Avascular necrosis (AVN) is a disorder leading to femoral head (FH) destruction, while BMPs are known for their osteogenic ability. In this study we analyzed BMP-2, BMP-4, BMP-6 and BMP-7 expression at the RNA and protein level in the normal and necrotic sites of the FHs.

Quantitative RT-PCR for BMP-2,-4,-6,-7 genes was performed in samples from the normal and necrotic sites of 52 FHs with AVN. Protein levels of BMP-2,-4,-6 were estimated by Western Blot analysis. Statistical analysis was performed using the t-test (p< 0.05).

BMP-2 and BMP-6 mRNA levels were higher in the normal than the necrotic site (BMP-2 and BMP-6, normal vs necrotic: 16.8 vs 7.5 and 2 vs 1.66, respectively). On the contrary, BMP-4 mRNA levels were higher in the necrotic (1.2) than the normal site (0.97), while BMP-7 mRNA levels were low in both sites. At the protein level, BMP-2 expressed higher in the normal (0.63) than the necrotic region (0.58), while BMP-4 and BMP-6 detected at higher levels in the necrotic site (BMP-4 and BMP-6, normal vs necrotic: 0.51 vs 0.61 and 0.52 vs 0.57, respectively).

Different mRNA levels between the normal and necrotic site, as well as discrepancies between the gene and protein BMPs expression levels suggest a different regulation mechanism between the two regions. Better understanding of the expression pattern of BMPs could lead to a more successful use of these molecules in the prevention and treatment of AVN

Periosteum is a specialized connective tissue that surrounds bone, containing progenitor cells that develop into osteoblasts. The osteo-progenitor cells along with growth factors, such as BMPs, play critical role in development, reconstruction and bone formation. Aim: to evaluate the expression of BMPs in human periosteum and in different subrgroups, including different donor sites, gender, and smoking habits.

Gene expression of BMPs 2,4,6,7 was performed in 60 periosteal samples using quantitative RT-PCR. Samples were obtained from 32 men/28 women, 22 smokers/38 non-smokers, 29 lower/31 upper extremities.

BMP2 gene expression was significantly higher (median: 12.02, p< 0.05) than the mRNA levels of BMPs 4,6,7 (median: 1.36, 2.55, 0.04) in all samples. BMP2 mRNA levels were higher in large compared to small bones (median: 13.4 vs 9.48), while BMPs 4,6,7 gene expression was similar (1.3 vs 1.4, 2.7 vs 2.1, 0.04 vs 0.03, respectively). In lower extremities, BMPs mRNA levels were higher than in the upper; the same was detected in non-smokers versus smokers group (BMPs2,4,6,7: 13.9 vs 1.5, 3.1 vs 0.048, 8.7 vs 1.06, 1.6 vs 0.026, respectively). mRNA transcripts of BMP2 were higher in men than women (median: 13.1 vs 10.8).

In our study, BMP2 expression is characteristically higher than that of BMP4, BMP6 and BMP7, highlighting the critical role that BMP2 plays in bone homeostasis. Furthermore, the elevated expression of BMP2 in men towards women, and of all BMPs of the lower extremity samples indicate the effect of hormones and mechanical factors in periosteal BMPs gene regulation; while the effect of smoking is reflected in the reduction of BMPs expression in smokers.

We evaluated the contribution of specific gene polymorphisms of IL-1a/IL-1R/IL-1RA/IL-4Ra/IL-1b/IL-12/γIFN/TGF-b/TNF-a/IL-2/IL-4/IL-6/IL-10 cytokines in patients with AVN.

DNA was extracted from 112 patients and 238 healthy Greek individuals. DNA analysis was performed by the PCR-SSP method and the use of the Protrans kit. Statistical analysis was performed by χ2 test.

In the patients, the TC frequency of the IL-1a (nt-889) was 52% while in normal was 40%. The C/G allele frequency of TGF-b codon 25 in patients was 9% C and 91% G vs 13% C and 87% G in normal. At position −238 of TNFa, 11% of the patients had the GA genotype in contrast to 1% of the controls. The GG/GG haplotype of TNFa gene promoter (nt. −308 and −238) was more frequent in both groups, while the GG/GA haplotype detected in 9% and 1% of the patients and controls, respectively. At the −1082 position of the IL-10 gene, the GG genotype was detected in 15% of the controls and 7% of the patients. Also, the GCC/GCC haplotype in IL-10 (positions -1082/-819/-592) was higher in the controls (15%) than the patients (7%).

