Rosamultin Attenuates Acute Hypobaric Hypoxia-Induced Bone Injuries by Regulation of Sclerostin and Its Downstream Signals.

Wang, Xing-Min, Hui Liu, Jian-Yu Li, Jin-Xia Wei, Xia Li, Yong-Liang Zhang, Ling-Zhi Li, and Xi-Zheng Zhang. Rosamultin attenuates acute hypobaric hypoxia-induced bone injuries by regulation of sclerostin and its downstream signals. High Alt Med Biol. 21:273-286, 2020. Background: Rosamultin, one of the compounds extracted from Potentilla anserina L., exhibited significant pharmacological activity against oxidative stress and hypoxic injury in our previous study. However, the effect of rosamultin on bone damage induced by acute hypobaric hypoxia (HH) has not been thoroughly studied. Methods: In this study, we first investigated the protective effect of rosamultin against bone damage in rats following acute exposure to simulated high-altitude hypoxia. Furthermore, we explored the detailed mechanism involved in the regulation of rat bone remodeling by rosamultin in an acute HH environment through analysis of sclerostin expression and the regulation of downstream signaling pathways. Results: Pretreatment with rosamultin significantly reduced HH-induced oxidative stress and inflammation, improved bone metabolic abnormalities, and alleviated the imbalance in bone remodeling in rats exposed to acute HH. Rosamultin markedly downregulated the expression of sclerostin, activated the Wnt/ß-catenin signaling pathway, and enhanced the ratio of osteoprotegerin/receptor activator of nuclear factor kappa B ligand to maintain the balance of bone formation and resorption. Conclusions: Rosamultin attenuates acute HH-induced bone damage and improves abnormal bone remodeling in rats by inhibition of sclerostin expression and activation of the Wnt/ß-catenin signaling pathway.

Mechanical property and biocompatibility of silk fibroin-collagen type II composite membrane.

Osteoarthritis is caused by injuries and cartilage degeneration. Cartilage tissue engineering provides new ideas for the treatment of osteoarthritis. Herein, the different ratios composite membranes of silk fibroin/collagen type II were constructed (SF50-50:50, SF70-70:30, SF90-90:10). The surface properties of the composite membranes and chondrocyte morphology were observed by SEM (scanning electron microscopy). Physical functionality as well as stability of composite membranes was evaluated from tensile mechanical properties, the percentage of swelling and degradation. The tensile mechanical behavior of SF70 composite membranes was also predicted based on the constitutive model established in this study, and it is found that the experimental results and predictions were in good agreement. Biocompatibility was evaluated using chondrocytes (ADTC-5) culture. Cell proliferation was analyzed and the treatment of live/dead double staining was performed to assess the viability on chondrocytes. To sum up, SF70 showed the suitable morphology, physical stability, and biological functionality to promote proliferation of chondrocytes. This indicates that the mixing ratio of SF70 shows promise in the future as a scaffold material for cartilage repair.

Effects of constrained dynamic loading, CKIP­1 gene knockout and combination stimulations on bone loss caused by mechanical unloading.

Mechanical stimulation plays an important role in maintaining the growth and normal function of the skeletal system. Mechanical unloading occurs, for example, in astronauts spending long periods of time in space or in patients on prolonged bed rest, and causes a rapid loss of bone mass. Casein kinase 2­interacting protein­1 (CKIP­1) is a novel negative bone regulation factor that has been demonstrated to reduce bone loss and enhance bone formation. The aim of this study was to investigate the effect of constrained dynamic loading (Loading) in combination with CKIP­1 gene knockout (KO) on unloading­induced bone loss in tail­suspension mice. The blood serum metabolism index [alkaline phosphatase (ALP) activity and osteocalcin (OCN) levels], tibia mechanical behavior (including bone trabecular microstructure parameters and tibia biomechanical properties), osteoblast­related gene expression [ALP, OCN, collagen I and bone morphogenetic protein­2 and osteoprotegerin (OPG)] and osteoclast­related gene expression [receptor activators of NF­kB ligand (RANKL)] were measured. The results demonstrated that mice experienced a loss of bone mass after four weeks of tail suspension compared with a wild type group. The mechanical properties, microarchitecture and mRNA expression were significantly increased in mice after Loading + KO treatment (P<0.05). Furthermore, compared with loading or KO alone, the ratio of OPG/RANKL was increased in the combined treatment group. The combined effect of Loading + KO was greater than that observed with loading or KO alone (P<0.05). The present study demonstrates that Loading + KO can counter unloading­induced bone loss, and combining the two treatments has an additive effect. These results indicate that combined therapy could be a novel strategy for the clinical treatment of disuse osteoporosis associated with space travel or bed rest.

