computed-tomography-optical-imaging-osteogenesis-angiogenesis

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

The aim of this study was to evaluate whether recombinant human insulin-like growth factor I (rhIGF-I) could attenuate or prevent diaphragm (DIA) fiber atrophy with corticosteroid (CS) administration to emphysematous (EMP) hamsters. DIA muscle IGF-I responses to CS administration with and without exogenous rhIGF-I administration were evaluated. Three groups were studied: 1) EMP; 2) EMP + triamcinolone (T; 0.4 mg.kg-1.day-1 im); and 3) EMP + T + IGF-I (600 microg/day by constant infusion). After 4 wk, the DIA was analyzed histochemically and biochemically (IGF-I mRNA levels by RT-PCR and endogenous and exogenous IGF-I peptide levels immunochemically). Body weights of EMP-T progressively decreased, while those of EMP and EMP-T-IGF-I remained stable despite similarly reduced food intake in both T groups. DIA weight was reduced with T but preserved with rhIGF-I infusion. DIA fiber proportions were similar among the groups. The cross-sectional areas of types I, IIa, and IIx fibers were reduced (17 to 31%) with T administration but unchanged with rhIGF-I infusion. DIA IGF-I mRNA levels were similar across all groups. By contrast, the endogenous DIA IGF-I levels were reduced (41%) in the EMP-T-IGF-I animals. Total DIA IGF-I levels (endogenous + exogenous) were still significantly reduced. IGF-I immunoreactivity confirmed this reduction in all DIA fibers. We conclude that DIA fiber atrophy with T was completely prevented by exogenous rhIGF-I administration. This effect was likely mediated by the pharmacological influences of exogenously administered rhIGF-I. We speculate that this results from increased bioavailability of free IGF-I to react with muscle receptors. Reduced endogenous IGF-I levels in the DIA likely reflect a negative-feedback influence. These results may have important clinical implications for treatment options to offset the adverse effects of CS on the respiratory muscles in patients with chronic lung disorders.

Insulin-like growth factor I prevents corticosteroid-induced diaphragm muscle atrophy in emphysematous hamsters.

With emphysema, diaphragm length adaptation results in shortened fibers. We hypothesize that passive diaphragm stretch occurring acutely after lung volume reduction surgery (LVRS) results in fiber injury. Bilateral LVRS was performed in emphysematous hamsters. Studies were performed 1 (D1) and 4 (D4) days after LVRS, and compared with sham-treated groups. Sarcolemmal rupture was evident in 10.9% of fibers in LVRS-D1 and reduced to 1.6% in LVRS-D4. Ultrastructural analysis revealed focal abnormalities in both LVRS-D1 and LVRS-D4 animals in over one-third of fibers. Myofibrillar disruption was not observed in sham-treated animals. Diaphragm insulin-like growth factor-I (IGF-I) was increased in LVRS-D4 compared with other emphysematous groups. Increased IGF-I immunoreactivity was localized to types IIA and I fibers. The abundance of the splice variant of IGF-I mRNA sensitive to muscle stretch (IGF-IEb) increased 3.2-fold in LVRS D-4 diaphragms, compared with emphysema-sham animals. The main form of IGF-I mRNA was unchanged. Marked force deficit was observed in the LVRS-D1 diaphragm, compared with emphysema-sham and emphysema (no surgery) animals. These data highlight a markedly compromised ventilatory pump acutely after LVRS. Acute fiber stretch predisposes to muscle fiber injury and may also be a necessary mechanotransductive stimulus for fiber remodeling as the diaphragm adapts to reduced lung volume.

Short-term influences of lung volume reduction surgery on the diaphragm in emphysematous hamsters.

