Hypoxia-cultured mouse mesenchymal stromal cells from bone marrow and compact bone display different phenotypic traits.

It has been suggested that the bone marrow microenvironment harbors two distinct populations of mesenchymal stromal cells (MSC), one with a perivascular location and other present in the endosteum. A better understanding of the biology of these MSC subsets has been pursued in order to refine its clinical application. However, most comparative characterizations of mouse MSC have been performed in normoxia. This can result in misleading interpretations since mouse MSC subsets with low/defective p53 activity are known to be selected during culture in normoxia. Here, we report a comprehensive in vitro characterization of mouse MSC isolated from bone marrow (BM-MSC) and compact bone (CB-MSC) expanded and assayed under hypoxia for their morphology, clonogenic efficiency and differentiation capacity. We found that, under hypoxia, compact bone is richer in absolute numbers of MSC and isolation of MSC from compact bone is associated with a reduced risk of hematopoietic cell carryover. In addition, CB-MSC have higher in vitro osteogenic capacity than BM-MSC, while adipogenic differentiation potential is similar. These findings reinforce the hypothesis of the existence of MSC in bone marrow and compact bone representing functionally distinct cell populations and highlight the compact bone as an efficient source of murine MSC under physiological oxygen concentrations.

Effect of gamma rays and accelerated electron beam on medullary lipids decomposition: influence of dose and irradiation temperature.

Ionizing radiation sterilization of non-defatted bone grafts has been found to deteriorate their quality and biocompatibility due to induction of lipid peroxidation products toxic for osteoblast-like cells. Therefore, the purpose of our study was to evaluate the effect of two types of ionizing radiation-gamma rays (G) or accelerated electron beam (EB) applied with two doses at different temperature conditions on hydrocarbons production, resulting from decomposition of palmitic and oleic acids-most abundant fatty acids in medullary lipids. Bone marrow samples isolated from femoral shafts of 6 male donors (aged 46-67 years) were irradiated with G or EB with doses of 25 or 35 kGy at different temperature conditions (ambient or deep freezing temperature). Fresh-frozen, non-irradiated samples served as control. Marrow lipids were extracted with n-hexane (Soxhlet's method), hydrocarbons fraction isolated on Florisil column chromatography, separated by gas chromatography and detected by mass spectrometry. Irradiation of bone marrow with sterilization doses of ionizing radiation (G and EB) was found to induce lipid radiolysis as measured by resulting hydrocarbons production. The effect was dose-dependent, whereas no marked influence of radiation type was observed. In contrast, irradiation temperature had a profound effect on lipids decomposition which was partially prevented while irradiation was performed in deep frozen state. Defatting of bone grafts prior to ionizing radiation sterilization seems essential for their biocompatibility, whereas irradiation in a deep-frozen state might compromise the effectiveness of sterilization and needs further studies.

Pax2 is essential for proliferation and osteogenic differentiation of mouse mesenchymal stem cells via Runx2.

Mesenchymal stem cells (MSCs) have been widely studied in the field of regenerative medicine with the potential to solve osteoporosis. Paired box 2 (Pax2), as a transcription factor, is the master regulator of embryogenesis and oncogenesis. However, the function of Pax2 in osteogenesis is unknown. Here, we reported for the first time that the expression of Pax2 gradually increased during osteogenic differentiation of mouse MSCs, and osteoprogenitor cells. However, detected in osteoblastic cells of mouse tibia, the expression of Pax2 in the embryonic stage was higher than that in adulthood. In C3H/10/T1/2 cells and compact bone-derived mouse MSCs (mMSCs), Pax2 knock-down inhibited the proliferation of these cells, down-regulated the expression of osteogenic marker genes, as well as repressed the ALP activity and mineralization. In addition, Pax2 enhanced the transcriptional activity of Runx2, and activated the MAPK pathway genes (ERK, JNK and p38). Furthermore, knock-down of Pax2 repressed the mMSCs-mediated bone regeneration in an ectopic bone formation model. In conclusion, Pax2 promotes osteogenesis of mouse MSCs, suggesting that Pax2 has a role in the pathophysiology of bone related diseases, and has potential application in bone tissue regeneration.

