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

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

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

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. 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

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity. This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes. PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity. This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions

The medial periosteal hinge plays a key role in fractures of the head of the humerus, offering mechanical support during and after reduction and maintaining perfusion of the head by the vessels in the posteromedial periosteum. We have investigated the biomechanical properties of the medial periosteum in fractures of the proximal humerus using a standard model in 20 fresh-frozen cadaver specimens comparable in age, gender and bone mineral density. After creating the fracture, we displaced the humeral head medial or lateral to the shaft with controlled force until complete disruption of the posteromedial periosteum was recorded. As the quality of periosteum might be affected by age and bone quality, the results were correlated with the age and the local bone mineral density of the specimens measured with quantitative CT. Periosteal rupture started at a mean displacement of 2.96 mm (. sd. 2.92) with a mean load of 100.9 N (. sd. 47.1). The mean maximum load of 111.4 N (. sd. 42.5) was reached at a mean displacement of 4.9 mm (. sd. 4.2). The periosteum was completely ruptured at a mean displacement of 34.4 mm (. sd. 11.1). There was no significant difference in the mean distance to complete rupture for medial (mean 35.8 mm (. sd. 13.8)) or lateral (mean 33.0 mm (. sd. 8.2)) displacement (p = 0.589). The mean bone mineral density was 0.111 g/cm. 3. (. sd. 0.035). A statistically significant but low correlation between bone mineral density and the maximum load uptake (r = 0.475, p = 0.034) was observed. This study showed that the posteromedial hinge is a mechanical structure capable of providing support for percutaneous reduction and stabilisation of a fracture by ligamentotaxis. Periosteal rupture started at a mean of about 3 mm and was completed by a mean displacement of just under 35 mm. The microvascular situation of the rupturing periosteum cannot be investigated with the current model

To test and evaluate the effectiveness of local injection of autologous fat-derived mesenchymal stem cells (MSCs) into fracture site to prevent non-union in a clinically relevant model. 5 male Wistar rats underwent the same surgical procedure of inducing non-union. A mid-shaft tibial osteotomy was made with 1mm non-critical gap. Periosteum was stripped around the two fracture ends. Then, the fracture was fixed by ante-grade intramedullary nail. The non-critical gap was maintained by a spacer with minimal effect on the healing surface area. At the same surgical time, subcutaneous fat was collected from the ipsilateral inguinal region and stem cells were isolated and cultured in vitro. Within three weeks postoperatively, the number of expanded stem cells reached 5×10. 6. and were injected into the fracture site. Healing was followed up for 8 weeks and the quality was measured by serial x-rays, microCT, mechanical testing and histologically. Quality of healing was compared with that of previously published allogenic, xenogeneic MSCs and Purified Buffered Saline (PBS) controls. All the five fractures united fully after 8 weeks. There was a progressive increase in the callus radiopacity during the eight-week duration, the average radiopacity in the autologous fat-MSC injected group was significantly higher than that of the allogeneic MSCs, xenogeneic MSCs and the control group, P < 0.0001 for treatment, time after injection, and treatment-time interaction (two-way repeated measure ANOVA). MicroCT, mechanical testing and histology confirmed radiological findings. The autologous fat-MSCs are effective in prevention of atrophic non-union by stimulation of the healing process leading to a solid union. The quality and speed of repair are higher than those of the other types of cell transplantation tested