Mit Emphysem Membran L&auml;nge Anpassung f&uuml;hrt zu verk&uuml;rzten Fasern. Wir vermuten, dass passive Membran-Strecke auch akut, nachdem Lunge Volume Reduzierung Chirurgie (LVRS) Faser Verletzungen f&uuml;hrt. Bilaterale LVRS erfolgte in emphysematous Hamster. Studien wurden durchgef&uuml;hrt, 1 (D1) und 4 (D4) Tage nach LVRS und Schein-behandelten Gruppen gegen&uuml;ber. Sarcolemmal Bruch zeigte sich in 10,9 % der Fasern in LVRS-D1 und auf 1,6 % im LVRS-D4 reduziert. Ultrastrukturelle Analyse ergab fokale Abnormalit&auml;ten in sowohl LVRS-D1 und LVRS-D4 Tiere in mehr als ein Drittel der Fasern. Myofibrill&auml;re St&ouml;rung wurde nicht in Schein-behandelten Tieren beobachtet. Membran Insulin-wie Wachstumsfaktor-ich (IGF-ich) wurde in LVRS-D4 im Vergleich mit anderen emphysematous Gruppen erh&ouml;ht. Erh&ouml;hte IGF-ich Immunoreactivity lokalisiert wurde, Typ IIA und ich Fasern. Die F&uuml;lle der Splice-Variante der IGF-ich mRNA empfindlich auf Muskeldehnung (IGF-IEb) erh&ouml;hte 3.2-fold in LVRS D-4-Membranen, Emphysem-Sham Tieren gegen&uuml;ber. Die wichtigste Form der IGF-ich mRNA war unver&auml;ndert. Deutliche Kraft Defizit wurde im Zwerchfell LVRS-D1, verglichen mit Emphysem-Sham und Emphysem (keine Operation) Tiere beobachtet. Diese Zahlen beleuchten eine merklich beeintr&auml;chtigte ventilatory Pumpe akut nach LVRS. Akute Faser Stretch pr&auml;disponiert Muskelfaser Verletzungen und kann auch die erforderlichen Mechanotransductive Impuls f&uuml;r Faser Umbau wie das Zwerchfell an reduzierten Lunge Lautst&auml;rke anpasst.

Kurzfristigen Einfl&uuml;ssen der Lunge Volume Reduzierung Chirurgie auf dem Zwerchfell in emphysematous Hamster.

Tumor necrosis factor (TNF)-alpha has been implicated in several muscle-wasting disorders, with increased levels of the cytokine reported in malnourished children. The role of TNF-alpha in mediating malnutrition-induced inhibition of diaphragm (DIA) muscle growth in young growing rats was evaluated. Three groups of rats were studied: 1) control (CTL); 2) nutritional deprivation (ND; 50% of normal food intake for 7 days); and 3) ND + rat specific anti-TNF-alpha antibody. DIA fiber cross-sectional areas were determined. Serum and muscle TNF-alpha levels were measured by real-time PCR, ELISA, and immunohistochemistry. Body weights decreased 20% in ND rats and increased 46% in CTL animals. Anti-TNF-alpha had no effect on body weight or on DIA mass in ND animals. ND significantly reduced cross-sectional areas of all fiber types (33-46%). Anti-TNF-alpha failed to attenuate ND-induced inhibition of DIA fiber growth. Serum TNF-alpha levels increased 2.6-fold in ND animals, with levels suppressed to below CTL values with anti-TNF-alpha. DIA TNF-alpha mRNA and protein levels increased two- to threefold in ND rats. Anti-TNF-alpha antibodies suppressed muscle levels of the cytokine in ND animals to near CTL values. TNF-alpha immunoreactivity in all DIA fibers revealed similar directions of change in both ND groups. Direction and magnitude of change in DIA phosphorylated p38 MAPK (a likely second messenger of TNF-alpha) tracked those of TNF-alpha. Muscle levels of IGF-I mRNA and phosphorylated Akt were markedly reduced in ND animals with no change following anti-TNF-alpha therapy. Thus rat anti-TNF-alpha at a dose known to neutralize the cytokine failed to attenuate or reverse ND-induced inhibition of DIA fiber growth in our model.

Tumor necrosis factor-alpha and malnutrition-induced inhibition of diaphragm fiber growth in young rats.

