Aims. Several artificial bone grafts have been developed but fail to achieve anticipated osteogenesis due to their insufficient neovascularization capacity and periosteum support. This study aimed to develop a vascularized bone-periosteum construct (VBPC) to provide better angiogenesis and osteogenesis for bone regeneration. Methods. A total of 24 male New Zealand white rabbits were divided into four groups according to the experimental materials. Allogenic adipose-derived mesenchymal stem cells (AMSCs) were cultured and seeded evenly in the collagen/chitosan sheet to form cell sheet as periosteum. Simultaneously, allogenic AMSCs were seeded onto alginate beads and were cultured to differentiate to endothelial-like cells to form vascularized bone construct (VBC). The cell sheet was wrapped onto VBC to create a vascularized bone-periosteum construct (VBPC). Four different experimental materials – acellular construct, VBC, non-vascularized bone-periosteum construct, and VBPC – were then implanted in bilateral L4-L5 intertransverse space. At 12 weeks post-surgery, the bone-forming capacities were determined by CT, biomechanical testing, histology, and immunohistochemistry staining analyses. Results. At 12 weeks, the VBPC group significantly increased new bone formation volume compared with the other groups. Biomechanical testing demonstrated higher torque strength in the VBPC group. Notably, the haematoxylin and eosin, Masson’s trichrome, and immunohistochemistry-stained histological results revealed that VBPC promoted neovascularization and new bone formation in the spine fusion areas. Conclusion. The tissue-engineered VBPC showed great capability in promoting angiogenesis and osteogenesis in vivo. It may provide a novel approach to create a superior blood supply and nutritional environment to overcome the deficits of current artificial bone graft substitutes. Cite this article: Bone Joint Res 2023;12(12):722–733

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

In both physiological and pathological processes, periosteum plays a determinant role in both bone formation and fracture healing. However, no specific reports are available so far focusing on the detailed structural and major cellular differences between the periostea covering different bone surface areas in relation to ageing. The aim of this study is to compare the structural and cellular differences in diaphyseal and epiphyseal periostea in different-aged rats using histological and immunohistochemical methods. Four female Lewis rats from each group of juvenile (7-week old), mature (7-month old) and aged groups (2-year old) were sacrificed and the right femur of each rat was retrieved, fixed, decalcified and embedded. 5μm thick serial sagittal sections were cut and stained with Hematoxylin and Eosin, Stro-1 (stem cell marker), F4/80 (macrophage marker), TRAP (osteoclast marker) and vWF (endothelial cell marker). 1mm length of middle diaphyseal and epiphyseal periosteum were selected for observation. The thickness, total cell number and positive cell number for each antibody in each periosteal area and different-aged groups were measured and compared. The results were subjected to ANOVA and SNK-q tests. The results showed that the thickness and cell number in diaphyseal periosteum decreased with age (p< 0.001). In comparison with diaphyseal area, the thickness and cell number in epiphyseal periosteum were much higher (p< 0.001). There were no significant differences between the juvenile and aged groups in the thickness and cell number in cambial layer of epiphyseal periosteum (p> 0.05). However, the juvenile rats had more Stro1+, F4/80+ cells and blood vessels and few TRAP+ cells in different periosteal areas compared with other groups(p< 0.001). The aged rats showed much less Stro1+ cells, but more F4/80+,TRAP+ cells and blood vessels in the cambial layer of epiphyseal periosteum (p< 0.001). In conclusion, the age-related structure and cell population in diaphyseal and epiphyseal periostea are different, especially in aged rats. The epiphyseal periosteum of aged rats seems more destructive than diaphyseal part and other age groups. Macrophages in the periosteum play a dual important role in osteogenesis and osteoclastogenesis