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

Objectives. We sought to determine if a durable bilayer implant composed of trabecular metal with autologous periosteum on top would be suitable to reconstitute large osteochondral defects. This design would allow for secure implant fixation, subsequent integration and remodeling. Materials and Methods. Adult sheep were randomly assigned to one of three groups (n = 8/group): 1. trabecular metal/periosteal graft (TMPG), 2. trabecular metal (TM), 3. empty defect (ED). Cartilage and bone healing were assessed macroscopically, biochemically (type II collagen, sulfated glycosaminoglycan (sGAG) and double-stranded DNA (dsDNA) content) and histologically. Results. At 16 weeks post-operatively, histological scores amongst treatment groups were not statistically different (TMPG: overall 12.7, cartilage 8.6, bone 4.1; TM: overall 14.2, cartilage 9.5, bone 4.9; ED: overall 13.6, cartilage 9.1, bone 4.5). Metal scaffolds were incorporated into the surrounding bone, both in TM and TMPG. The sGAG yield was lower in the neo-cartilage regions compared with the articular cartilage (AC) controls (TMPG 20.8/AC 39.5, TM 25.6/AC 33.3, ED 32.2/AC 40.2 µg sGAG/1 mg respectively), with statistical significance being achieved for the TMPG group (p < 0.05). Hypercellularity of the neo-cartilage was found in TM and ED, as the dsDNA content was significantly higher (p < 0.05) compared with contralateral AC controls (TM 126.7/AC 71.1, ED 99.3/AC 62.8 ng dsDNA/1 mg). The highest type II collagen content was found in neo-cartilage after TM compared with TMPG and ED (TM 60%/TMPG 40%/ED 39%). Inter-treatment differences were not significant. Conclusions. TM is a highly suitable material for the reconstitution of osseous defects. TM enables excellent bony ingrowth and fast integration. However, combined with autologous periosteum, such a biocomposite failed to promote satisfactory neo-cartilage formation. Cite this article: E. H. Mrosek, H-W. Chung, J. S. Fitzsimmons, S. W. O’Driscoll, G. G. Reinholz, J. C. Schagemann. Porous tantalum biocomposites for osteochondral defect repair: A follow-up study in a sheep model. Bone Joint J 2016;5:403–411. DOI: 10.1302/2046-3758.59.BJR-2016-0070.R1

This study was undertaken to elucidate the mechanism of biological repair at the tendon-bone junction in a rat model. The stump of the toe flexor tendon was sutured to a drilled hole in the tibia (tendon suture group, n = 23) to investigate healing of the tendon-bone junction both radiologically and histologically. Radiological and histological findings were compared with those observed in a sham control group where the bone alone was drilled (n = 19). The biomechanical strength of the repaired junction was confirmed by pull-out testing six weeks after surgery in four rats in the tendon suture group. Callus formation was observed at the site of repair in the tendon suture group, whereas in the sham group callus formation was minimal. During the pull-out test, the repaired tendon-bone junction did not fail because the musculotendinous junction always disrupted first. In order to understand the factors that influenced callus formation at the site of repair, four further groups were evaluated. The nature of the sutured tendon itself was investigated by analysing healing of a tendon stump after necrosis had been induced with liquid nitrogen in 16 cases. A proximal suture group (n = 16) and a partial tenotomy group (n = 16) were prepared to investigate the effects of biomechanical loading on the site of repair. Finally, a group where the periosteum had been excised at the site of repair (n = 16) was examined to study the role of the periosteum. These four groups showed less callus formation radiologically and histologically than did the tendon suture group. In conclusion, the sutured tendon-bone junction healed and achieved mechanical strength at six weeks after suturing, showing good local callus formation. The viability of the tendon stump, mechanical loading and intact periosteum were all found to be important factors for better callus formation at a repaired tendon-bone junction