The Combination of icariin and constrained dynamic loading stimulation attenuates bone loss in ovariectomy-induced osteoporotic mice.

Osteoporosis is a disease characterized by low bone mass and progressive destruction of bone microstructure, resulting in increasing the risk of fracture. Icariin (ICA) as a phytoestrogen shows osteogenic effects, and the mechanical stimulation has been demonstrated the improving effect on osteoporosis. The objective of this study was to investigate the effect of ICA in combination with constrained dynamic loading (CDL) stimulation on osteoporosis in ovariectomized (OVX) mice. The serum hormone levels, bone turnover markers, trabecular architecture, ulnar biomechanical properties, and the expression of osteoblast-related gene (alkaline phosphatase, ALP; osteocalcin, OCN; bone morphogenetic protein-2, BMP-2; Collagen I (α1), COL1; osteoprotegerin, OPG) and osteoclast-related genes (receptor activators of NF-κB ligand, RANKL; tartrate-resistant acid phosphatase, TRAP) were analyzed. The results showed that ICA + CDL treatment could increase the osteocalcin (20.85%), estradiol levels (20.61%) and decrease the TRAP activity (26.27%) significantly than CDL treatment. The combined treatment attenuated bone loss and biomechanical decrease more than single use of CDL treatment. ICA + CDL treatment significantly up-regulated the level of osteoblast-related gene expression and down-regulated the osteoclast-related genes expression; moreover, the combined treatment increased the ratio of OPG/RANKL significantly compared to ICA (72.83%) or CDL (65.63%) treatment alone. The present study demonstrates that icariin in combination with constrained dynamic loading treatment may have a therapeutic advantage over constrained dynamic loading treatment alone for the treatment of osteoporosis, which would provide new evidence for the clinical treatment of osteoporosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1415-1424, 2018.

Ratcheting behavior of articular cartilage under cyclic unconfined compression.

The ratcheting deformation of articular cartilage can produce due to the repeated accumulations of compressive strain in cartilage. The aim of this study was to investigate the ratcheting behavior of articular cartilage under cyclic compression. A series of uniaxial cyclic compression tests were conducted for online soaked and unsoaked cartilage samples and the effects of stress variation and stress rate on ratcheting behavior of cartilage were investigated. It is found that the ratcheting strains of online soaked and unsoaked cartilage samples increase rapidly at initial stage and then show the slower increase with cyclic compression going on. On the contrary, the ratcheting strain rate decreases quickly at first and then exhibits a relatively stable and small value. Both the ratcheting strain and ratcheting strain rate increase with stress variation increasing or with stress rate decreasing. Simultaneously, the optimized digital image correlation (DIC) technique was applied to study the ratcheting behavior and Young's modulus of different layers for cartilage under cyclic compression. It is found that the ratcheting behavior of cartilage is dependent on its depth. The ratcheting strain and its rate decrease through the depth of cartilage from surface to deep, whereas the Young's modulus increases.

A comparative study of mechanical strain, icariin and combination stimulations on improving osteoinductive potential via NF-kappaB activation in osteoblast-like cells.