DNA preservation in compact and trabecular bone.

Significant variation exists in the molecular structure of compact and trabecular bone. In compact bone full dissolution of the bone powder is required to efficiently release the DNA from hydroxyapatite. In trabecular bone where soft tissues are preserved, we assume that full dissolution of the bone powder is not required to release the DNA from collagen. To investigate this issue, research was performed on 45 Second World War diaphysis (compact bone)-epiphysis (trabecular bone) femur pairs, each processed with a full dissolution (FD) and partial dissolution (PD) extraction method. DNA quality and quantity were assessed using qPCR PowerQuant analyses, and autosomal STRs were typed to confirm the authenticity of isolated DNA. Our results support different mechanisms of DNA preservation in compact and trabecular bone because FD method was more efficient than PD method only in compact bone, and no difference in DNA yield was observed in trabecular bone, showing no need for full dissolution of the bone powder when trabecular bone tissue is processed. In addition, a significant difference in DNA yield was observed between compact and trabecular bone when PD was applied, with more DNA extracted from trabecular bone than compact bone. High suitability of trabecular bone processed with PD method is also supported by the similar quantities of DNA isolated by FD method when applied to both compact and trabecular bone. Additionally similar quantities of DNA were isolated when compact bone was extracted with FD method and trabecular bone was extracted with PD method. Processing trabecular bone with PD method in routine identification of skeletonized human remains shortens the extraction procedure and simplifies the grinding process.

Multiscale engineered artificial compact bone via bidirectional freeze-driven lamellated organization of mineralized collagen microfibrils.

Bone, renowned for its elegant hierarchical structure and unique mechanical properties, serves as a constant source of inspiration for the development of synthetic materials. However, achieving accurate replication of bone features in artificial materials with remarkable structural and mechanical similarity remains a significant challenge. In this study, we employed a cascade of continuous fabrication processes, including biomimetic mineralization of collagen, bidirectional freeze-casting, and pressure-driven fusion, to successfully fabricate a macroscopic bulk material known as artificial compact bone (ACB). The ACB material closely replicates the composition, hierarchical structures, and mechanical properties of natural bone. It demonstrates a lamellated alignment of mineralized collagen (MC) microfibrils, similar to those found in natural bone. Moreover, the ACB exhibits a similar high mineral content (70.9 %) and density (2.2 g/cm3) as natural cortical bone, leading to exceptional mechanical properties such as high stiffness, hardness, and flexural strength that are comparable to those of natural bone. Importantly, the ACB also demonstrates excellent mechanical properties in wet, outstanding biocompatibility, and osteogenic properties in vivo, rendering it suitable for a broad spectrum of biomedical applications, including orthopedic, stomatological, and craniofacial surgeries.

The biomechanical and biological effect of supercooling on cortical bone allograft.

BACKGROUND: The need for a storage method capable of preserving the intrinsic properties of bones without using toxic substances has always been raised. Supercooling is a relatively recently introduced preservation method that meets this need. Supercooling refers to the phenomenon of liquid in which the temperature drops below its freezing point without solidifying or crystallizing. OBJECTIVES: The purpose of this study was to identify the preservation efficiency and applicability of the supercooling technique as a cortical bone allograft storage modality. METHODS: The biomechanical effects of various storage methods, including deep freezing, cryopreservation, lyophilization, glycerol preservation, and supercooling, were evaluated with the three-point banding test, axial compression test, and electron microscopy. Additionally, cortical bone allografts were applied to the radial bone defect in New Zealand White rabbits to determine the biological effects. The degree of bone union was assessed with postoperative clinical signs, radiography, micro-computed tomography, and biomechanical analysis. RESULTS: The biomechanical properties of cortical bone grafts preserved using glycerol and supercooling method were found to be comparable to those of normal bone while also significantly stronger than deep-frozen, cryopreserved, and lyophilized bone grafts. Preclinical research performed in rabbit radial defect models revealed that supercooled and glycerol-preserved bone allografts exhibited significantly better bone union than other groups. CONCLUSIONS: Considering the biomechanical and biological superiority, the supercooling technique could be one of the optimal preservation methods for cortical bone allografts. This study will form the basis for a novel application of supercooling as a bone material preservation technique.