Summary Statement. A coupled finite element - analytical model is presented to predict and to elucidate a clinical healing scenario where bone regenerates in a critical-sized femoral defect, bounded by periosteum or a periosteum substitute implant and stabilised via an intramedullary nail. Introduction. Bone regeneration and maintenance processes are intrinsically linked to mechanical environment. However, the cellular and subcellular mechanisms of mechanically-modulated bone (re-) generation are not fully understood. Recent studies with periosteum osteoprogenitor cells exhibit their mechanosensitivity in vitro and in situ. In addtion, while a variety of growth factors are implicated in bone healing processes, bone morphogenetic protein-2 (BMP-2) is recognised to be involved in all stages of bone regeneration. Furthermore, periosteal injuries heal predominantly via endochondral ossification mechanisms. With this background in mind, the current study aims to understand the role of mechanical environment on BMP-2 production and periosteally-mediated bone regeneration. The one-stage bone transport model [1] provides a clinically relevant experimental platform on which to model the mechanobiological process of periosteum-mediated bone regeneration in a critical-sized defect. Here we develop a model framework to study the cellular-, extracellular- and mechanically-modulated process of defect infilling, governed by the mechanically-modulated production of BMP-2 by osteoprogenitor cells located in the periosteum. Methods. Material properties of the healing callus and periosteum contribute to the strain stimulus sensed by osteoprogenitor cells therein. Using a mechanical finite element model, periosteal surface strains are first predicted as a function of callus properties. Strains are then input to a mechanistic mathematical model, where mechanical regulation of BMP-2 production mediates rates of cellular proliferation, differentiation and extracellular matrix (ECM) production, to predict healing outcomes. A parametric approach enables the spatial and temporal prediction of tissue regeneration via endochondral ossification. Predictions are compared with experimental, micro-computed tomographic and histologic, measures of cartilage and mineralised bone tissue regenerates. Model Predictions in Light of Experimental Case Studies: A validated baseline model predicts defect healing via cellular egression, extracellular matrix production and endochondral ossification, using parameters optimised to mimic experimental outcome measures at initial and final stages of healing. To elucidate which predictive model paramenters result in the intrinsic differences in experimental outcomes between defects bounded by either periosteum in situ or a periosteum substitute implant, model parameters are then varied by orders of magnitude to determine which factors exert dominant influence on achievement of experimentally relevant ECM area outcomes. Considering the complete set of parameters relevant to healing, the rate of osteoprogenitor to osteoblast differentiation, as well as rates of chondrocyte and osteoblast proliferation must be reduced and ECM production by chondrocytes must be increased from baseline, to achieve healing outcomes analogous to those observed in experiments. Discussion/Conclusion. The novel model framework presented here integrates a mechanistic feedback system, based on the mechanosensitivity of periosteal osteoprogenitor cells, which allows for modeling and prediction of tissue regeneration on multiple length and time scales

Introduction and Objective. Objectives: To determine the effectiveness of LIA compared to ACB in providing pain relief and reducing opiates usage in hamstring graft ACL reconstructions. Materials and Methods. In a consecutive series of hamstring graft ACL reconstructions, patients received three different regional and/or anaesthetic techniques for pain relief. Three groups were studied: group 1: general anaesthetic (GA)+ ACB (n=38); group 2: GA + ACB + LIA (n=31) and group 3: GA+LIA (n=36). ACB was given under ultrasound guidance. LIA involved infiltration at skin incision site, capsule, periosteum and in the hamstring harvest tunnel. Analgesic medications were similar between the three groups as per standard multimodal analgesia (MMA). Patients were similar in demographics distribution and surgical technique. The postoperative pain and total morphine requirements were evaluated and recorded. The postoperative pain was assessed using the visual analogue scores (VAS) at 0hrs, 2hrs, 4hrs, weight bearing (WB) and discharge (DC). Results. There was no statistically significant difference in opiates intake amongst the three groups. When comparing VAS scores; there were no statistical difference between the groups at any of the time intervals that VAS was measured. However, the GA+LIA group hospital's LOS (m=2.31hrs, SD=0.75) was almost half that of GA+ACB group (m=4.24hrs, SD=1.08); (conditions t(72)=8.88; p=0.000). There was no statistical significance in the incidence of adverse effects amongst the groups. Conclusions. The LIA technique provided equally good pain relief following hamstring graft ACL reconstructions when compared to ACB, while allowing for earlier rehabilitation, mobilisation and discharge