BACKGROUND: The combination of drugs and exercise was the effective treatment in bone injure and rebuilding in clinic. As mechanical strain has potential in inducing the differentiation of osteoblasts in our previous study, the further research to investigate the combination of mechanical strain and icariin stimulation on inducing osteoblast proliferation, differentiation and the possible mechanism in MC3T3-E1 cell line. METHODS: A whole cell enzyme-linked immunosorbent assay that detects the bromodeoxyuridine incorporation during DNA synthesis was applied to evaluate the proliferation. The mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), type I collagen (Col I), bone morphogenetic protein-2 (BMP-2) and BMP-4 was detected by real-time reverse-transcription polymerase chain reaction. The activity of ALP was analyzed by ELISA and the protein expression of OCN, Col I and BMP-2 was assessed by western blot. Moreover, the activity of nuclear transcription factor kappa-B (NF-κB) signaling pathway was investigated with the expression of inhibitor of κB (IκB) α, phosphorylation of IκB-α (P-IκB-α), p65, P-p65 by western blot. RESULTS: We observed that compared to single mechanical strain or icariin stimulation, the mRNA and protein expressions of ALP (P < 0.05 or P < 0.01), OCN (P < 0.01) and Col I (P < 0.05 or P < 0.01) were increased significantly by the combination of mechanical strain and icariin stimulation. Moreover, the combination of mechanical strain and icariin stimulation could up-regulate the expression of BMP-2 (P < 0.01) and BMP-4 compared to single mechanical strain or icariin stimulation. The combination of mechanical strain and icariin stimulation could activate NF-κB signaling pathway by increasing the expression of IκB α, P-IκB-α, p65, P-p65 (P < 0.01). CONCLUSION: The combination of mechanical strain and icariin stimulation could activate the NF-κB pathway to improve the proliferation, differentiation of osteoblast-like cells.

Reduction of the pro-inflammatory response by tetrandrine-loading poly(L-lactic acid) films in vitro and in vivo.

Inflammatory response of implantable biomaterials and drug delivery vehicles, driven by the reaction of macrophages to foreign body particles released from the implant, is an urgent problem to resolve. Despite this, little is known about the inflammatory molecular mechanism following the implantation of biomaterials and the evaluation of anti-inflammatory biomaterials. In this study, tetrandrine (TET) was loaded into poly (l-lactic acid) (PLLA) films to assess the anti-inflammatory effects in vitro and in vivo. The water contact angle measurement indicated the variation of hydrophilicity and the electron spectroscopy for chemical analysis (ESCA) data suggested that TET was loaded into PLLA films, which were marked as enriched with nitrogen atoms. TET-loading PLLA films had satisfactory sustained releasing behavior in salicylic acid solution with accelerating release. RAW 264.7 macrophages cultured in TET-loading PLLA films maintained lower levels of chemokines, cytokines, and enzymes involved in the inflammatory process, such as NO, TNF-α, IL-6, iNOS, COX-2 than control PLLA films, suggesting that TET-loading PLLA films could regulate the mRNA expression and protein expression to reduce the inflammatory response in macrophages. The degree of inflammatory reaction for the implant with the TET-loading PLLA films was significantly less severe than that close to control PLLA films in 4, 12 weeks after operation in rats. The present study will provide a new method to evaluate and treat the biocompatibility related to inflammatory response for implanted biomaterials and drug delivery system.

Extracellular matrix of mechanically stretched cardiac fibroblasts improves viability and metabolic activity of ventricular cells.

