE: The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.

Prevascularisation with endothelial progenitor cells improved restoration of the architectural and functional properties of newly formed bone for bone reconstruction.

PURPOSE: The aim of this study was to examine whether the addition of endothelial progenitor cells (EPCs) contributes to restoring the architectural and functional properties of newly formed bone for reconstruction of bone defects. METHODS: Bone marrow-derived EPCs and mesenchymal stem cells (MSCs) were co-seeded onto demineralized bone matrix (DBM) as a prevascularized tissue-engineered bone (TEB) for the repair of segmental bone defects to evaluate the effects of prevascularization of TEB on ameliorating morphological, haemodynamic and mechanical characteristics. RESULTS: The restoration of the intraosseous vasculature and medullary cavity was improved markedly compared to the non-prevascularized groups. The blood supply, biomechanical strength, and bone mineral density of the prevascularized group were significantly higher than those of the non-prevascularized groups during bone reconstruction. CONCLUSIONS: The present study indicates that EPC-dependent prevascularization contributes to bone healing with structural reconstruction and functional recovery and may improve the understanding of correlation between angiogenesis and osteogenesis.