Multiscale theoretical model shows that aging-related mechanical degradation of cortical bone is driven by microstructural changes in addition to porosity.

This study aims to gain mechanistic understanding of how aging-related changes in the microstructure of cortical bone drive mechanical consequences at the macroscale. To that end, cortical bone was modeled as a bundle of elastic-plastic, parallel fibers, which represented osteons and interstitial tissue, loaded in uniaxial tension. Distinct material properties were assigned to each fiber in either the osteon or interstitial fiber "families." Models representative of mature (20-60 yrs.) bone, and elderly (60+) bone were created by modeling aging via the following changes to the input parameters: (i) increasing porosity from 5% to 15%, (ii) increasing the ratio of the number of osteon fibers relative to interstitial fibers from 40% to 50%, and (iii) changing the fiber material properties from representing mature bone samples to representing elderly bone samples (i.e., increased strength and decreased toughness of interstitial fibers together with decreased toughness of osteon fibers). To understand the respective contributions of these changes, additional models isolating one or two of each of these were also created. From the computed stress-strain curve for the fiber bundle, the yield point (ϵy, σy), ultimate point (ϵu, σu), and toughness (UT) for the bundle as a whole were measured. We found that changes to all three input parameters were required for the model to capture the aging-related decline in cortical bone mechanical properties consistent with those previously reported in the literature. In both mature and elderly bundles, rupture of the interstitial fibers drove the initial loss of strength following the ultimate point. Plasticity and more gradual rupture of the osteons drove the remainder of the response. Both the onset and completion of interstitial fiber rupture occurred at lower strains in the elderly vs. mature case. These findings point to the importance of studying microstructural changes beyond porosity, such as the area fraction of osteons and the material properties of osteon and interstitial tissue, in order to further understanding of aging-related changes in bone.

Extended Finite Element models of introcortical porosity and heterogeneity in cortical bone.

Due to changes in the bone quality during ageing the fracture risk increases. The influence of the different parameters affecting bone quality is not well understood. The Finite Element method offers the opportunity to determine the individual contribution of a parameter by changing single parameters. In this study, the ABAQUS extended Finite Elements Method (xFEM) was applied to simulate the crack propagation in compact bone samples using the quadratic nominal stress as crack criterion. Micro computed tomography images of compact-tension samples machined from a 19 and an 81 years old donor were used to generate Finite Element meshes consisting of linear tetrahedrons via Mimics. Cavities were modelled only in the estimated crack area to avoid a high number of degrees of freedom. Crack area was meshed with a higher number of smaller elements. The other areas were meshed with a small number of larger elements. The changes in the material constants due to the simplification of the model were taken into account by using effective material parameters in these partitions. Our results show that age-related loss in bone toughness results from increased porosity and loss in heterogeneity of material level properties.

Strength-fracture toughness synergy strategy in ostrich tibia's compact bone: Hierarchical and gradient.

Ostriches are the fastest bipeds in the world, but their tibias are very thin. How the thin tibia can withstand the huge momentum impacts of the heavy body during running? The present work revealed that the combination of hierarchical and gradient design strategies was the main reason for their high strength and fracture toughness. The microstructure of ostrich's tibias compact bone was self-assembled into the 6-level hierarchical structure from the hydroxyapatite (HAP) crystals, collagen fiber (sub-nano), mineralized collagen fiber (nano-), mineralized collagen fiber bundle (sub-micro), lamellae (micro-) and osteon (macro-scales). The most distinctive design in the ostrich compact bone was that the HAP crystals were embedded in collagen fibers as well as wrapped in the outer layer of mineral collagen fibers (MCFs) in the form of HAP nanocrystals, thus achieving a high degree of soft and hard combination from the nanoscale. The bending strength was gradient-structure dependent and up to 787.2 ± 40.5 MPa, 4 times that of a human's compact bone. The fracture toughness (KJc) is 5.8 ± 0.1 MPa m1/2. Several toughening mechanisms, such as crack deflection/twist, bridging, HAP fibers pulling-out, and fracture of the MCF bundles were found in the compact bone.