BACKGROUND: In heart, the extracellular matrix (ECM), produced by cardiac fibroblasts, is a potent regulator of heart's function and growth, and provides a supportive scaffold for heart cells in vitro and in vivo. Cardiac fibroblasts are subjected to mechanical loading all the time in vivo. Therefore, the influences of mechanical loading on formation and bioactivity of cardiac fibroblasts ECM should be investigated. METHODS: Rat cardiac fibroblasts were cultured on silicone elastic membranes and stimulated with mechanical cyclic stretch. After removing the cells, the ECMs coated on the membranes were prepared, some ECMs were treated with heparinase II (GAG-lyase), then the collagen, glycosaminoglycan (GAG) and ECM proteins were assayed. Isolated neonatal rat ventricular cells were seeded on ECM-coated membranes, the viability and lactate dehydrogenase (LDH) activity of the cells after 1-7 days of culture was assayed. In addition, the ATPase activity and related protein level, glucose consumption ratio and lactic acid production ratio of the ventricular cells were analyzed by spectrophotometric methods and Western blot. RESULTS: The cyclic stretch increased collagen and GAG levels of the ECMs, and elevated protein levels of collagen I and fibronectin. Compared with the ECMs produced by unstretched cardiac fibroblasts, the ECMs of mechanically stretched fibroblasts improved viability and LDH activity, elevated the Na⁺/K⁺-ATPase activity, sarco(endo)plasmic reticulum Ca²âº-ATPase (SERCA) activity and SERCA 2a protein level, glucose consumption ratio and lactic acid production ratio of ventricular cells seeded on them. The treatment with heparinase II reduced GAG levels of these ECMs, and lowered these metabolism-related indices of ventricular cells cultured on the ECMs. CONCLUSIONS: Mechanical stretch promotes ECM formation of cardiac fibroblasts in vitro, the ECM of mechanically stretched cardiac fibroblasts improves metabolic activity of ventricular cells cultured in vitro, and the GAG of the ECMs is involved in regulating metabolic activity of ventricular cells.

Counter-effect of constrained dynamic loading on osteoporosis in ovariectomized mice.

In recent years, dynamic mechanical loading has been shown to effectively enhance bone remodeling. The current study attempted to research the counter-effect of constrained dynamic loading on osteoporosis (OP) in ovariectomized (OVX) mice. Female Kunming (KM) mice were randomly divided into 2 groups: SHAM and OVX. The right ulnas of the OVX mice were subjected to a 4-week constrained dynamic loading protocol, and the mechanical properties, trabecular micromorphology parameters and biochemical indices of osteogenesis were evaluated. We detected higher levels of tissue alkaline phosphatase (AKP) and serum bone gamma-carboxyglutamic-acid-containing proteins (BGPs), better trabecular micromorphology parameters and ulnar mechanical properties in the loading group than in the nonloading group. In summary, constrained dynamic loading could prevent ovariectomy-induced osteoporosis by facilitating osteogenesis, improving trabecular microstructure and enhancing bone mechanical properties.

Cardiac fibroblast-derived extracellular matrix produced in vitro stimulates growth and metabolism of cultured ventricular cells.

Cardiac fibroblasts (CFs) produce extracellular matrix (ECM) which is a potent regulator of heart cell function and growth, and provides a supportive microenvironment for heart cells. Therefore, CF-derived ECM produced in vitro is very suitable for heart-cell culturing and cardiac tissue engineering. The aim of this study was to investigate the effect of CF-derived ECM produced in vitro on the growth and metabolism of cultured ventricular cells. CF-derived ECM-coated cell culture dishes were prepared by culturing rat CFs and then decellularizing the cultures. Isolated neonatal rat ventricular cells were seeded on ECM-coated, collagen I-coated or uncoated dishes, and the growth of cells after 1-5 days of culture was assayed with MTT reagent. In addition, cellular metabolic activity was analyzed by spectrophotometric methods and protein levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) by Western blotting. The relative growth of ventricular cells was better on ECM-coated than on uncoated or collagen I-coated dishes. Furthermore, the glucose consumption ratio, lactic acid production ratio, Na(+)/K(+)-ATPase activity, SERCA activity and protein levels of SERCA2a were all higher in cells on the ECM-coated dishes. In conclusion, cardiac fi broblast-derived ECM produced in vitro stimulates the growth and metabolism of cultured ventricular cells. This study indicates that the bioactivity of the ECM supports heart cell growth in vitro, and this might be useful for cardiac tissue engineering.

Mechanical stimulus inhibits the growth of a bone tissue model cultured in vitro △.