Introduction and Objective. Heterotopic ossification is the formation of extraskeletal mineralized tissue commonly associated with either trauma or surgery. While several mouse models have been developed to better characterize the pathologic progression of HO, no model currently exists to study HO of the hip, the most common location of acquired HO in patients. Owing to the unique biological mechanisms underpinning the formation of HO in different tissues, we sought to develop a model to study the post-surgical HO of the hip. Materials and Methods. Wild-type mice C57BL/6J mice were used to study the procedure outcomes, while Pdgfra-CreERT2;mT/mG and Scx-GFP reporter animals were used for the lineage tracing experiments (total n=16 animals, male, 12 weeks old). An anterolateral approach to the hip was performed. Briefly, a 2 cm incision was made centered on the great trochanter and directed proximal to the iliac crest and distally over the lateral shaft of the femur. The joint was then reached following the intermuscular plane between the rectus femoris and gluteus medius muscles. After the joint was exposed, the articular cartilage was removed using a micropower drill with a 1.2 mm reamer. The medius gluteus and superficial fascia were then re-approximated with Vicryl 5-0 suture (Ethicon Inc, Somerville, NJ) and skin was then closed with Ethilon 5-0 suture (Ethicon Inc). Live high resolution XR imaging was performed every 2 wks to assess the skeletal tissues (Faxitron Bioptics, Tucson, AZ). The images were then scored using the Brooker classification. Ex-vivo microCT was conducted using a Skyscan 1275 scanner (Bruker-MicroCT, Kontich, Belgium). 3D reconstruction and analysis was performed using Dragonfly (ORS Inc., Montreal, Canada). For the histological analysis of specimens, Hematoxylin and Eosin (H&E), modified Goldner's Trichrome (GMT) stainings were performed. Reporter activity was assessed using fluorescent imaging. Results. Substantial periarticular heterotopic bone was seen in all cases. A periosteal reaction and an initial formation of calcified tissue within the soft tissue was apparent starting from 4 wks after surgery. By XR, progressive bone formation was observed within the periosteum and intermuscular planes during the subsequent 8 weeks. Stage 1 HO was observed in 12.5% of cases, stage 2 in 62.5% of cases, and stage 3 HO in 25% of cases. 3D microCT reconstructions of the treated hip joints demonstrated significant de novo heterotopic bone in several location which phenocopy human disease. Heterotopic bone was observed in an intracapsular location, periosteal location involving the iliac bone and proximal femur, and intermuscular locations. Histological analyses further confirmed these findings. To assess the cells which gave rise to HO in this model, an inducible PDGFRα and constitutive Scx-GFP reporter mice were used. A dramatic increase in mGFP reporter activity was noted PDGFRα within the HO injury site, including in areas of new cartilage and bone formation. Scx-associated reporter activity increased in the soft tissue and periosteal periacetabular areas of injured hips. Conclusions. HO has a diverse set of pathologies, of which joint associated HO after elective surgery is the most common. Here, we present the first mouse model of hip dislocation and acetabular reaming that mimics elements of human periarticular HO. The diverse locations of HO after acetabular reaming (intracapsular, intermuscular and periosteal) suggests the activation of different and specific HO program after surgery. Such a field effect would be consistent with local trauma and inflammation, which is a well-studied contributor to HO genesis. Not surprisingly, joint-associated HO significantly derives from PDGFRα-expressing cells, which has been shown to similarly give rise to intramuscular and intratendinous HO