Ontogenetic growth and the development of a unique fibrocartilage entheses in Macropus fuliginosus.

Here we examine the bone histology of the femora and humeri of the Western Grey Kangaroo, Macropus fuliginosus. Our results reveal that bone modelling in response to ontogenetic growth and the development of tuberosities on the femur, and especially in the humerus, lead to a highly complex histology. We propose that the alternating fast and slow rates of bone deposition are seasonal, and are likely correlated with heterothermy related to ecological constraints during the summer months. In females, after the fourth growth mark in the femur, there is a distinctive change to a more lamellar textured bone deposition with sparse vascularisation, directly indicating a slowdown in growth. However, in males, the zones remain woven textured and well vascularised, which is indicative of continued fast growth. Here we also report the novel occurrence of a fibrocartilaginous entheses for the attachment of the m. quadratus femoris to the caudal femoral tuberosity. Using a combination of methodologies, we show that perimeter measurements of growth marks provide a reasonable estimation of the age of kangaroos. Additionally, we observed large individuals that have ceased diaphyseal appositional growth of the femur and the humerus, as well as fusion of the distal epiphyses of both bones, though the proximal epiphyses may remain unfused.

Accelerated Bone Induction of Adult Rat Compact Bone Plate Scratched by Ultrasonic Scaler Using Acidic Electrolyzed Water.

Fresh compact bone, the candidate graft material for bone regeneration, is usually grafted for horizontal bone augmentation. However, the dense calcified structure inhibits the release of growth factors and limits cellular and vascular perfusion. We aimed to create mechano-chemically altered dense skull bone by ultrasonic treatment, along with partial demineralization using commercially available acidic electrolyzed water (AEW). The parietal skull bone of an 11-month-old Wistar rat was exposed and continuously treated with a piezoelectric ultrasonic scaler tip for 1 min, using AEW (pH 2.3) or distilled water (DW, pH 5.6) as irrigants. Treated parietal bone was removed, cut into plates (5 × 5 × 1 mm3), grafted into the back subcutaneous tissues of syngeneic rats, and explanted at 1, 2, and 3 weeks. AEW bone showed an irregular surface, deep nano-microcracks, and decalcified areas. SEM-EDS revealed small amounts of residual calcium content in the AEW bone (0.03%) compared to the DW bone (0.86%). In the animal assay, the AEW bone induced bone at 2 weeks. Histomorphometric analysis showed that the area of new bone in the AEW bone at 2 and 3 weeks was significantly larger. This new combination technique of AEW-demineralization with ultrasonic treatment will improve the surface area and three-dimensional (3D) architecture of dense bone and accelerate new bone synthesis.

Cryopreserved Spontaneous Spheroids from Compact Bone-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering.

Spontaneously formed spheroids from mouse compact bone-derived mesenchymal stromal cells (CB-MSCs) possess enhanced stemness and superior plasticity. In this study, the effect of cryopreservation on viability, stemness, and osteogenic differentiation capability of spontaneous CB-MSC spheroids were investigated. CB-MSCs were isolated from mouse femur and tibia. Spheroids were cryopreserved with various concentrations of dimethyl sulfoxide (DMSO). After thawing, the number of living and dead cells was measured. The expression levels of stem cell markers and osteogenic marker genes were analyzed. The cryopreserved and noncryopreserved spheroids were transplanted in mice with a beta-tricalcium phosphate as a scaffold to evaluate the in vivo bone-forming capability. The percentage of living cells was highest when 5% DMSO was used as a cryoprotectant, confirmed by the number of dead cells. The expression of stem cell marker genes and osteogenic differentiation capability were maintained after cryopreservation with 5% DMSO. The cryopreserved spontaneous CB-MSC spheroids showed remarkable new bone formation in vivo, identical to that of the noncryopreserved spheroids even without osteogenic induction. The cryopreserved spontaneous CB-MSC spheroids retained stemness and osteogenic differentiation capability and highlight the utility of spontaneous CB-MSC spheroids as ready-to-use tissue-engineered products for bone tissue engineering.