OBJECTIVES: To construct the cancellous bone explant model and a method of culturing these bone tissues in vitro, and to investigate the effect of mechanical load on growth of cancellous bone tissue in vitro. METHODS: Cancellous bone were extracted from rabbit femoral head and cut into 1-mm-thick and 8-mm-diameter slices under sterile conditions. HE staining and scanning electron microscopy were employed to identify the histomorphology of the model after being cultured with a new dynamic load and circulating perfusion bioreactor system for 0, 3, 5, and 7 days, respectively. We built a three-dimensional model using microCT and analyzed the loading effects using finite element analysis. The model was subjected to mechanical load of 1000, 2000, 3000, and 4000 µÎµ respectively for 30 minutes per day. After 5 days of continuous stimuli, the activities of alkaline phosphatase (AKP) and tartrate-resistant acid phosphatase (TRAP) were detected. Apoptosis was analyzed by DNA ladder detection and caspase-3/8/9 activity detection. RESULTS: After being cultured for 3, 5, and 7 days, the bone explant model grew well. HE staining showed the apparent nucleus in cells at the each indicated time, and electron microscope revealed the living cells in the bone tissue. The activities of AKP and TRAP in the bone explant model under mechanical load of 3000 and 4000 µÎµ were significantly lower than those in the unstressed bone tissues (all P<0.05). DNA ladders were seen in the bone tissue under 3000 and 4000 µÎµ mechanical load. Moreover, there was significant enhancement in the activities of caspase-3/8/9 in the mechanical stress group of 3000 and 4000 µÎµ(all P<0.05). CONCLUSIONS: The cancellous bone explant model extracted from the rabbit femoral head could be alive at least for 7 days in the dynamic load and circulating perfusion bioreactor system, however, pathological mechanical load could affect the bone tissue growth by apoptosis in vitro. The differentiation of osteoblasts and osteoclasts might be inhibited after the model is stimulated by mechanical load of 3000 and 4000 µÎµ.

Mechanical strain promotes osteoblast ECM formation and improves its osteoinductive potential.

BACKGROUND: The extracellular matrix (ECM) provides a supportive microenvironment for cells, which is suitable as a tissue engineering scaffold. Mechanical stimulus plays a significant role in the fate of osteoblast, suggesting that it regulates ECM formation. Therefore, we investigated the influence of mechanical stimulus on ECM formation and bioactivity. METHODS: Mouse osteoblastic MC3T3-E1 cells were cultured in cell culture dishes and stimulated with mechanical tensile strain. After removing the cells, the ECMs coated on dishes were prepared. The ECM protein and calcium were assayed and MC3T3-E1 cells were re-seeded on the ECM-coated dishes to assess osteoinductive potential of the ECM. RESULTS: The cyclic tensile strain increased collagen, bone morphogenetic protein 2 (BMP-2), BMP-4, and calcium levels in the ECM. Compared with the ECM produced by unstrained osteoblasts, those of mechanically stimulated osteoblasts promoted alkaline phosphatase activity, elevated BMP-2 and osteopontin levels and mRNA levels of runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), and increased secreted calcium of the re-seeded MC3T3-E1 cells. CONCLUSION: Mechanical strain promoted ECM production of osteoblasts in vitro, increased BMP-2/4 levels, and improved osteoinductive potential of the ECM. This study provided a novel method to enhance bioactivity of bone ECM in vitro via mechanical strain to osteoblasts.

Differential effects of mechanical strain on osteoclastogenesis and osteoclast-related gene expression in RAW264.7 cells.