Introduction and Objective. Found in bone-associated prosthesis, Cutibacterium acnes (C. acnes) is isolated in more than 50% of osteoarticular prosthesis infections, particularly those involving shoulder prostheses. Ongoing controversies exist concerning the origin of C. acnes infection. Few reports construct a reasonable hypothesis about probable contaminant displaced from the superficial skin into the surgical wound. Indeed, despite strict aseptic procedures, transecting the sebaceous glands after incision might result in C. acnes leakage into the surgical wound. More recently, the presence of commensal C. acnes in deep intra-articular tissues was reported. C. acnes was thus detected in the intracellular compartment of macrophages and stromal cells in 62.5% of the tested patients who did not undergo skin penetration. Among bone stromal cells, mesenchymal stem cells (MSCs) are predominantly found in bone marrow and periosteum. MSCs are the source of osteogenic lines of cells capable of forming bone matter. In this study, the pathogenicity of C. acnes in bone repair context was investigated. Materials and Methods. Human bone marrow derived MSCs were challenged with C. acnes clinical strains harvested from non-infected bone site (Cb). The behaviour of Cb strain was compared to C. acnes took from orthopaedic implant-associated infection (Ci). The infective capabilities of both strains was determined following gentamicin-based antibiotic protection assay. The morphology and ultrastructural analysis of infected MSCs was performed respectively through CLSM pictures of Phalloidin. ®. stained MSCs cytoskeleton and DAPI labelled Cb, and transmission and scanning electron microscopies. The virulence of intracellular Ci and Cb (Ci-MSCs and Cb-MSCs) was investigated by biofilm formation on non-living bone materials; and the immunomodulatory response of infected MSCs was investigated (PGE-2 and IDO secretion detected by ELISA). Bone cells (osteoblasts and PMA differentiated macrophages) were then challenged with Cb-MSCs and Ci-MSCs. Intracellular accumulation of ROS within infected macrophages was assessed by flow cytometry after 2 h of infection and the catalase production by Cb-MSC and Ci-MSC was evaluated. Statistical analyses were performed using Mann & Whitney test. Results. Following MSCs infection by C. acnes, the rate of viable bacteria inside MSCs was about 4% and 6% for Cb and Ci, respectively. Cb showed however a lower invasiveness in comparison to Ci (0.6-fold, p=0.01), confirming the higher pathogenicity of Ci. The ultrastructural and morphology analysis of infected MSCs confirmed the presence of bacteria free in MSCs cytoplasm, localized between F-actin fibers of MSCs, which preserved their elongated morphology. Considering the high level of secreted immunomodulatory mediators (PGE-2 and IDO), our results suggest that Cb-infected MSCs could promote a transition of macrophages from a primarily pro-inflammatory M1 to a more anti-inflammatory M2 phenotype. In comparison with Cb, Cb-MSCs increased significantly the formation of biofilm on TA6V and PEEK but reduced the biofilm formation on 316L SS. Ci-MSCs showed a significant increase in biofilm formation on PEEK vs Ci, while no difference in biofilm formation was noticed on TA6V and 316L SS. Regarding the ability of MSCs bacteria to infect osteoblasts, our results showed a higher infective capabilities of Cb-MSCs versus Cb (>2-fold, p=0.02), while no difference was noticed between Ci and Ci-MSCs. Along with an increase in catalase production by Cb-MSCs, we noticed its higher persistence to macrophage degradation. Conclusions. Taken together, our results demonstrate a shift in commensal Cb to pathogenic following infection. Indeed, Cb- MSCs acquires features that (i) increase biofilm formation on orthopedic based materials, (ii) increase the osteoblast infection and (iii) develop resistance to the macrophage degradation, through the increase of catalase production. Overall, these results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during implant-associated infections