Compact osteoma of the maxilla: A rare case report.

Osteomas of the facial bones are a rare entity and very few cases have been reported in the literature. Osteoma is a benign osteogenic lesion with a very slow growth, characterized by proliferation of either cancellous or compact bone. This paper describes a case of a 27 year old male seeking treatment for a slowly enlarging lesion in the maxillary right anterior region. Surgical excision of the lesion was done and the histopathologic evaluation revealed dense compact bone with osteocytes in the lacunae suggestive of compact osteoma. One year followup showed no evidence of recurrence. To best of our knowledge this is the twelfth case of maxillary osteoma reported in English language literature.

Isolation and characterization of mesenchymal stem cells in orthopaedics and the emergence of compact bone mesenchymal stem cells as a promising surgical adjunct.

The potential clinical and economic impact of mesenchymal stem cell (MSC) therapy is immense. MSCs act through multiple pathways: (1) as "trophic" cells, secreting various factors that are immunomodulatory, anti-inflammatory, anti-apoptotic, proangiogenic, proliferative, and chemoattractive; (2) in conjunction with cells native to the tissue they reside in to enhance differentiation of surrounding cells to facilitate tissue regrowth. Researchers have developed methods for the extraction and expansion of MSCs from animal and human tissues. While many sources of MSCs exist, including adipose tissue and iliac crest bone graft, compact bone (CB) MSCs have shown great potential for use in orthopaedic surgery. CB MSCs exert powerful immunomodulatory effects in addition to demonstrating excellent regenerative capacity for use in filling boney defects. CB MSCs have been shown to have enhanced response to hypoxic conditions when compared with other forms of MSCs. More work is needed to continue to characterize the potential applications for CB MSCs in orthopaedic trauma.

The impact of burial period on compact bone microstructure: Histological analysis of matrix loss and cell integrity in human bones exhumed from tropical soil.

Human bone histological analysis is a useful tool to assess post mortem diagenesis and to predict successful nuclear DNA typing of forensic material. This study is part of a series of studies developed by the authors intended to improve the understanding of post mortem diagenesis and to develop applications for DNA analysis of skeletal species from tropical soils, in order to optimize genetic and anthropological protocols. The aim of this study was to analyze the impact of burial period on the integrity of exhumed compact bone microstructure from tropical climate. In fragments of exhumed human femora from 39 individuals from the same cemetery (exhumed group) and 5 fresh femora from routine autopsies (control group), sections stained by hematoxylin-eosin were analyzed in order to measure bone microstructural integrity. We found that bone integrity index in exhumed group was negatively influenced by the period of burial (r = -0.37, p < 0.05) and highly significantly decreased (p < 0.0001) in comparison to control group. The period of burial and nitric acid decalcification time was positively correlated (r = 0.51; p < 0.01), leading to imply a bone petrification process during inhumation. Exhumed group showed higher level of matrix bone loss (p < 0.001), as expected, and 87% of cases analyzed were "tunneled" as described by Hackett. Bone integrity index and bone matrix tend to decrease in bones buried in tropical soil between 8-14 years of inhumation. This period is short if we consider cases in which there are preserved bones interred for longer periods in other environments. These data must be considered in cases where genetic identification of exhumed skeletons from tropical environment is required. The diagenesis in these bones and the variations of results found are discussed, clarifying some challenges for forensic laboratories, especially in DNA analysis.