Mechanical strain plays a critical role in the formation, proliferation and maturation of bone cells. However, little is known about the direct effects of different magnitudes of mechanical strain on osteoclast differentiation. The aim of the present study was to investigate how the fusion and activation of osteoclasts can be regulated by mechanical strain magnitude using the RAW264.7 mouse monocyte/macrophage cell line as an osteoclast precursor. Mechanical strain (substrate stretching) was applied via a 4-point bending system when RAW cells were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL) for an indicated period of time. The numbers of tartrate-resistant acid phosphatase-positive (TRAP+) and apoptotic cells were counted. The expression of TRAP, matrix metalloproteinase-9 (MMP-9), RANK, cathepsin K and carbonic anhydrase II (CAII) was measured by semi-quantitative RT-PCR, and immunocytochemistry staining for RANK was performed. We found that the number of nuclei per osteoclast derived from RAW cells decreased under low magnitude mechanical strain and increased under high magnitude strain within physiological load with an enhanced fusion of TRAP+ osteoclasts, compared to the control with no mechanical strain. The expression of RANK mRNA was downregulated by low magnitude strain and beyond physiological load, while it was upregulated by high magnitude strain within physiological load, correlating with the increased expression of RANK examined by immunocytochemistry, suggesting the mechanical regulation of RANK expression. There was also an increase in the expression of MMP-9 mRNA in the groups subjected to a mechanical strain of 2,000 and 2,500 µÎµ. No significant differences were detected in the expression of TRAP mRNA, cathepsin K and CAII under mechanical strain compared to the control under no strain (0 µÎµ). These findings indicate that low-magnitude strain suppresses osteoclast fusion and activation, while high-magnitude strain within physiological load promotes osteoclast fusion and activation related to a mechanical magnitude-dependent response of RANK expression. These data, therefore, provide a deeper understanding of how different magnitudes of mechanical strains exert their effects on osteoclastogenesis.

Mechanical strain regulates osteoblast proliferation through integrin-mediated ERK activation.

Mechanical strain plays a critical role in the proliferation, differentiation and maturation of bone cells. As mechanical receptor cells, osteoblasts perceive and respond to stress force, such as those associated with compression, strain and shear stress. However, the underlying molecular mechanisms of this process remain unclear. Using a four-point bending device, mouse MC3T3-E1 cells was exposed to mechanical tensile strain. Cell proliferation was determined to be most efficient when stimulated once a day by mechanical strain at a frequency of 0.5 Hz and intensities of 2500 µÎµ with once a day, and a periodicity of 1 h/day for 3 days. The applied mechanical strain resulted in the altered expression of 1992 genes, 41 of which are involved in the mitogen-activated protein kinase (MAPK) signaling pathway. Activation of ERK by mechanical strain promoted cell proliferation and inactivation of ERK by PD98059 suppressed proliferation, confirming that ERK plays an important role in the response to mechanical strain. Furthermore, the membrane-associated receptors integrin ß1 and integrin ß5 were determined to regulate ERK activity and the proliferation of mechanical strain-treated MC3T3-E1 cells in opposite ways. The knockdown of integrin ß1 led to the inhibition of ERK activity and cell proliferation, whereas the knockdown of integrin ß5 led to the enhancement of both processes. This study proposes a novel mechanism by which mechanical strain regulates bone growth and remodeling.

Involvement of p38MAPK/NF-κB signaling pathways in osteoblasts differentiation in response to mechanical stretch.

Bone morphogenetic proteins (BMPs) are known to be important in osteoblasts' response to mechanical stimuli. BMPs/Smad signaling pathway has been demonstrated to play a regulatory role in the mechanical signal transduction in osteoblasts. However, little is currently known about the Smad independent pathway in osteoblasts differentiation in mechanical loading. In this study, MC3T3-E1 cells were subjected to mechanical stretch of 2000 micro-stain (µÎµ) at 0.5 Hz, in order to investigate the involvement of p38MAPK and NF-κB signaling pathways in mechanical response in osteoblasts. We found BMP-2/BMP-4 were up-regulated by mechanical stretch via the earlier activation of p38MAPK and NF-κB signaling pathways, which enhanced osteogenic gene expressions including alkaline phosphatase (ALP), collagen type I (Col I) and osteocalcin (OCN), and the expressions of these osteogenic genes were remarkably decreased with Noggin (an inhibitor for BMPs signals) pretreatment. Furthermore, BMP-2/BMP-4 expressions were suppressed by PDTC, an inhibitor of NF-κB pathway and SB203580, an inhibitor of p38MAPK pathway, respectively, leading to the declined levels of ALP, Col I and OCN. Interestingly, blocking in p38MAPK pathway can also cause the inactivation of NF-κB pathway in mechanical stretch. Collectively, the results indicate during mechanical stretch p38MAPK and NF-κB signaling pathways are activated first, and then up-regulate BMP-2/BMP-4 to enhance osteogenic gene expressions. Moreover, p38MAPK and NF-κB signals have cross-talk in regulation of BMP-2/BMP-4 in mechanical response.