Tumor-Nekrose-Faktor (TNF)-Alpha hat verwickelt wurde, in mehreren Muskelschwund St&ouml;rungen, mit einem erh&ouml;hten die Cytokine in unterern&auml;hrte Kinder gemeldet. Die Rolle von TNF-Alpha bei der Vermittlung von Unterern&auml;hrung-induzierte Hemmung der Membran (DIA) Muskel-Wachstum bei jungen wachsenden Ratten wurde ausgewertet. Drei Gruppen von Ratten wurden untersucht: 1) Kontrolle (CTL); 2) Ern&auml;hrung Entbehrung (ND, 50 % der normalen Nahrungsaufnahme f&uuml;r 7 Tage); und 3) ND + Ratte spezifischen Anti-TNF-Alpha-Antik&ouml;rper. DIA Faser Querschnittsfl&auml;che wurden ermittelt. Serum und Muskel TNF-Alpha Stufen wurden von Real-Time PCR, ELISA und Immunhistochemie gemessen. K&ouml;rper Gewichte verringerte sich um 20 % bei ND Ratten und 46 % bei CTL Tieren erh&ouml;ht. Anti-TNF-Alpha hatte keine Auswirkungen auf K&ouml;rpergewicht oder DIA-Masse in ND-Tiere. ND hat die Querschnittsfl&auml;che der alle Fasertypen (33-46 %) deutlich reduziert. Anti-TNF-Alpha konnte ND-induzierte Hemmung der DIA Faser Wachstum d&auml;mpfen. TNF-Alpha-Serumspiegel erh&ouml;ht 2.6-fold in ND-Tiere, mit Ebenen unterdr&uuml;ckt unter CTL-Werten mit Anti-TNF-Alpha. DIA TNF-Alpha-mRNA und Protein-Stufen erh&ouml;ht zwei-bis dreifachen in ND-Ratten. Anti-TNF-Alpha-Antik&ouml;rper unterdr&uuml;ckt Muskel Ebenen der Cytokine ND Tiere auf CTL-Werte in der N&auml;he. TNF-Alpha-Immunoreactivity in allen DIA Fasern ergab &auml;hnliche Richtungen Ver&auml;nderungen in beiden Gruppen ND. Richtung und Gr&ouml;&szlig;e der &Auml;nderung in DIA phosphoryliert p38 MAPK (ein wahrscheinlich zweite Bote von TNF-Alpha) verfolgt die TNF-alpha. Muskel-Ebenen der IGF-ich mRNA und phosphorylierten Akt wurden ND Tiere ohne &Auml;nderung nach Anti-TNF-Alpha-Therapie deutlich reduziert. Somit Ratte Anti-TNF-Alpha in einer Dosis, die bekannterma&szlig;en die Cytokine nicht d&auml;mpfen oder umkehren ND-induzierte Hemmung der DIA-Faser-Wachstum in unserem Modell zu neutralisieren.

Tumor-Nekrose-Faktor-Alpha und Unterern&auml;hrung-induzierte Hemmung der Membran-Faser-Wachstum bei jungen Ratten.

The aim of this study was to evaluate the effect of nutritional deprivation (ND) on signal transduction pathways influencing the translational apparatus in the diaphragm muscle. Male rats were divided into two groups: 1) 20% of usual food intake for 4 days (ND) with water provided at libitum and 2) free-eating control (Ctl). Total protein and RNA were extracted from the diaphragm. Insulin-like growth factor I mRNA was analyzed by RT-PCR. Protein analyses of key cytoplasmic proteins for three signaling pathways deemed important in influencing protein turnover [phosphatidylinositol 3-kinase- Akt-mammalian target of rapamycin, P13K/Akt/glycogen synthase kinase (GSK)-3, and MAPK-ERK] were performed by Western blot. Body weight decreased 30% in ND and increased 17% in Ctl animals. Diaphragm mass decreased 29% in ND animals. Muscle insulin-like growth factor I mRNA abundance was reduced 63% in ND animals. ND resulted in a 55% reduction in phosphorylated (Ser473) Akt. Phosphorylation of mammalian target of rapamycin at Ser2448 was reduced by 85% in ND animals. Downstream effectors important in translation initiation were also affected by ND. Phosphorylated (Thr389) 70-kDa ribosomal protein S6 kinase was significantly reduced (35%) by ND. ND also resulted in significant dephosphorylation of the translational repressor initiation factor 4E-binding protein 1. Phosphorylation of GSK-3alpha (Ser21) and GSK-3beta (Ser9) was increased 55 and 45%, respectively, with ND. Phosphorylation of ERK1 (Thr202) and ERK2 (Tyr204), p44 and p42, respectively, was reduced 64 and 55%, respectively, with ND. Total protein concentration for all signaling intermediates of the three pathways was preserved. We conclude that short-term ND altered the phosphorylation states of key proteins of several pathways involved in protein turnover. This forms the framework for future studies aimed at identifying therapeutic targets in the management of short-term nutritionally induced cachectic states.

Effect of severe short-term malnutrition on diaphragm muscle signal transduction pathways influencing protein turnover.