The genotypes TC (nt-889) of IL-1a, GC (codon 25) of TGF-b, GC (nt-1082) of IL-10 and GA (nt −238) of TNFa, are more prevalent in the patients than the healthy individuals (p< 0.05). Based on our results, the presence of one of the above mentioned polymorphisms or the simultaneous carriage of more than one may contribute to the risk for osteonecrosis

Introduction: Osteoporosis is a common skeletal disease characterized by a combination of low bone mass and altered bone microarchitecture with a consequent increase of fragility. The human CER1 is a novel candidate gene for osteoporosis that can bind directly to bone morphogenetic proteins and inhibit their activity. In this study we evaluated the contribution of five novel gene single-nucleotide polymorphisms (SNPs) of CER1 in blood samples from osteoporotic and control groups.

Materials and Methods: Peripheral blood samples from 100 postmenopausal women with osteoporosis and 50 healthy Greek women, between 45 and 85 years of age, were collected and DNA was extracted. CER1 polymorphisms genotyping was carried out by PCR and sequencing of the whole gene. Bone mineral density (BMD) was examined by DXA. Statistical analysis was performed using Pearson χ2 or Fisher’s exact test in order to compare allelic frequency distribution.

Results: Genetic analysis of the CER1 gene revealed five SNPs at the positions 239C> G (rs3747532), 1058G> T (rs1494360), 2160A> G, 2355A> G (rs17289263), and 2749T> C of the CER1 gene. The above genotypes were distributed differently among osteoporotic and controls. In osteoporotic patients, the SNPs frequencies were: 78.6% heterozygotes and 3.6% homozygotes for 239C> G SNP, 66.7% and 4.3% heterozygotes and homozygotes, respectively, with T allele at the position 1058, 52.4% heterozygotes and 9.5% homozygotes for the polymorphic site A> G nt.2160, 51.2% heterozygotes and 2.4% homozygotes for the G allele at 2355 position of the CER1 gene, whereas only heterozygotes (38.9%) for the 2749T> C polymorphic site were determined (P< 0.001). However, in the control group the polymorphisms were detected only in heterozygosity and the overall distributions of the polymorphisms 239C> G, 1058G> T, 2160A> G, 2355A-> G, and 2749T> C, were 38.9%, 31.3%, 15.6%, 9.4%, 6.9% (P< 0.001), respectively.

Discussion: All the above polymorphisms, except the SNP rs3747532, are correlated with osteoporotic patients for the first time. Allele frequencies of the control group are significantly lower than those of osteoporotic for any of the five polymorphisms. These data provide the first evidence of an association (and most possible significant cumulative contribution) between the aforementioned genotypes in CER1 gene and the risk for osteoporosis in postmenopausal women.

Introduction: Avascular necrosis (AVN) of the femoral head (FH) is a painful disorder of the hip that leads to hip collapse. The pathology of AVN involves ischemic events leading to the death of bone. Several biological substances participate in the balance between osteoclasts and osteoblasts, like osteoprotegerin, RANK and RANKL. The expression of these genes affects the maturation and function of osteoblasts and osteoclasts and determines the rate of bone remodeling. In this study, we investigate the expression of OPG, RANK and RANKL in osteonecrotic FHs derived from 44 patients with AVN.

Methods and Materials: RNA and proteins were isolated from both necrotic and normal site of FHs of 44 patients diagnosed with AVN.

Quantitative RT-PCR was performed for OPG, RANKL and RANK molecules by using the Light Cycler FastStart DNA Master Hybridization Probes kit (Roche).

Western Blotting: 22 bone tissues were run on 4–12% NuPAGE gel (Invitrogen). Anti-OPG, anti-RANKL and anti-actin antibodies were used and membranes were immersed in ECL.

Results: Quantitative RT-PCR: The mRNA levels of OPG were higher in the necrotic (median: 5.25) than the normal site (median: 4.19) of the FHs and their difference was statistically significant (p< 0.05). The expression of RANK and RANKL was significantly lower than that of OPG following a similar pattern between the necrotic and normal site. The mRNA values of RANK and RANKL were higher in the necrotic sites [necrotic median: 1.0/normal median: 0.85, necrotic median: 0.8, normal median: 0.3, respectively] than the normal, although they were not statistically significant.

Western Blotting analysis: Normal sites from all FHs showed comparable OPG protein levels (median: 0.57) which were similar to those of normal (median: 0.63). Similar pattern to that of OPG was observed also for RANKL protein expression, where the median value for RANKL/F-actin ratio was 0.49 and 0.5 in normal and necrotic sites of FHs, respectively.