Bone fractures are highly observed clinical situation in orthopaedic treatments. In some cases, there might be non-union problems. Therefore, recent studies have focused on tissue engineering applications as alternative methods to replace surgical procedures. Various biopolymer based scaffolds are produced using different fabrication techniques for bone tissue engineering applications. In this study, hydroxyapatite (HAp) and loofah containing carboxymethyl chitosan (CMC) scaffolds were prepared. In this regard, first 4 ml of CMC solution, 0.02 g of hydroxyapatite (HAP) and 0.06 g of poly (ethylene glycol) diglycidyl ether (PEGDE) were mixed in an ultrasonic bath until the HAp powders were suspended. Next, 0.04 g of loofah was added to the suspension and with the help of PEGDE as the cross-linking agent, then, the mixture was allowed to cross-link at 40. o. C overnight. Finally, the three-dimensional, porous and sponge-like scaffolds were obtained after lyophilization (TELSTAR - LyoQuest −85) at 0.1 mbar and −25°C for 2 days. Morphologies, chemical structures and thermal properties of the scaffolds were characterized by scanning electron microscopy (SEM), Fourier Transform infrared spectroscopy (FT-IR) and thermogravimetric differential thermal analysis (TGA/DTA), respectively. In addition, swelling behavior and mechanical properties of the scaffolds under compression loading were determined. In order to investigate biocompatibility of the scaffolds, WST-1 colorimetric assay at days 0, 1, 3, 5 and 7 was conducted by using human dermal fibroblast. Also, histological and morphological analysis were performed for cell attachment at day 7. In conclusion, the produced scaffolds showed no cytotoxic effect. Therefore, they can be considered as a candidate scaffold for bone tissue regeneration. Further studies will be performed by using bone marrow and periosteum derived mesenchymal stem cells with these scaffolds

Numerous implanted hip and knee joint arthroplasties have to be replaced due to early or late loosening of the implant, a failure of osteointegration with fibrous tissue at the bone-implant-interface. This could be counteracted by ensuring that cells which attach to the implant surface differentiate towards bone cells afterwards. For this reason, human mesenchymal stem cells (hMSCs) will be included in this study. These cells are naturally available at the bone-implant-interface, multipotent and therefore ideal to study the osteoinductivity of a material. The goal of this pilot study was to test the cell response towards three different titanium grades with a novel surface structuring, as a first step towards achieving an improved implant surface for enhanced osteointegration. Disk-shaped titanium scaffolds with a diameter of 12 mm and a height of 1.2 mm were used. The surface topography (500 µm × 500 µm × 300 µm pores) was generated via laser treatment of the surface. By using nanosecond pulsed laser technique, a rough surface with micro- and nanostructural (titanium droplets) features was automatically formed. Three different batches made of commercially pure titanium grades 1 and 2 (Ti1/Ti2) or Ti6Al4V alloy grade 5 (Ti5) were produced. Four cell types were analysed on these batches: primary hMSCs from one donor (m, 25 y), periosteum derived cells (PDCs), human osteoblasts (hOBs) and periodontal ligament cells (PDLs). Cells were seeded on Ti1, Ti2 and Ti5 scaffolds in triplicates. Resazurin assay to examine cell viability was conducted with all cell types. Measurements were executed on several days after seeding, from day one up to day 14. Actin staining as well as live/dead staining was performed with hMSCs cultured on titanium for 1, 3, 5 or 7 days. The cell viability assay revealed early turning points of growth for osteogenic hOBs (day 3) and PDCs (day 7). HMSCs grew steadily on the material and non-osteogenic PDLs stayed in plateau throughout the cultivation period. With respect to the material, cells demonstrated better proliferation on Ti1 and Ti2 than on Ti5. Live/dead staining showed a high survival rate of hMSCs at each time point and on all three titanium grades, with a neglectable number of dead cells. Actin staining confirmed an enhanced spreading and stretching of hMSCs on Ti1 and Ti2 compared to hMSCs on Ti5. Our pilot data indicates that cells react to different titanium compositions, revealed by increased proliferation on commercially pure titanium (Ti1/2). Furthermore, our results demonstrate that osteogenic cells prefer the novel surface structuring in comparison to non-osteogenic PDL cells, which stayed in plateau. The turning points of growth (hOBs/PDCs) suggest an osteosupportiveness of the surface. Although hMSCs did not show a turning point in growth, their growth was steady and resulted in the highest number of cells along with a well stretched morphology. Due to their good proliferation and response to the material, hMSCs are currently being used for evaluating the osteogenic potential of the novel scaffolds