Periosteum is important for bone homoeostasis through the release of bone morphogenetic proteins (BMPs) and their effect on osteoprogenitor cells. Smoking has an adverse effect on fracture healing and bone regeneration. The aim of this study was to evaluate the effect of smoking on the expression of the BMPs of human periosteum. Real-time polymerase chain reaction was performed for BMP-2,-4,-6,-7 gene expression in periosteal samples obtained from 45 fractured bones (19 smokers, 26 non-smokers) and 60 non-fractured bones (21 smokers, 39 non-smokers). A hierarchical model of BMP gene expression (BMP-2 > BMP-6 > BMP-4 > BMP-7) was demonstrated in all samples. When smokers and non-smokers were compared, a remarkable reduction in the gene expression of BMP-2, -4 and -6 was noticed in smokers. The comparison of fracture and non-fracture groups demonstrated a higher gene expression of BMP-2, -4 and -7 in the non-fracture samples. Within the subgroups (fracture and non-fracture), BMP gene expression in smokers was either lower but without statistical significance in the majority of BMPs, or similar to that in non-smokers with regard to BMP-4 in fracture and BMP-7 in non-fracture samples. In smokers, BMP gene expression of human periosteum was reduced, demonstrating the effect of smoking at the molecular level by reduction of mRNA transcription of periosteal BMPs. Among the BMPs studied, BMP-2 gene expression was significantly higher, highlighting its role in bone homoeostasis

Summary Statement. The purpose of this experimental imaging study is to determine the Poisson's ratio of ovine periosteum, using strain mapping data from an imaging study designed to elucidate the mechanical environment of periosteal progenitor cells in situ during stance shift loading. Introduction. Periosteum is a composite, so-called “smart” or stimuli responsive material that provides a niche for pluripotent cells that exhibit mechanosensitivity in their proliferative and differentiation behavior. The overarching aim of this research program is to explore, understand, and exploit the mechanical signals that promote cell lineage commitment and de novo bone generation during embryonic development and postnatal healing. Further, our working hypothesis is that periosteum derived progenitor cells are highly sensitive to their local mechanical milieu, which guides their proliferation, motility and differentiation behavior. As a first step toward understand the role of periosteum anisotropy on defining the local mechanical milieu of a given progenitor cell, the objective of the current study is to determine the Poisson's ratio of ovine periosteum and its sensitivity to near, mid- and long-range strains. Methods. The Poisson's ratio for the ovine periosteum was determined using strain mapping data from an high resolution imaging study designed to elucidate the mechanical environment of periosteal progenitor cells in situ. The Poisson's ratio of long bone periosteum is given by the relative ratio of strain in the transverse direction to strain in the axial or longitudinal direction. Given high resolution video imaging data, digital image correlation is used to calculate the average strain and Poisson's ratio between three closest neighbors (nearest neighbor), between 25 points in a 50–150 pixel distance (short range, SR), and between 25 points in a 200–400 pixel distance (long range, LR) of the periosteum during an ex vivo loading setup designed to mimic stance shift loading. Results. Short and long range strains vary with spatial location and time during a given gait cycle. Calculations based on nearest neighbor, SR and LR show maximum strain at different time points in the gait cycle, different ranges of strains, as well as a non-uniform strain field that exhibits both spatial and temporal variation. Hence, the Poisson's ratio is highly dependent on location and time. Follow on studies at lower length scales allowing for subcellular length scale strain measurement are underway to accurately account for the in situ mechanical environment of a given periosteal progenitor cell, e.g. in order to relate its functional loading environment to its biological (proliferation, migration, and differentiation) behavior. Discussion/Conclusion. These results underscore the imperative not only to carry out high resolution imaging measurements but also to elucidate the structure-function relationships at smaller length scales, as these are necessary to elucidate both the origins of emergent, advanced material properties of the periosteum as well as mechanically modulation of progenitor cell proliferation, migration and differentiation

Aim. We evaluated the effect of the intact periosteum on the biomechanical properties of the rat long bones. Materials-Methods. The biomechanical properties of both femora and tibiae of 30 male, 4-month old Wistar rats have been evaluated in three-point bending testing. In one bone of each pair of femora or tibiae the periosteum was preserved intact, while in the contra-lateral bone the periosteum was stripped off. Ultimate strength,stiffness,energy absorption and deflection were derived automatically from the load-deformation curve recorded for each bone. Results. As regards the femur, the periosteum-covered bones displayed statistically significant higher values for all parameters measured compared to the periosteum-stripped bones. In the tibia, only energy absorption and deflection were significantly higher in the periosteum-covered bones. The fracture pattern was also different in these two groups. The periosteum-stripped femora and tibiae failed catastrophically, while in the periosteum-covered bones the two bone parts remained in close apposition stabilized by the periosteal membrane. Conclusion. The periosteum exacerbates the biomechanical capacity of intact rat long bones examined in bending, probably taking advantage of its fibrous composition and elastic properties