Relationship between surgical time and postoperative complications in senile patients with hip fractures.

OBJECTIVE: To elucidate the relationship between surgical time and postoperative complications in senile patients with hip fractures, and try to find out other factors which are related to these complications. METHODS: Sixty-two patients, 28 males aged from 65 to 72 years with a mean age of 76.3 years and 34 females aged from 65 to 95 years with a mean age of 78.1 years, who had undergone orthopedic surgery because of hip fractures, were enrolled in a retrospective cohort study. The surgical time and pattern, the type of fracture, preoperative comorbidities, American Society of Anesthesiologists (ASA) score and the volume of blood transfusion during operation were obtained from these patients who were followed up by telephone calls for postoperative complications. All the patients were followed up at least for 1 year and were divided into subgroups according to their clinical characteristics and the results were analyzed by the Statistical Analysis System software. RESULTS: There was no significant difference in the morbidity of postoperative complications with the gender, age, surgical time and pattern, or ASA score. There was significant difference in the morbidity of postoperative complications related to preoperative comorbidities and the volume of blood transfusion. There was a significant causality between preoperative comorbidities and postoperative complications. The morbidity of postoperative complications was 1.651 times higher in patients with preoperative comorbidities than those without. CONCLUSIONS: There is no relationship between the surgical time and postoperative complications in senile patients who received surgery for hip fracture within 1 year. No correlation is found between the postoperative complications and gender, age, type of fracture, surgical pattern, ASA score and the volume of blood transfusion. Preoperative comorbidities are an independent predictor for postoperative complications.

Culturing of ventricle cells at high density and construction of engineered cardiac cell sheets without scaffold.

In natural heart tissue, cell density is about 1.0 x 108/cm3, and the cell metabolism is very active. Therefore, culturing heart cells in 3-dimensions at high density and construction of engineered cardiac tissue in vitro is very difficult. The aim of this study was to simulate 3-dimensional culturing of cardiac cells and pursue a novel method to construct engineered cardiac tissue in vitro. The isolated neonatal rat ventricle cells were cultured at a high seeding density of 1 x 10(6)/cm2. The cells at high density metabolized actively; the glucose consumption and lactic acid production of ventricle cells were much greater than those of fibroblasts cultured at the same density. The pH value of the culture medium of ventricle cells consistently decreased more rapidly. These cultured ventricle cells contained vascular endothelial cells, cardiomyocytes, and smooth muscle cells that appeared close to each other, and had overlapping nuclei and plenty of extracellular collagen. The cells at high density were treated with 0.2% trypsin to construct engineered cardiac cell sheets without scaffold. The engineered cardiac cell sheets could beat and roll up spontaneously, each sheet was 3 to 5 cells thick, and contained abundant cardiomyocytes and extracellular collagen. In conclusion, cells cultured at high-density in vitro grew well in a 2-dimensional culturing environment, formed "quasi 3-dimension" culturing, and engineered cardiac cell sheets comprised of several layers of cells were constructed. This study provides some guidance for cardiac tissue engineering and a novel method to construct engineered cardiac tissue without scaffold.

The establishment of a mechanobiology model of bone and functional adaptation in response to mechanical loading.

