Acute lung inflammatory response and injury after hemorrhagic shock are more severe in postpartum rabbits.

OBJECTIVE: The acute respiratory distress syndrome may complicate postpartum hemorrhagic shock and resuscitation, but its mechanisms are not yet well defined. We studied the lung inflammatory response to postpartum hemorrhagic shock and resuscitation in a rabbit model and the role of the nuclear factor-κB pathway. DESIGN: Randomized, controlled, prospective study. SETTING: University hospital laboratory. SUBJECTS: Nonobstetric (not pregnant nor postpartum) and obstetrical (within 2 hrs postpartum) rabbits. INTERVENTIONS: Nonobstetric and obstetric female New Zealand white rabbits underwent fixed-pressure or fixed-volume hemorrhagic shock for 30 mins and then were rapidly resuscitated with the shed blood and Ringer's solution. Finally, they were either monitored for survival time or euthanized by exsanguination for lung tissue examination 24 hrs after hemorrhage. MEASUREMENTS AND MAIN RESULTS: After hemorrhagic shock and resuscitation, median survival time in obstetric rabbits (3 days) was significantly shorter (p<.05) than that in nonobstetric rabbits (5 days). Compared with nonobstetric rabbits, obstetric rabbits had more severe lung injury as indicated by alveolar and interstitial fluid accumulation and marked neutrophil sequestration and greater lung injury score, myeloperoxidase activity, expression of intercellular adhesion molecule-1, serum tumor necrosis factor-α levels, and nuclear factor-κB activation, and lower serum interleukin-10 levels (p<.05 for all). CONCLUSIONS: After hemorrhage and resuscitation, obstetric rabbits had significantly shorter survival time and more severe lung injury than nonobstetric rabbits. The mechanism may be through upregulation of the signal transductions of the nuclear factor-κB pathways.

Growth/differentiation factor-5 induces osteogenic differentiation of human ligamentum flavum cells through activation of ERK1/2 and p38 MAPK.

Ossification of ligamentum flavum (OLF) is a pathological ectopic ossification in the spinal ligament, leading to spinal canal stenosis, but little was known about its pathogenesis. A previous study has found growth/differentiation factor (GDF)-5 expression at ossified sites of the ligaments from OLF patients. This study aimed to investigate the osteogenic effects of GDF-5 on cultured human ligamentum flavum cells (LFCs). LFCs were isolated from human spinal ligamentum flavum, and treated with or without recombinant human (rh) GDF-5. Alkaline phosphatase (ALP) activity was measured. Expression of osteocalcin was assessed by reverse transcriptase-PCR, Western blotting and immunofluorescence. Matrix mineralization was assessed by alizarin red staining. Activation of mitogen-activated protein kinases (MAPK) ERK1/2, p38 and JNK were detected by Western blotting. We found that rhGDF-5 treatment increased ALP activity and osteocalcin expression in a time- and dose-dependent manner, and induced mineralized nodule form. In addition, rhGDF-5 challenge mediated the ERK1/2 and p38 activation but not JNK. Inhibiting this activation pharmacologically, using U0126, a ERK1/2 inhibitor, or SB203580, a p38 inhibitor, resulted in significantly lower ALP activity and osteocalcin protein expression. The present study shows that rhGDF-5 induces osteogenic differentiation of human LFCs through activation of ERK1/2 and p38 MAPK. These findings give some new insight into the pathogenesis of OLF.

Does whole-body vibration with alternative tilting increase bone mineral density and change bone metabolism in senior people?