Discussion: OPG, RANK and RANKL are key genes for maintaining the balance between osteoblasts and osteoclasts. Our results show marked differences in the expression of OPG between the necrotic and the normal sites of the FHs; however, mRNA levels of RANKL varied insignificantly between normal and necrotic part of FH while mRNA levels of RANK gene remain similar in both sides of FHs. In contrast, the production of OPG and RANKL at the protein level showed no remarkable divergence. This indicates that the expression and production pattern of RANK may play the key role in the maintenance of the balance between osteoblasts and osteoclasts in AVN.

Introduction: Periosteum is a tissue with pluripotential mesenchymal cells (MSCs). During fracture repair several growth factors are released from periosteum, including bone morphogenetic proteins (BMPs), which induce the differentiation of bone marrow stromal cells towards the osteoblastic lineage, therefore increasing the pool of mature bone forming cells and enhance the differentiated function of osteoblasts.

The purpose of our study is to evaluate the expression of periosteal BMPs mRNA from fracture samples, collected within 24 hours of fracture and to compare it with BMPs expression from periosteal samples of normal (non-fractured) bones.

Materials and Methods: Periosteum samples were collected from 25 patients with recent fracture (during the past 24 hours) (age: 12–80) and 25 individuals without fracture (age: 10–73). BMPs (BMP2, BMP4, BMP6) mRNA levels were analysed by Real Time RT-PCR by using the Light Cycler machine and PBGD as a housekeeping gene.

Results: BMP2 mRNA levels were significantly higher (p< 0.05) in normal samples (median:12.15) than in fracture (median:4.39). BMP6 and BMP4 mRNA expression followed similar pattern to that of BMP2 but in significant lower levels. In normal samples, BMP4 mRNA median levels were 1.99, while in fracture samples the levels were significantly lower (median:0.35), (p< 0.05). BMP6 mRNA levels were also higher in normal samples (median:2.21) than in fractures (median:1.87) (p> 0.05). Furthermore, the decrease of BMPs mRNA levels in fracture samples was higher for BMP4 followed by BMP2 and BMP6.

Discussion: Our results indicate high BMP2 mRNA levels expressed from periosteal cells. In recent fractures there is a significant reduction of BMP2 compared to normal samples; however, the expression of BMP2 remains more elevated in comparison to the other BMPs highlighting the potential role of BMP2 at the initiation of healing process of fractures. BMP6 and BMP4 expression was similar among normal periosteal cells while levels of BMP6 were higher than BMP4 in fracture periosteal cells. The suppression of BMP6 expression was minimum and less significant than BMP2 and BMP4 suppression indicating the potential role of BMP6 at the early stages of MSCs differentiation in periosteum. On the other hand, BMP4 remains in low levels in any confrontation and seems that plays a minor role in early healing process of fracture. BMPs are considered to play central role in fracture response and bone remodelling but further investigation has to be done as much in their correlation and toward other growth factors as in their expression levels during bone fracture repair process.

Aims: Previous work at this institution has demonstrated that perfusion beneath circumferential negative pressure wound therapy (NPWT) is decreased, which conflicts with most studies on NPWT and perfusion. This study investigates perfusion beneath non-circumferential NPWT in humans and also discusses the potential methodology flaw common to all previous research in this field.

Methods: Tests were conducted on both lower limbs of six volunteers (N=12). Volunteers were sequentially randomised into two groups, which would receive different suction pressures (−400 mmHg and −125 mmHg). A doughnut shaped NPWT dressing was placed over the shin of each leg. The central hole allowed for measurement of the transcutaneous partial pressure of oxygen (tcpO2), an indirect measure of perfusion. Readings were taken every five minutes throughout the experiment. After acquiring readings for 15 minutes to establish a baseline, suction was switched on and readings were taken for another 15 minutes. Suction was then disconnected and readings were taken for 15 minutes. Suction was then reapplied and the sequence was repeated. Data were analysed using the Wilcoxon and Mann-Whitney tests.

Results: On applying suction pressures of −400 mmHg, there was a significant reduction of the tcpO2 (mean reduction 7.35 mmHg, SD 7.4, p< 0.0005). At −125 mmHg, there was also a significant reduction of the tcpO2 (mean reduction 5.10 mmHg, SD 7.4, p< 0.0005). Although there was a tendency for greater reductions in the −400 mmHg group this was not significantly different to the −125 mmHg group (p=0.07).

Conclusion: NPWT reduces tissue perfusion, with higher suction pressure resulting in greater reductions in perfusion. Studies on perfusion using laser Doppler, which report findings contrary to these, may be flawed due to the measuring technique of this device. The compressive forces of NPWT are likely to result in false increased readings on application of suction when using the laser Doppler.

This represents a paradigm shift in our understanding of NWPT and that these dressings should be used with caution on tissues with compromised perfusion.