We recently reported increased leg lean mass and strength in men with chronic obstructive pulmonary disease (COPD) receiving 10 wk of testosterone (T) and leg resistance training (R) (Casaburi R, Bhasin S, Cosentino L, Porszasz J, Somfay A, Lewis M, Fournier M, Storer T. Am J Respir Crit Care Med 170: 870-878, 2004). The present study evaluates the role of muscle IGF and related factors as potential mechanisms for our findings, using quadriceps muscle biopsies from the same cohort. Patient groups were 1) weekly placebo (P) injections + no R; 2) P and R; 3) weekly injections of T + no R; and 4) T + R (TR). Muscle fibers were classified histochemically, and their cross-sectional areas (CSAs) and fiber density (number of fibers per unit area) were determined. Gene transcripts were determined by real-time PCR and protein expression by RIA. While no significant changes in fiber CSAs were noted across groups, increased trends were observed after 10 wk, and significant decrements in muscle fiber density were noted in all treated groups. A global increase in all myosin heavy chain (MyHC) mRNA isoforms was observed in TR patients. Muscle IGF-IEa and IGF-IEc mRNAs were significantly increased with TR group. Muscle IGF-I protein was increased in all intervention groups (greatest in TR). While TR IGF-II mRNA was increased, protein levels were unaltered. IGF binding protein-4 mRNA was increased with TR. Myogenin mRNA was increased in both T groups, while MyoD and myostatin were unchanged. Muscle atrophy F-box mRNA tended to increase with TR. Our data suggest that the combined interventions produced an enhanced local anabolic milieu driven in large part by the muscle IGF system, despite potentially negative biochemical influences present in COPD patients.

Skeletal muscle adaptations to testosterone and resistance training in men with COPD.

Increased expression of forkhead box O (Foxo) transcription factors were reported in cultured myotubes and mouse limb muscle with corticosteroid (CS) treatment. We previously reported that administration of CS to rats resulted in muscle fiber atrophy only by day 7. The aim of this study, therefore, was to evaluate the time-course changes in the expression of Foxo transcription factors and muscle-specific ubiquitin E3 ligases in rat limb muscle following CS administration. Triamcinolone (TRI; 1 mg x kg(-1) x day(-1) im) was administered for 1, 3, or 7 days. Control (CTL) rats were given saline. Muscle mRNA was analyzed by real-time RT-PCR. Compared with CTL, body weights of TRI-treated animals decreased by 3, 12, and 21% at days 1, 3, and 7, respectively. Muscle IGF-1 mRNA levels decreased by 33, 65, and 58% at days 1, 3, and 7 in TRI-treated rats compared with CTL. Levels of phosphorylated Akt were 28, 50, and 36% lower in TRI animals at these time points. Foxo1 mRNA increased progressively by 1.2-, 1.4-, and 2.5-fold at days 1, 3, and 7 in TRI animals. Similar changes were noted in the expression of Foxo3a mRNA (1.3-, 1.4-, and 2.6-fold increments). By contrast, Foxo4 mRNA was not significantly changed in TRI animals. With TRI, muscle atrophy F box/Atrogin-1 increased by 1.8-, 4.1-, and 7.5-fold at days 1, 3, and 7 compared with CTL rats. By contrast, muscle RING finger 1 increased only from day 7 (2.7-fold). Gradual reduction in IGF-I expression with TRI over the time series paralleled that of Akt. These findings are consistent with a progressive stimulus to muscle protein degradation and the need to process/remove disassembled muscle proteins via the ubiquitin-proteasome system. Elucidating the dynamic catabolic responses to CS challenge is important in understanding the mechanisms underlying muscle atrophy and therapeutic measures to offset this.

Time course expression of Foxo transcription factors in skeletal muscle following corticosteroid administration.

Vertebral compression fractures (VCFs), the most common fragility fractures, account for approximately 700,000 injuries per year. Since open surgery involves morbidity and implant failure in the osteoporotic patient population, a new minimally invasive biological solution to vertebral bone repair is needed. Previously, we showed that adipose-derived stem cells (ASCs) overexpressing a BMP gene are capable of inducing spinal fusion in vivo. We hypothesized that a direct injection of ASCs, designed to transiently overexpress rhBMP6, into a vertebral bone void defect would accelerate bone regeneration. Porcine ASCs were isolated and labeled with lentiviral vectors that encode for the reporter gene luciferase (Luc) under constitutive (ubiquitin) or inductive (osteocalcin) promoters. The ASCs were first labeled with reporter genes and then nucleofected with an rhBMP6-encoding plasmid. Twenty-four hours later, bone void defects were created in the coccygeal vertebrae of nude rats. The ASC-BMP6 cells were suspended in fibrin gel (FG) and injected into the bone void. A control group was injected with FG alone. The regenerative process was monitored in vivo using microCT, and cell survival and differentiation were monitored using tissue specific reporter genes and bioluminescence imaging (BLI). The surgically treated vertebrae were harvested after 12 weeks and subjected to histological and immunohistochemical (against porcine vimentin) analyses. In vivo BLI detected Luc-expressing cells at the implantation site over a 12-week period. Beginning 2 weeks postoperatively, considerable defect repair was observed in the group treated with ASC-BMP6 cells. The rate of bone formation in the stem cell-treated group was two times faster than that in the FG-treated group, and bone volume at the end point was 2-fold compared to the control group. Twelve weeks after cell injection the bone volume within the void reached the volume measured in native vertebrae. Immunostaining against porcine vimentin indicated that the ASC-BMP6 cells contributed to new bone formation. Here we show the potential of injections of BMP-modified ASCs to repair vertebral bone defects in a rat model. Our results could pave the way to a novel approach for the biological treatment of traumatic and osteoporosis-related vertebral bone injuries.

