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

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