BACKGROUND AND AIMS: Whole-body vibration (WBV) presents as osteogenic in animal models and young patients, but the effect remains unclear in senior people. The use of alternative tilting during WBV to ameliorate bone mass and bone metabolism, particularly in senior people, has not previously been reported. This study assessed changes in bone mineral density (BMD) and bone metabolism in senior people after six-month treatment of whole-body vibration with alternative tilting (WBVAT). METHODS: Fifty-three senior people (11M/42F, >65 yrs, mean age 77) and 15 adults (4M/11F, 50-60 yrs, mean age 53) were enrolled and assigned randomly to WBVAT (senior: n=27; adult: n=7) and control groups (senior: n=26; adult: n=7), respectively. The WBVAT groups were subjected to vertical vibration (0.5-0.8 g, 45-55 Hz) and alternative tilting (2° tilting angle or 8 mm displacement at 0.4 Hz) 20 minutes per day, 3 days a week, for 6 months. BMD in the lumbar spine and femoral neck was measured at 0, 3 and 6 months, respectively, as well as biochemical markers of bone metabolism, including serum calcium, phosphorus, alkaline phosphatase (ALP), osteocalcin and tartrate resistance acid phosphatase at 0, 1, 3 and 6 months, respectively. RESULTS: After 6-month WBVAT treatment, BMD in the lumbar spine and femoral neck increased significantly by 2.52% and 3.22% for senior people, and 1.63% and 2.05% for adults, respectively. The 6-month WBVAT treatment increased BMD in the senior people, both with and without osteoporosis (OP) and in both men and women, but led to a BMD gain greater in people with OP (p<0.01) and women (p<0.01), respectively. The serum ALP level increased significantly by a net 24.4% in seniors after WBVAT treatment at 3 months; other biochemical markers showed non-significant differences between the WBVAT and control groups. CONCLUSIONS: WBVAT treatment may increase BMD in senior people, particularly those with OP and women. Changes in bone metabolism after WBVAT treatment were not observed in most cases.

[Effects of bioactive modification of poly-D,L-lactide acid scaffolds on the biological behaviors of the seed cells].

OBJECTIVE: To study the changes in the biological behavior of bone marrow mesenchymal stem cells (BMSCs) transfected with red fluorescent protein by lentivirus (RFP-BMSCs) seeded on in poly-D, L-lactide acid (PDLLA) scaffolds with bioactive modification by ammonia plasma and Gly-Arg-Gly-Asp-Ser (GRGDS) in vitro. METHODS: Circular sheets of PDLLA scaffolds (8 mm in diameter and 1 mm in thickness) were prepared and aminated with PDLLA (group A) or modified with the peptide conjugate A/PDLLA (group PA), with untreated PDLLA as the control (group P). The RFP-BMSCs were seeded on the scaffold materials and their proliferation and metabolic activity were detected using CyQuant NF and Alamar blue staining. The mineralization on the scaffolds was observed using calcein fluorescent dye under a fluorescent microscope. The adhesion and proliferation of RFP-BMSCs were observed by fluorescent microscope, and scanning electron microscope (SEM) was used to confirm the observed adhesion of the seed cells. RESULTS: The RFP-BMSCs seeded on the 3 scaffolds all showed proliferative activity at different time points after cell seeding, and the cell numbers decreased significantly in the order of PA>A>P (P<0.001). The cell number was significantly greater in group PA than in group A at all the time points except for days 10 (P=0.077) and 12 (P=0.491), and gradually became similar with the passage of time. The metabolic changes of the cells follow a similar pattern of cell proliferation. RFP-BMSCs showed more active proliferation in group A and group PA than in group P. On days 14 and 21, the intensity of green fluorescence decreased in the order of group PA, A and P. The RFP-BMSCs showed better adhesion in group PA than in group A, and the cells in group P appeared more scattered under scanning electron microscope. CONCLUSION: Bioactive modification of PDLLA by ammonia treatment and conjugation with GRGDS peptides may promotes the adhesion, proliferation, metabolism and mineralization of RFP-BMSCs seeded on PDLLA scaffolds.

[Histology and proliferative capability of thoracic vertebral body growth plates of rats at different ages].