Autologous chondrocytes transplantation (ACT) was first used in humans in 1987 and is based on a surgical technique where cells are injected under a periosteal flap. Due to the sometimes tricky surgical isolation and suture of the periosteum and complications with hypertrophy of periosteal tissue (5 – 10% of the cases) that in some cases requires a second arthroscopic trimming ‘easier’ transplantation techniques based on cells cultured on scaffolds and membranes have been suggested. However, the standard ACT technique creates a unique in vivo bioreactor where chondrocytes and periosteum form a unique local environment. If live periosteum and chondrocytes are transplanted to a defect in the rabbit patellae a cartilage repair tissue is formed in contrast to treatment with ‘dead’ periosteum and live chondrocytes were no repair tissue is demonstrated. The unique environment formed by the periosteum and chondrocytes might be responsible for the unique in vivo induction of early embryological development patterns seen in limb formation in the foetus: We have found that the transplanted chondrocytes are expressing early developmental genes e.g Sox 9 and wnt14 and fibroblast growth factor 3 receptors (FGFR3), a marker of chondrocytes progenitor cells. Furthermore, we have found that the articular chondrocytes are able to demonstrate a phenotypic expressivity with an additional ability of bone and adipose tissue formation. Changes to the transplantation procedure must address these unique features of the ACT technology in order to maintain the long term clinical outcome

This study aimed to explore the relationship between the geometry of the tuberosity located superior to the Achilles tendon enthesis and the thickness of its fibro-cartilaginous periosteum. The tuberosity acts as a pulley for the tendon during dorsiflexion of the foot and is thus compressed by the overlying tendon. This can result in pressure-related injuries which account for a significant number of Achilles-related problems among sportsmen or women. We postulated that variations in the contact area between the tendon and the tuberosity (and consequently the pressure exerted by the tendon) affects the periosteum thickness. Here, we report four methods of portraying the two dimensional geometry of the superior tuberosity. Material was obtained from 10 elderly dissecting room cadavers donated to the Cardiff University for anatomical examination and prepared for routine histology. Serial sagittal sections were collected at 1 mm intervals, and stained with Masson’s trichrome, toluidine blue and haematoxylin & eosin. In the first method, the area of the bursal cavity was measured between the deep surface of the tendon and the tuberosity within a 9mm radius of the proximal part of the attachment site. The second technique was similar, though used the long axis of the tendon as a reference, rather than its deep surface. The third technique measured the area of the tuberosity within 20 degrees of the tendon long axis. The final technique measured the cumulative gradient of the first 5 mm of the tuberosity, with reference to the tendon long axis. The periosteum thickness was measured at 500 μm intervals from the proximal part of the enthesis and mean values calculated. A good correlation was seen between all techniques, with the tuberosities having the most localised area of contact with the tendon, showing the thickest periosteum

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

Introduction: Normal adult periosteum and cortical and produces no signal with typical bone has a short T. 2. Magnetic Resonance pulse sequence echo times available in clinical practice. We wished to assess the value of using pulse sequences with a very short echo time to detect signal from periosteum and cortical bone. Materials and Methods: Ultrashort echo time (UTE) pulse sequences (TE = 0.08 msec) were used with and without preceding fat suppression and/or long T. 2. component suppression pulses. Later echo images and difference images produced by subtracting these from the first echo image were also obtained. Two volunteers and ten patients were examined, four of whom had contrast enhancement with intravenous Gadodiamide. Two sheep tibiae were also examined before and after stripping of the periosteum. The separated periosteum was also examined. Results: The periosteum was seen on the sheep tibiae before stripping but there was only a faint signal adjacent to cortical bone afterwards and the removed tissue produced a high signal when examined separately. High signal regions were observed adjacent to cortical bone in the femur, tibia, spine, calcaneus, radius, ulna and carpal bones. Fat suppression and long T. 2. suppression generally increased the conspicuity of these regions. The high signal regions were more obvious with contrast enhancement. Periosteum could generally be distinguished from susceptibility artifacts on difference images by its high signal on the initial image and its failure to increase in extent with images with increased TE’s. Signal in cortical bone was detected with UTE sequences in normal adults and patients. This signal was usually made more obvious by subtracting a later echo image from the first provided that the SNR was sufficiently high. Normal mean adult T. 1. ’s ranged from 140 msec to 260 msec, and mean T. 2. ’s ranged from 0.42 to 0.50 msec. Increased signal was observed after contrast enhancement in a normal volunteer and in all three patients in whom it was administered. Changes in signal in short T. 2. components were seen in acute fractures in cortical bone and after fracture malunion. In a case of osteoporosis, bone volume and signal were reduced. Furthermore, in fractures increased signal was seen in the periosteum and this showed marked enhancement. Three weeks after fracture, tissue with properties consistent with periosteum was seen displaced from the bone by callus. Discussion: The normal adult periosteum and cortex can be visualized with ultrashort TE sequences. Conspicuity is usually improved by fat suppression and the use of difference images. Use of subtraction images was useful for selectively demonstrating periosteal and cortical contrast enhancement and separating this from enhancement of surrounding blood. Obvious periosteal and cortical enhancement was seen after fractures. This novel MRI sequence images for the first time the soft tissue component of cortical bone and enables visualization of different haemodynamic situations