Early Visualization of Optic Canal for Safe Anterior Clinoidectomy: Operative Technique and Supporting Computed Tomography Findings.

BACKGROUND: Anterior clinoidectomy is considered difficult because important structures are adjacent to but hidden behind the anterior clinoid process (ACP). We sought to make anterior clinoidectomy a simpler procedure. METHODS: A modification of anterior clinoidectomy is presented. To obtain a radiologic basis, original images from 357 consecutive sets of computed tomography angiography data were assessed. RESULTS: Anterior clinoidectomy was performed in 77 consecutive patients, as follows. By making a small window in the lateral wall of the ACP and curetting out the content inside, the lateral wall of the optic canal was easily exposed and functioned as a landmark for removal of the remaining bone. In addition, by removing the posterior edge and thinning the lateral part, the ACP was fractured and removed. The optic canal was opened if needed. No complications due to anterior clinoidectomy were observed. Among the 689 sides evaluated, 641 sides showed cancellous bone and 29 showed well-developed pneumatization inside the ACP. In each ACP examined, we observed the presence of compact bone facing the optic nerve, which constitutes the lateral wall of the optic canal. On 180 sides, part of the compact bone between the clinoid process and the internal carotid artery was absent; in other cases, thin compact bone was present. CONCLUSIONS: Visualizing the optic canal in the early stage of the procedure allows anterior clinoidectomy to be performed safely.

Changes in the microstructure of compact and trabecular bone tissues of mice subchronically exposed to alcohol.

BACKGROUND: Alcohol is one of the most commonly consumed neurotoxins by humans. Its negative effect on bone health is known for a long time. However, its impact on qualitative and quantitative 2D characteristics of the compact bone is still unclear. Therefore, the aim of this study was to investigate in detail the effects of subchronic alcohol exposure on compact and trabecular bone tissues microstructure of laboratory mice using 2D and 3D imaging methods. Ten clinically healthy 12 weeks-old mice (males) were randomly divided into two groups. Animals from experimental group (group E; n = 5) drank a solution composed of 15% ethanol and water (1.7 g 100% ethanol kg-1 b.w. per day) for 8 weeks, while those from control group (group C; n = 5) drank only water. RESULTS: Subchronic exposure to alcohol leads to several changes in qualitative 2D characteristics of the compact bone such as the presence of primary vascular radial bone tissue in pars anterior of endosteal border and a higher number of resorption lacunae (five times more) in the middle part of substantia compacta. Morphometrical 2D evaluations of the compact bone showed significantly increased sizes of primary osteons' vascular canals (p < 0.05) in mice from the experimental group (E group). Sizes of Haversian canals and secondary osteons were not affected by alcohol consumption. In mice from the E group, significantly lower values for relative bone volume and bone mineral density of the compact bone were observed. In the trabecular bone, decreased values for bone volume, trabecular number, trabecular thickness and bone surface (p < 0.05) were documented. CONCLUSIONS: Alcohol decreased not only bone volume and density of the compact bone, but it also reduced trabecular bone volume and leads to trabecular thinning. It caused vasodilation of primary osteons' vascular canals and increased porosity in the compact bone.

Comparison of ground sections, paraffin sections and micro-CT imaging of bone from the epiphysis of the porcine femur for morphometric evaluation.