OBJECTIVE: To compare the histological features of the thoracic vertebral body growth plates (VBGPs) of rats at different ages and assess their proliferative capability. METHODS: The thoracic VBGPs obtained from rats aged 1 day and 1, 4, 8, 16 and 28 weeks were identified using safranin O-fast green staining, and the height of the hypertrophic zone, proliferative zone, and resting zone were measured. The chondrocytes were isolated from these VBGPs with a modified trypsin-collagenase type II digestion method for primary culture in vitro. The expressions of proliferating cell nuclear antigen (PCNA) mRNA and protein was detected by real time-PCR and Western blotting, respectively. RESULTS: The 1-day- and 1-week-old rats showed significantly greater hypertrophic zone and proliferative zone in the VBGPs than older rats (P<0.01); the proliferative zone was significantly greater in rats aged 4 weeks than in those aged 28 weeks (P<0.05). The resting zone was obviously greater in rats aged 1 day and 1 week than in older rats (P<0.05), and also greater in rats aged 4 weeks than in those aged 16 and 28 weeks (P<0.05). Obvious ossification in the resting zone occurred at 16 weeks, and most of the resting zone became ossified at 28 weeks. The expression of PCNA decreased at both the mRNA and protein levels as the rats grew. CONCLUSION: The 3 zones of VBGPs are greater in rats aged 1 day and 1 week than in older ones. Ossification in the resting zone begins at 16 weeks, and till 28 weeks, most of the resting zone is ossified. The proliferation ability of VBGP chondrocytes decreases with the increase of age of the rats.

Hypertrophy of ligamentum flavum in lumbar spine stenosis associated with the increased expression of connective tissue growth factor.

Hypertrophy of the ligamentum flavum (LF) contributes to lumbar spinal stenosis (LSS), and results mainly from fibrosis. Connective tissue growth factor (CTGF) is a profibrotic factor involved in the fibrotic process. This study aimed to evaluate CTGF expression in hypertrophied lumbar LF and the involvement of CTGF in LF hypertrophy. Ten patients with LSS were enrolled in this study. The control group included 10 patients with lumbar disc herniation. LF thickness was measured on the preoperative axial T1-weighted MRI. LF samples were collected during surgery. LF fibrosis was scored by Masson's trichrome staining. CTGF expression was determined by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Correlation between LF thickness and CTGF expression was analyzed. Human LF cells were cultured and treated with recombinant human (rh) CTGF. Expression of types I and III collagen was determined by real-time PCR and ELISA. The thickness and fibrosis scores of LF in the LSS group were higher than that in the control group (all P < 0.001). CTGF was expressed in the extracellular matrix of all ligament samples, and was significantly higher in the LSS group than that in the control group (P < 0.001). The increase of CTGF expression was positive correlation with the LF thickness (r = 0.969, P = 0.000). rhCTGF treatment increased the mRNA expression and protein synthesis of types I and III collagen of the LF cells (all P < 0.001). Our results suggest that the increased expression of CTGF is associated with hypertrophy of the LF in patients with LSS.

Amide-linkage formed between ammonia plasma treated poly(D,L-lactide acid) scaffolds and bio-peptides: enhancement of cell adhesion and osteogenic differentiation in vitro.

The surface characteristics of scaffolds for bone tissue engineering must support cell adhesion, migration, proliferation, and osteogenic differentiation. In the study, poly(D,L-lactide acid) (PDLLA) scaffolds were modified by combing ammonia (NH(3) ) plasma pretreatment with Gly-Arg-Gly-Asp-Ser (GRGDS)-peptides coupling technologies. The x-ray photoelectron spectroscopy (XPS) survey spectra showed the peak of N1s at the surface of NH(3) plasma pretreated PDLLA, which was further raised after GRGDS conjugation. Furthermore, N1s and C1s in the high-resolution XPS spectra revealed the presence of -C=N(imine), -C-NH-(amine), and -C=O-NH- (amide) groups. The GRGDS conjugation increased amide groups and decreased amine groups in the plasma-treated PDLLA. Confocal microscope and high performance liquid chromatography verified the anchored peptides after the conjugation process. Bone marrow mesenchymal stem cells were co-cultured with scaffolds. Fluorescent microscope and scanning electron microscope photographs revealed the best cell adhesion in NH(3) plasma pretreated and GRGDS conjugated scaffolds, and the least attachment in unmodified scaffolds. Real-time PCR demonstrated that expression of osteogenesis-related genes, such as osteocalcin, alkaline phosphatase, type I collagen, bone morphogenetic protein-2 and osteopontin, was upregulated in the single NH(3) plasma treated and NH(3) plasma pretreated scaffolds following GRGDS conjugation. The results show that NH(3) plasma treatment promotes the conjugation of GRGDS peptides to the PDLLA scaffolds via the formation of amide linkage, and combination of NH(3) plasma treatment and peptides conjugation may enhance the cell adhesion and osteogenic differentiation in the PDLLA scaffolds. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 682-694, 2011.