Summary Statement. MCP-1/ CCR2 axis at the early phase plays a pivotal role in the fracture healing. Inflammation plays a pivotal role in fracture healing. Among them, chemokines play key roles in inflammation. Monocyte chemotactic protein-1 (MCP-1), via its receptor C-C chemokine receptor 2 (CCR2), acts as a potent chemoattractant for various cells to promote migration from circulation to inflammation site. Thus, the importance of MCP-1/CCR2 axis in fracture healing has been suggested. However, the involvement of MCP-1/CCR2 axis tofracture site is not fully elucidated. Results. PCR Array: The expression of MCP-1 and MCP-3 had increased on day 2 than 0 or 7 in the rib fracture healing. Immunohistochemistry Staining: To verify the localization of MCP-1 expression, we examined the Wild type (WT)-mouse rib fracture healing. We observed high expression of MCP-1 and MCP-3 at the periosteum and the endosteum on post-fracture day 3. In vivo Antagonist Study: To elucidate whether MCP-1/CCR2 axis is involved during the early phase of fracture healing, we continuously administered RS102895, CCR2 antagonist, before or after rib fracture. Micro-CT analysis showed delayed fracture healing in the before-group compared with both the control and after-group. On day 21, the hard callus volume in the before-group was significantly smaller than that in the control-group. Histological analysis showed that fractures in both the control and the after-groups were healed by day 21. In contrast, less of cartilage in the callus was observed in the before-group on day 7. Gain of Function: To examine the roles of MCP-1 at the periosteum and the endosteum during the fracture healing, we created a segmental bone graft exchanging model. The bone grafts were transplanted from MCP-1. −/−. mice to another MCP-1. −/−. mice (KO-to-KO). Micro-CT analysis showed that KO-to-KO transplantation led to the delay of fracture healing on day 21. Next, we created exchanging-bone graft models between MCP-1. −/−. and WT mice, in which a segmental bone derived from a WT mouse was transplanted into a host MCP-1. −/−. mouse (WT-to-KO). In contrast to KO-to-KO bone graft transplantation, the transplantation of WT-derived graft into host KO mouse resulted in a significant increase of new bone formation on day 21. Histological analysis revealed that marked and localised induction of MCP-1 expression in the periosteum and the endosteum around the WT-derived graft was observed in the host MCP-1. −/−. mouse. Loss of Function: To validate whether MCP-1 is a crucial chemokine for fracture healing, we created WT-to-WT and KO-to-WT bone graft models. When WT-donor graft was transplanted into WT-host, abundant new bone formation was observed around a WT-derived graft on day 21. In contrast, transplantation of KO-derived graft into WT-host resulted in a marked reduction of periosteal bone formation on a donor graft. Discussion. In this study, we demonstrated that MCP-1/ CCR2 axis at the early phase modulates the fracture healing. Furthermore, we showed that MCP-1 in the periosteum and the endosteum promotes the fracture healing in vivo. Thus, these results clearly suggest that MCP-1 in the periosteum and the endosteum at the early inflammatory phase is an essential component for successful fracture healing