Summary Statement. Progenitor cells from the periosteal niche are of great clinical interest due to their remarkable regenerative capacity. Here we report on progenitor cells from arthritic patients whose femoral neck periosteum was resected over the course of hip replacement. Introduction. This study aims to determine whether periosteum derived cells (PDCs) can be isolated from tissue resected in the normal course of hip arthroplasty. Further, it aims to determine how different isolation protocols affect PDC behavior (surface marker expression, proliferation, and differentiation). In addition, the study aims to characterise the populations of PDCs, isolated through either enzymatic digestion or migration, and their relative capacity to differentiate down multiple capacities; direct comparison with commercially available human marrow-derived stromal cells cultured under identical conditions will enable the placement of the PDC data in context of the current state of the field. Methods. Proximal femoral head/neck explants (n=4) were acquired from patients within 8 h of hip replacement surgery (IRB 12–335, Cleveland Clinic), after examination and diagnosis by pathology. To isolate digested PDCs (dPDCS), the minced tissue is suspended in 3 mg/ml collagenase II (Gibco) solution in alpha-MEM with Glutamax (Invitrogen) with 1% antibioticantimycotic (Invitrogen) overnight in a 37°C incubator. Any undigested tissue is filtered from the cells using a 100 μm filter, and isolated cells are cultured in standard culture media. To isolate migrated PDCs (mPDCs), the minced tissue is directly plated into tissue culture flasks in alpha-MEM with Glutamax supplemented with 10% FBS (Invitrogen), 1% antibioticantimycotic overnight, and cultured in standard culture media. The cells are left to egress from the tissue for one week. Finally, validated bone marrow derived hMSCs (BMSCs) are purchased from four independent vendors (Lonza, PromoCell, ScienCell, Cell Applications) as standards for comparison. Cell cohorts are compared using proliferation assays, cell population analysis using flow cytometry, and quantitative adipogenesis, osteogenesis and chondrogenesis assays. Results. PDCs and BMSCs exhibit similar proliferation rate, morphology, and surface receptor expression. PDCs showed no significant differences to BMSCs with regard to osteo- and adipogenic differentiation capacity. Chondrogenic assay of PDCs showed increased pellet size with pellets exhibiting staining indicative of hyaline cartilage for BMSC, and immature fibrocartilage for dPDCs and mPDCs. This study demonstrates, for the first time to our knowledge, the feasibility of isolating PDCs from the femoral neck of patients undergoing joint replacement surgery. PDCs and BMSCs exhibit similar surface marker expression and equivalent adipogenic and osteogenic capacity. Interestingly, cell cohorts from the same tissue albeit different isolation protocols show intrinsic differences in their differentiation capacities. Additionally, all cell types display marked intraindividual differences in their capacity to differentiate down different lineages, with no significant correlation to age. Discussion/Conclusion. The femoral neck periosteum offers a feasible source for isolation and a possible source for banking of autologous multipotent cells with similar potential as BMSCs. Further in vivo studies will determine the regenerative capacity of these cells in a more physiological context

Bone formation proceeds through two distinct processes. One involves the deposition of bone by osteoblasts (intramembranous ossification) and another through the remodeling of an intermediate cartilaginous matrix formed by chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs) aggregates – a process called endochondral ossification (EO). EO is responsible for formation of long bones during development and most prevalent during facture repair upon callus formation. In adult bone injuries MSCs from periosteum form bone via EO whereas MSCs from bone marrow are primarily differentiate to osteoblast in vivo. We hypothesized that the unique biophysical and biochemical properties of bone mineral phase has a role in programming MSCs. Using a biomimetic bone like apatite (BBHAp) as surrogate for bone mineral phase, we studied the chondrogenic differentiation of human marrow derived MSCs and observed that the BBHAp dictates MSCs fate and strictly dictates the pathway of bone formation in vivo. Through exhaustive dissection of the signaling pathways at play, a prominent role of PTH1R in modulating the effects imposed by the BBHAp has been unraveled. These fundamental insights gained in how bone microenvironment might alter fate of MSCs has important implications for bone repair and regeneration therapies