BACKGROUND: Mechanical stimuli affected bone adaptation, however, the mechanism on a dose-response relationship between mechanical stimuli and bone response is unclear. Therefore, we established a mechanobiology model to evaluated the adaptive response of bone to strain deformation at high-frequencies (5-15 Hz) of externally applied strain. METHODS: The ulnae of adult female rats were subjected to dynamic axial loading in vivo using Instron materials-testing machine. The applied loading at frequencies of 5 Hz, 10 Hz, and 15 Hz for 10 min with a haversine, low-magnitude waveform for a 2 weeks period, the peak strains is 2000 muepsilon and 3000 muepsilon. Strain was recorded using strain gauge conditioner and compared to physiological values obtained after testing. FINDINGS: At frequencies of 10 Hz, 15 Hz groups, loading promoted obviously secreted of osteocalcin and collagen; a relative benefit in Bone Mineral Density (BMD) was found compare to the control (P < 0.05) followed the decline of material mechanical properties (modulus of elasticity, ultimate stress) (P < 0.01). INTERPRETATION: These data show that a mechanobiology model of the axial ulna loading technique had been established successfully in rat. A short daily period of low-magnitude, high-frequency mechanical stimuli results in an osteogenic response related to peak strain magnitude, which do not result in significant differences in mechanical properties between the groups.

[The establishment of a new mechanobiology model of bone and functional adaptation studies in vivo].

OBJECTIVES: to study the functional adaptation in response to artificial loading in vivo. METHODS: A single element strain gauge of < 2 mm x 3 mm in size was attached in longitudinal alignment to the medial surface of the ulnar midshaft, in vivo recordings of ulnar strains during locomotion were obtained. The ulnae of natural female rats were subjected to dynamic axial loading in vivo simulate strains during locomotion using INSTRON materials-testing machine. The left ulna of adult female rats were subjected to applied loading at frequencies of 5 Hz, 10 Hz, 15 Hz for 10 min/d with a haversian, low-magnitude (1mm peak to peak) waveform for a two weeks period, the peak strains at the Left ulnar midshaft is 2000 microepsilon and 3000 microepsilon, the right ulna of each rat served as a paired internal control. Dual Energy X-ray Absorptiometry (DXA) was used to measure bone mineral density (BMD) at the ulnar; 3-point bending was used to test mechanical characteristics; the ulna's response to loading was traced by subcutaneously injecting each rat twice with 7.5 mg/kg calcein and 30 mg/kg Tetracycline Hcl on days 3 and 12 of the loading period, and analyzed by histomorphometry; immunohistochemistry as an effect of elevated strain in the bone matrix. RESULTS: at frequencies of 10 Hz, 15 Hz groups, loading promoted obviously secreted of alkaline phosphatase (ALP), osteocalcin (OCN) and collagen I; a relative benefit in BMD was found compare to the control (P < 0.05) followed the decline of material mechanical properties (modulus of elasticity, ultimate stress) (P < 0.01). CONCLUSION: These data show that a new bionics mechanobiology model of the axial ulna loading technique had be established successfully in rat. A short daily period of low-magnitude, high-frequency mechanical stimuli results in an osteogenic response related to peak strain magnitude.

Construction of a unidirectionally beating 3-dimensional cardiac muscle construct.

BACKGROUND: Cardiac tissue engineering aims to construct cardiac tissue with characteristics similar to those of the native tissue. Engineered cardiac tissues (ECTs) can be constructed using synthetic scaffold or liquid collagen. We report an initial study using our own newly designed cardiac muscle device to construct heart tissue. We investigated the effects of cell seeding density and collagen quantity on the formation of liquid collagen-based cardiac muscle. METHODS: We obtained cardiac myocytes from neonatal rats mixed with collagen type I and matrix factors cast in circular molds to form circular strands. Cell densities (0.1 x 10(7) to 6 x 10(7)) and collagen quantity (0.3 to 1.0 ml/ECT) were tested. Cell gross morphology, cell orientation, spatial distribution and ultrastructure were evaluated using histologic analyses, confocal laser scanning microscopy and transmission electron microscopy. RESULTS: Histologic analyses of ECTs revealed that cardiac cells reconstituted longitudinally oriented, cardiac bundles with morphologic features characteristic of the native tissue. Confocal and electron microscopy demonstrated that, using optimized cell density and collagen quantity, we made ECTs with characteristic features similar to those of native differentiated myocardium. CONCLUSIONS: ECTs comparable to native cardiac tissue can be engineered under optimized conditions. This construct is a first step in the development of cardiac tissue engineered in vitro, and may be used as a basis for studies of cardiac development, drug testing and tissue replacement therapy.