Summary Statement. The Dkk3-derived cells represent a branch of the periosteal mesenchymal lineage that produces fibrocartilage as well as regenerating the periosteal structures. Introduction. Mesenchymal progenitor cells are capable of generating a wide variety of mature cells that constitute the connective tissue system. Our Laboratory has been developing SMAA GFP reporter mice to prove to be an effective tool for identifying these cells prior to the expression of markers of differentiation characteristic of bone, fat, muscular blood vessels or fibrocartilage. Dkk3 was chosen as a candidate reporter because microarray of SMAA-sorted cells culture indicated high expression of this non-canonical anti-Wnt factor, which was not anticipated in a culture with strong osteogenic potential. Material and Methods. Fracture healing process was evaluated in 12 week old male mice at 3, 5, 7, 14, 21 and 28days post fracture. A 3 color reporter mouse was generated by crossing SMAA-GFPcherry × Col3.6GFPcyan × Dkk3-eGFP and subjected to tibial fracture. A closed transverse fracture was performed by Einhorn device under isoflurane anesthesia after insertion of intramedullary pinning. Longitudinal 5 mm non-calcified cryosections were stabilised with Cryofilm tape. Results. Three days post fracture, the proliferating SMAA-red cells were also beginning to express either Dkk3 or Col3.6. By day 5 the two populations had diverged with the Dkk3 cells being on the outer surface of the developing callus while the Col3.6 cells were forming bone at the base of the callus. By day 7 when the callus is filled with cartilage, Dkk3 is active in cells that are in transition from elongated cells on the external surface of the callus to fibrocartilagenous cells that now express low levels of Col3.6. The zone of cells that express Dkk3 appear to block the passage of the surrounding vasculature into the underlying cartilage and does not deposit fibronectin. By day 14–21 when the cartilage core is resorbed, the only remaining Dkk3 is located in the newly formed periosteum external to the active endocortical bone forming activity associated with the inward remodeling of the outer cortical shell. Discussion. We interpret these findings that Dkk3 marks a non-osteogenic limb of the SMAA progenitor population that within the fracture partitions the osteogenic signals away from the surrounding skeletal muscle and the underlying differentiating fibrocartilage. It is a progenitor to cells that form fibrocartilage in the fracture zone as well as the tenascin C positive cells that populate the fibrous zone of the periosteum, and it resides in the cambial zone of the periosteum. Knowing the biological and molecular function of these cells should lead to a fuller appreciation of the pro- and anti-osteogenic factors that regulate skeletal repair

Aim: This study aimed to investigate the ability of vascularized periosteum to induce bone formation under functional loading in vivo. Method: Sixteen juvenile mini pigs were used, assigned in 4 different groups. In goup A, a 1,4 cm rib gap was internally þxated and the periosteum ßap was entirely preserved and sutured in situ. In group B the same method was followed, but the periosteum adjacent to the gap was completely excised. In group C, the periosteum was preserved; þxation was used and in addition to these, a biologically inert cement was used to obliterate the marrow cavities at the osteotomy sites. Finally, group D (control) included animals in which the gap was left without þxation and periosteum was completely removed. Specimens were harvested at 8 weeks and were evaluated macroscopically, radiologically and histopathologically. Data was analyzed using Fisherñs exact test and non-parametric statistics. Results: Results of this study showed that all gaps created in group A and 10 of 11 in group C demonstrated complete bone formation, bridging the entire defect. No traces of bone formation were observed in groups B and D. Conclusion: Rib periosteum has extremely high osteogenic capacity and can bridge large defects in vivo under the following conditions: a) its vascular supply is preserved and b) rigid þxation and functional loading is applied

Autologous chondrocyte implantation (ACI) has been used for many years for the treatment of symptomatic defects in articular joints, predominantly the knee. Traditionally, cells were implanted behind a periosteal membrane, but in more recent times Chondrogide, a membrane consisting of porcine collagens I and III, has been used. There have been trials comparing the clinical outcome of these two groups of patients; in this study we compare the histological outcome using the two different patch types. In a study of 100 patients having received ACI treatment of cartilage defects in the knee, 41 received Chondrogide (ACI-C) and 59 received periosteum (ACI-P). All of these patients had a post-operative biopsy taken at a mean of 16.9±9.2 months and 20.8±23.2 months for ACI-C and ACI-P respectively for histology using the ICRS II scoring system. Lysholm scores, a measure of knee function, were obtained pre- and post-operatively at the time of biopsy and statistical differences tested for via a Mann-Whitney U-test. The mean age of the two groups at treatment was 37±8 and 35±10 years, the size of defect treated was 6.1±5.4 and 4.4±2.7 cm2 and the biopsy follow-up time was 50.6±22.2 and 81.2±34.8 months for ACI-C and ACI-P patients respectively. Both groups exhibited a significant improvement in Lysholm score from pre-operative to the time of biopsy (14.3±25.7; n=100), although there was no significant difference in improvement in Lysholm score between the two patch types. There was no significant difference between the histology score of the two groups, nor was the score found to correlate with the Lysholm score at that time. The individual components of the ICRS II score did not differ significantly with patch type (even for the surface architecture) apart from cellular morphology which was 6.5±3 and 8.2±1.6 for ACI-C and ACI-P respectively. The histological quality of repair tissue formed with ACI-C differed little from that seen with ACI-P, despite the former group being biopsied ∼4 months sooner after treatment and being used to treat defects which were 39% larger. Hence Chondrogide appears just as suitable as periosteum for use as a patch in the procedure of ACI