The aim of this study was to compare data on the volume fraction of bone and the thickness of the cortical compact bone acquired during microcomputed tomography (micro-CT) analysis with data acquired from identical samples using stereological analysis of either decalcified paraffin sections or ground sections. Additionally, we aimed to compare adjacent tissue samples taken from the major trochanter of the porcine femur to map the basic biological variability of trabecular bone. Fifteen pairs of adjacent tissue blocks were removed from the major trochanter of the proximal epiphyses of porcine femurs (female pigs aged 24-39 months, weight=59.16±8.15kg). In each sample, the volume of the cortical compact bone, the volume of the trabecular bone, and the thickness of the cortical compact bone was assessed using micro-CT. Afterwards, half of the samples were decalcified and processed using paraffin histological sections. Another half was processed into ground sections. The volume and thickness of bone was assessed in histological sections using stereological techniques. There were no significant differences in the bone volumes and thicknesses measured by micro-CT and the corresponding values quantified in decalcified sections. Similarly, there were no differences between the results from micro-CT and the analysis of the corresponding ground sections. Histomorphometric studies based on relatively low numbers of undecalcified ground sections or demineralized paraffin sections of bone yield data on bone volume and the thickness of cortical compact bone that is comparable with three-dimensional micro-CT examination. The pilot data on the variability of cortical compact bone and trabecular bone volumes in the porcine major trochanter provided in this study aim for planning experiments in the field of bone healing and implantology.

Trace and macro elements in the femoral bone as indicators of long-term environmental exposure to toxic metals in European brown bear (Ursus arctos) from Croatia.

We explored the long-term accumulation of aluminium, strontium, cadmium and lead in the compact and trabecular bone of the femoral epiphysis, metaphysis and diaphysis in 41 brown bears (Ursus arctos) from Croatia. Also, we assessed their influence on macro and trace elements (sodium, magnesium, phosphorus, potassium, calcium, manganese, iron, cobalt, copper, zinc and barium) in bears' bone. There were no sex differences in element levels in general, while age was associated with bone length and levels of all elements, except for cadmium. Elements had different levels depending on the part of the bone sampled. More pronounced differences were observed between the compact and trabecular regions, with higher levels of majority of elements found in compact bone. Moderate to high associations (Spearman coefficient, rS = 0.59-0.97) were confirmed between calcium and potassium, magnesium, phosphorus, manganese, cobalt, zinc, strontium and lead. Lead levels in the bone were below those known to cause adverse health effects, but in 4 of 41 animals they exceeded baseline levels for domestic animals. The femoral bone of the brown bear reflected the accumulative nature of lead and strontium well, as it did the impairment of bone-forming essential element levels associated with these two elements. However, the distribution pattern of elements along the bone was not uniform, so additional care should be taken when choosing on the part of the bone sampled.

Growth differentiation factor­5 induces tenomodulin expression via phosphorylation of p38 and promotes viability of murine mesenchymal stem cells from compact bone.

Growth differentiation factor (GDF)­5 serves a role in tissue development and tenomodulin serves an important role in the development of tendons. The effects of GDF­5 on mesenchymal stem cells (MSCs), particularly with regards to tendon bioengineering, are poorly understood. The present study aimed to investigate the effects of GDF­5 on cell viability and tenomodulin expression in MSCs from murine compact bone. MSCs were isolated from murine compact bones and confirmed by flow cytometric analysis. In addition, the adipogenic, osteoblastic and chondrocyte differentiation capabilities of the MSCs were determined. MSCs were treated with GDF­5 and the effects of GDF­5 on MSC viability were determined. The mRNA and protein expression levels of tenomodulin were detected by reverse transcription­quantitative polymerase chain reaction and western blotting, respectively. MSCs from murine compact bone were successfully isolated. GDF­5 had optimal effects on cell viability at 100 ng/ml (+36.9% of control group without GDF­5 treatment, P<0.01) and its effects peaked after 6 days of treatment (+56.6% of control group, P<0.001). Compared with the control group, treatment with 100 ng/ml GDF­5 for 4 days enhanced the mRNA expression levels of tenomodulin (3.56±0.94 vs. 1.02±0.25; P<0.05). In addition, p38 was activated by GDF­5, as determined by enhanced expression levels of phosphorylated p38 (p­p38). The GDF­5­induced protein expression levels of p­p38 and tenomodulin were markedly inhibited following treatment with SB203580, an inhibitor of p38 mitogen­activated protein kinase. These results suggested that GDF­5 treatment may increase tenomodulin protein expression via phosphorylation of p38 in MSCs from murine compact bone. These findings may aid the future development of tendon bioengineering.