Summary Statement. Thickness and cellularity of human periosteum are important parameters both for engineering replacement tissue as well as for surgeons looking to minimise tissue damage while harvesting the most viable periosteum possible for autologous regenerative therapies. This study provides a new foundation for understanding the basic structural features of middiaphyseal periosteum from femora and tibiae of aged donors. Introduction. A number of recent studies describe mechanical, permeability and regenerative properties of periosteal tissue and periosteum derived cells in a variety of animal models [1,2]. However, due to lack of access in healthy patients, the structural properties underlying human periosteum's inherent regenerative power and advanced material properties are not well understood. Periosteum comprises a cellular cambium layer directly apposing the outer surface of bone and an outer fibrous layer encompassed by the surrounding soft tissues. As a first step to elucidate periosteum's structural and cellular characteristics in human bone, the current study aims to measure cambium and fibrous layer thickness as well as cambium cellularity in human femora and tibiae of aged donors. Methods. Five cm segments of the mid-diaphysis were harvested from the left and right tibiae and femora of formalin-fixed cadavers donated to the Department of Anatomy at the Ludwig Maximilians University of Munich. Overlying skin and musculature was preserved during embedding to avoid disruption of periosteal tissue. A total of 29 mid-diaphyseal samples were collected from eight donors, aged between 68 and 99. Cambium layer thickness, fibrous thickness and cambium cell number were measured at regular 100 μm intervals from the centroidal axis along the bone's outer surface (ImageJ 1.42q). The major and minor centroidal axes (CA) serve as automated reference points in cross sections of cadaveric mid-diaphyseal femora and tibiae. Results. Based on the results of this study, within a given individual, the cambium layer of the major CA of the tibia is significantly thicker and more cellular than the respective layer of the femur. These significant intraindividual differences do not translate to significant interindividual differences. Further, mid-diaphyseal periosteal measures including cambium and fibrous layer thickness and cellularity do not correlate significantly with age or body mass. Finally, qualitative observations of periosteum in amputated and contralateral or proximal long bones of the lower extremity exhibit stark changes in layer organization, thickness, and cellularity. Discussion/Conclusion. In a translational context, these unprecedented data, though inherently limited by availability and accessibility of human mid-diaphyseal periosteum tissue, provide important reference values for use of periosteum in context of facilitated healing and regeneration of tissue

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

Vascular Endothelial Growth Factor (VEGF) has been shown to stimulate angiogenesis in a number of tissues and, in addition, to possess direct vasoactive properties. Stimulation of blood flow and angiogenesis are important features of the fracture healing process, particular in the early phases of healing. Inadequate vascularity has been associated with delayed union after fracture. The periosteum, and in particular its osteogenic cambial layer, has been shown to be very reactive to fracture and to contribute substantially to fracture healing. Fracture haematoma contains a considerable concentration of VEGF and enhanced plasma levels are observed in patients with multiple trauma. VEGF has been suggested to play a role during new bone formation possibly providing an important link between hypertrophic cartilage, angiogenesis and consequent ossification. However, the expression of VEGF in normal periosteum and in periosteum close to a fracture has not been previously reported. We hypothesise that the expression of VEGF in long bone periosteum will show a distinct response to fracture. We investigated the expression of VEGF in vivo in human periosteum, using immunocytochemistry to detect the expression of Factor VIII and VEGF protein respectively. Under prior approval from the local Ethics Committee, biopsies of periosteal tissues were collected from two distinct groups (1) control and (2) following long bone fracture. Patient age range was 16 – 45 years for both groups. Group 1 consisted of patients (n = 5) who underwent an elective orthopaedic procedure during which periosteum was disrupted. Group 2 patients (n = 8) had long bone fractures from which periosteal tissue was harvested close to the fracture site during internal fixation at various time points following fracture (24 hours to nine days). In Group 1 the periosteum showed abundant but delicate blood vessels staining throughout for VEGF but there was no other visible staining of other structures or cells. In Group 2 the vasculature in the periosteum close to the fracture site demonstrated a characteristic, time-dependent course of expression of VEGF. At 24 and 48h following fracture the vasculature showed a heterogenous picture. The vessels in periosteum showed signs of activation: thickened endothelia and dilated lumina, but did not express VEGF. At 60h the vessels began to show signs of the presence of VEGF protein and by 4 days most periosteal vessels expressed VEGF. Also at this time, VEGF staining was visible in some of the stromal cells of the periosteum that was not seen in any of the earlier times. At 9 days VEGF was visible not only in the omnipresent vasculature, but now consistently in spindle shaped cells of fibroblastic appearance and chondrocytes throughout the early callus. This study, though limited by the number of patients, shows for the first time the expression of VEGF in normal periosteum as well as in periosteum during fracture healing. Interestingly, activated vessels in the early healing phase show little expression of VEGF; however it is known that the fracture haematoma contains VEGF in abundance. It is possible that the vasoactive role of VEGF prevails in these early days. There may be a critical time point at around 48h post fracture following which angiogenesis begins and VEGF is expressed in the endothelium throughout the vessel wall. The study suggests an important role for VEGF in the regulation of fracture healing. VEGF is not only expressed in endothelial cells within the periosteum but also in fibroblast-like stem cells and chondrocytes throughout the early callus suggesting it may play an important role in both osteo- and angiogenesis

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.

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.

Periosteal mesenchymal stem cells (PMSC) are an emerging niche of stem cells to enhance bone healing by tissue engineering process. They have to be differentiated into osteoprogenitors in order to synthesize new bone matrix. In vitro differentiation with specific differentiation medium (DM) is not exactly representative of what occurs in vivo. The interaction between PMSC and growth factors (GF) present in biological matrix is somewhat less understood. The goal of this study is to explore the possibility of spontaneous PMSC differentiation in contact with different biological matrices without DM. 500.000 porcine PMSC were seeded on 6-well plates and cultured with proliferation medium (PM). When reaching 80% confluence, biological samples (n=3) of demineralized bone matrix (DBM), decellularized porcine bone allograft (AOp), human bone allograft (AOh), human periosteum (HP) and human fascia lata (HFL) were added. Negative and positive control wells included cells with only PM or DM, respectively. The differentiation progress was assessed by Alizarin Red staining at days 7, 14 and 21. Bone morphogenetic protein content (BMP 2, 4, 5, 6, 7, 8, 9 and 11) of each sample was also investigated by western blot. Alizarin red highlighted bone nodules neoformation on wells containing AOp, AOh and DBM, like positive controls. HP and HFL wells did not show any nodules. These results are correlated to a global higher BMP expression profile in AOp than in HP and HFL but not statistically significant (p=0.38 and p>.99, respectively). The highest expression in each tissue was that of BMP2 and BMP7, which play an important role in osteoinduction. PMSC are well known to participate to bone formation but, despite BMP presence in HP and HFL, they did not permit to achieve osteogenesis alone. The bone contact seems to be essential to induce in vitro differentiation into osteoprogenitors

Aims

Cigarette smoking has a negative impact on the skeletal system, causes a decrease in bone mass in both young and old patients, and is considered a risk factor for the development of osteoporosis. In addition, it disturbs the bone healing process and prolongs the healing time after fractures. The mechanisms by which cigarette smoking impairs fracture healing are not fully understood. There are few studies reporting the effects of cigarette smoking on new blood vessel formation during the early stage of fracture healing. We tested the hypothesis that cigarette smoke inhalation may suppress angiogenesis and delay fracture healing.