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

We hypothesise that the Masquelet induced membrane used for the reconstruction of large bone defects were likely to involve mesenchymal stem cells (MSCs), given the excellent resultant skeletal repair. This study represents the first characterisation in humans of the induced membrane formed as a result of the Masquelet technique. Methods. Induced membranes and matching periosteum were harvested from 7 patients. Cytokines (BMP2, VEGF, SDF1) and cell lineage markers (CD31, CD271, CD146) were studied by immunohistochemisty. Flow cytometry was used to measure the cellularity and cellular composition. MSCs were enumerated using a colony forming unit fibroblast assay. In expanded cultures, a 96-gene array card was used to assess their transcriptional profile. Alkaline phophatase, alizarin red and calcium assays were employed to measure their in vitro osteogenic potential. Results. Membrane was more cellular(p=0.028), had more MSC phenotype(p=0.043) compared to matched periosteum. The molecular profiles were similar, except for 2-fold abundance of SDF-1 in membrane (p=0.043)compared to periosteum. Membrane and periosteum had a similar proportion of endothelial cells and CFU-F colonies; expanded MSCs from both sources were highly osteogenic. Discussion. These results indicate that the induced membrane possesses a rich source of MSC and therefore our findings support the view that the induced membrane plays an active role in bone regeneration

INTRODUCTION. The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted hypertrophic differentiation of the generated tissue due to endochondral ossification. Continuing on our earlier studies, our goal is to further improve the engineering of hyaline cartilage for the treatment of a cartilage defect in our in vivo model for subperiosteal generation of cartilage, by tuning the differentiation status of the generated cartilage and prevent hypertrophic differentiation. As a healthy cartilage matrix contains high amounts of aggrecan we hypothesise that aggrecan supplementation of the bio-gel used in the generation of the subperiosteal cartilage, mimics the composition of the extracellular matrix environment of cartilage with potential beneficial properties for the engineered cartilage. METHODS. A 2% (m/v) low melting agarose was injected between the bone and periosteum at the upper medial side of the tibia of both legs of New Zealand white rabbits (DEC 2012–151). The agarose was left unloaded (n=7) or supplemented (n=7) with 2% (w/v) bovine aggrecan (Sigma-Aldrich). After 14 days, rabbits were euthanised. Generated subperiosteal cartilage tissue was analysed for weight, GAG and DNA content. In addition, RT-qPCR and (immuno)histochemistry was performed for key markers of different phases of endochondral ossification. RESULTS. The nett weight of the generated subperiosteal cartilage tissue was not significantly different between groups, nor was the GAG content different. No significant differences in chondrogenic marker expression (COL2A1, SOX9, ACAN and PTHrP) were detected. Interestingly, gene expression levels of hypertrophic markers COL10A1 and ALPL were significantly decreased. COL1A1 expression was not significantly different between groups. DISCUSSION. In summary, generation of subperiosteal cartilage was successful when an agarose bio-gel was injected beneath the periosteum. The addition of aggrecan to the bio-gel did not result in differences in weight or GAG content in cartilage samples between conditions. However, lower levels of hypertrophic markers were observed, while leaving chondrogenic marker expression unaltered. These data show the potential of aggrecan to favourably influence the subperiosteal microenvironment for the in vivo generation of hyaline cartilage for the optimisation of cartilage regenerative medicine approaches