Gene-modified adult stem cells regenerate vertebral bone defect in a rat model.

Skeletal muscle dysfunction contributes to exercise limitation in COPD. The role of the nitric oxide synthase (NOS) system in muscle dysfunction is ill defined. Reduced levels of endothelial NOS (eNOS) and elevated levels of inducible NOS (iNOS) in the skeletal muscle of COPD patients have been recently reported. We hypothesized that resistance exercise training (R) and/or testosterone supplementation (T) would alter the transcription and expression of the NOS isoenzymes in COPD skeletal muscle.

Testosterone and resistance training effects on muscle nitric oxide synthase isoforms in COPD men.

Attenuation of muscle wasting has been reported with eicosapentaenoic acid (EPA) use in cachectic states. Pathways mediating muscle proteolysis with severe short-term nutritional deprivation (ND)±EPA were evaluated, including diaphragm fiber-specific cross-sectional areas, mRNA (real-time PCR) and protein expression (Western blot). Rats were divided into three groups: (1) free-eating controls, (2) ND and (3) ND+EPA. ND significantly influenced multiple proteolytic pathways. EPA significantly reduced mRNA abundances for most genes to control levels with ND. However, discordant muscle protein expression of many genes was noted with the use of EPA, as protein levels failed to fall. EPA had no impact on diaphragm muscle atrophy, despite the impressive mRNA and some protein results. We conclude that EPA does not attenuate diaphragm muscle atrophy with severe levels of ND. Postulated mechanisms include reduction in muscle protein synthesis and persistent ongoing stimuli for proteolysis. Our study provides unique data on proteolytic signals with ND and has important implications for future studies using EPA.

Effects of malnutrition with or without eicosapentaenoic acid on proteolytic pathways in diaphragm.

Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones. Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and microcomputed tomography (μCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes. Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The μCT scans revealed a significant increase in bone formation in allograft + PTH treated mice comparing to allograft + PBS treated mice. The osteogenic genes osteocalcin (Oc/Bglap) and integrin binding sialoprotein (Ibsp) were upregulated in the allograft + PTH treated animals. In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.

PTH promotes allograft integration in a calvarial bone defect.

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the US alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination intravenous MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (p<0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to intravenous MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.Molecular Therapy (2015); doi:10.1038/mt.2015.211.

Article	Year
Insulin-like growth factor I prevents corticosteroid-induced diaphragm muscle atrophy in emphysematous hamsters. American journal of physiology. Regulatory, integrative and comparative physiology\| PubMed ID: 12689851	2003
Short-term influences of lung volume reduction surgery on the diaphragm in emphysematous hamsters. American journal of respiratory and critical care medicine\| PubMed ID: 15201133	2004
Tumor necrosis factor-alpha and malnutrition-induced inhibition of diaphragm fiber growth in young rats. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 16002775	2005
Effect of severe short-term malnutrition on diaphragm muscle signal transduction pathways influencing protein turnover. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 16484360	2006
Skeletal muscle adaptations to testosterone and resistance training in men with COPD. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 17673568	2007
Time course expression of Foxo transcription factors in skeletal muscle following corticosteroid administration. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 19850732	2010
Gene-modified adult stem cells regenerate vertebral bone defect in a rat model. Molecular pharmaceutics\| PubMed ID: 21834548	2011
Testosterone and resistance training effects on muscle nitric oxide synthase isoforms in COPD men. Respiratory medicine\| PubMed ID: 21843929	2012
Effects of malnutrition with or without eicosapentaenoic acid on proteolytic pathways in diaphragm. Respiratory physiology & neurobiology\| PubMed ID: 22019487	2012
PTH promotes allograft integration in a calvarial bone defect. Molecular pharmaceutics\| PubMed ID: 24131143	2013
PTH induces systemically administered mesenchymal stem cells to migrate to and regenerate spine injuries. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 26585691	2015

Xiaoyu Da

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