The histology and mechanics of leg lengthening by callus distraction were studied in 27 growing rabbits. Tibial diaphyses were subjected to subperiosteal osteotomy, held in a neutral position for 10 days and then slowly distracted at 0.25 mm/12 hours, using a dynamic external fixator. Radiographs showed that the gap became filled with callus having three distinct zones. Elongation appeared to occur in a central radiolucent zone; this was bounded by two sclerotic zones. Histologically, the radiolucent zone consisted of longitudinally arranged cartilage and fibrous tissue while the sclerotic zones were formed by fine cancellous bone. New bone occasionally contained islands of cartilage, suggesting it had been formed by endochondral ossification. After completion of distraction, the two sclerotic zones fused, shrank and were eventually absorbed, leaving tubular bone with a new cortex. When the periosteum had been removed at the operation, callus formation was markedly disturbed and there was failure of bone lengthening. Scraping of endosteum, in contrast, did not have a pronounced effect. These results suggest that the preservation of periosteum is essential if bone lengthening by callus distraction is to succeed, and that preservation of the periosteum is more important than careful corticotomy

This study aimed to investigate the ability of vascularized periosteum to induce bone formation under functional loading in vivo. To achieve this, a gap was created in the ribs of mini pigs while functional loading was provided by the respiratory movements. Sixteen juvenile mini pigs were used, assigned in 4 different groups. In group A, a 1,4 cm rib gap was internally fixated (KLS Martin LP 2,0 mm mini plates and screws) and the periosteum flap was entirely preserved and sutured in situ. In group B the same method was followed, but the periosteum adjacent to the gap was completely excised. In group C, the periosteum was preserved; fixation was used and in addition to these, a biologically inert cement was used to obliterate the marrow cavities at the osteotomy sites. Finally, group D (control) included animals in which the gap was left without fixation and periosteum was completely removed. Specimens were harvested at 8 weeks and were evaluated macroscopically, radiologically and histopathologically. Data was analyzed using Fisher’s exact test and non-parametric statistics. Results of this study showed that all gaps created in group A and 10 in 11 in group C demonstrated complete bone formation, bridging the entire defect. No traces of bone formation were observed in groups B and D. These results indicate that rib periosteum has extremely high osteogenic capacity and can bridge large defects in vivo under the following conditions: a) its vascular supply is preserved and b) rigid fixation and functional loading is applied

Background: Conventional magnetic resonance pulse sequence echo times (TEs) produces no signal of cortical bone. In this pilot study we wished to explore the value of a novel pulse sequence with an ultrashort echo time (UTE), which is able to detect signal from cortical bone and periosteum (Ref.). The signal obtained using an UTE sequence from cortical bone reflects the soft tissue component of cortical bone including its vasculature. We hypothesized that conditions, which alter the soft tissue component and vascularity of bone, show a change in signal. We have examined the lower limb in patients and volunteers of different age and at different time points following fracture of the tibia. Subjects and Methods: Seven volunteers (aged 29 – 85 years) and eight patients with acute fractures of the tibia (aged 18 – 56 years) were examined at different time points (2 days – 16 weeks) following fracture, in three of the patients serial scans were obtained. Three patients were examined years following bone injury: one patient with a hypertrophic mal-union at 5 years, one patient with polio 14 years following a tibial osteotomy and one patient 28 years following a tibial fracture. Ultra-short echo time pulse sequences (TE = 0.07 or 0.08 ms) were used with and without preceding fat suppression and / or long T2 component suppression pulses. Intravenous gadolinium (0.3 mmol/kg) was administered to one volunteer and three of the patients. Mean signal intensity (AU) was plotted against time following contrast enhancement. T1 and T2* values for cortical bone were determined and T1 was plotted against age. Results A signal was obtained of cortical bone, periosteum and callus in all subjects. The injection of contrast enhanced the signal in all of these tissues. Distribution curves of gadolinium in cortical bone showed enhanced signal intensity following fracture. The signal was dependent on the type and severity of fracture and the time following fracture. There was a marked increase in signal in a hypertrophic mal-union 5 years following fracture and a moderate increase in signal was still detectable 28 years following fracture. Osteoporosis associated with polio reduced volume and signal of bone. T1 echo times ranged from 140 – 260 ms and increased significantly with age (P < 0.01). T2* ranged from 0.42 – 0.50 ms. Fat suppression and long T2 suppression increased the conspicuity of the periosteum. Conclusion: Magnetic resonance imaging using UTE sequences is able to detect a signal from cortical bone for the first time. Cortical bone, callus and adult periosteum show a distinct signal following fracture with a characteristic time course. Measurements reflect the organic matrix rather than the inorganic crystals of bone. The T1 of cortical bone is very short and changes with age. The distribution curve of gadolinium can be established in cortical bone and is understood to reflect changes in blood flow. We present a pilot study to introduce a new MRI sequence, which at present a research tool, has potential for selected clinical application