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In bone tissue, nerves are primarily located in the periosteum and play an indispensable role in bone defect repair. However, most bone tissue engineering approaches ignored the reconstruction of the nerve network. Herein, we aimed to develop a bilayer hydrogel simulating periosteum-bone structure to induce innervated bone regeneration. The bottom "bone" layer consisted of gelatin methacryloyl (GelMA), poly(ethylene glycol) diacrylate (PEGDA), and nano-hydroxyapatite (nHA), whereas the upper "periosteum" layer consisted of GelMA, sodium alginate (SA) and MgCl 2 . The mechanical properties of the upper and bottom hydrogels were designed to be suitable for neurogenesis and osteogenesis, respectively. Besides, Mg 2+ from the "periosteum" layer released at the early stage (within 7 d), which aligned with the optimal time window for nerve regeneration and osteogenic related neuropeptide release. Simultaneously, the prevention of long-term Mg 2+ release (after 7 d) could avoid osteogenic inhibition caused by prolonged Mg 2+ exposure. Additionally, the incorporation of nHA in the bottom "bone" layer supported the long-term osteogenesis due to its osteoconductivity and slow degradation. In vitro biological experiments revealed that the bilayer hydrogel (GS@Mg/GP@nHA) promoted neurite growth and calcitonin gene-related peptide (CGRP) expression in rat dorsal root ganglion (DRG) neurons, as well as the osteogenesis of rat bone-derived mesenchymal stem cells (BMSCs). Moreover, the in vivo experiments demonstrated that the GS@Mg/GP@nHA hydrogel efficiently promoted nerve network reconstruction and bone regeneration of rat calvarial bone defects. Altogether, the bilayer hydrogel GS@Mg/GP@nHA could promote innervated bone regeneration, providing new insights for biomaterial design for bone tissue engineering.

Bone fractures often require internal fixation using plates or screws. Normally, these devices are made of permanent metals like titanium providing necessary strength and biocompatibility. However, they can also cause long-term complications and may require removal. An interesting alternative are biocompatible degradable devices, which provide sufficient initial strength and then degrade gradually. Among other materials, biodegradable magnesium alloys have been developed for craniofacial and orthopaedic applications. Previously, we tested implants made of magnesium hydroxide and RS66, a strong and ductile ZK60-based alloy, with respect to biocompatibility and degradation behaviour. Here, we compare the effects of dissolving magnesium hydroxide and RS66 cylinders on bone regeneration and bone growth in rabbit condyles using microtomographical and histological analysis. Both magnesium hydroxide and RS66 induced a considerable osteoblastic activity leading to distinct but different spatio-temporal patterns of cancellous and periosteal bone growth. Dissolving RS66 implants induced a prominent periosteal bone formation on the medial surface of the original condyle whereas dissolving magnesium hydroxide implants enhance mainly cancellous bone formation. Especially periosteal bone formation was completed after 6 and 8 weeks, respectively. The observed bone promoting functions are in line with previous reports of magnesium stimulating cancellous and periosteal bone growth and possible underlying signalling mechanisms are discussed. STATEMENT OF SIGNIFICANCE: Biodegradable magnesium based implants are promising candidates for use in orthopedic and traumatic surgery. Although these implants are in the scientific focus for a long time, comparatively little is known about the interactions between degrading magnesium and the biological environment. In this work, we investigated the effects of two degrading cylindrical magnesium implants (MgOH 2 and RS66) both on bone regeneration and on bone growth. Both MgOH 2 and RS66 induce remarkable osteoblastic activities, however with different spatio-temporal patterns regarding cancellous and periosteal bone growth. We hypothesize that degradation products do not diffuse directionless away, but are transported by the restored blood flow in specific spatial patterns which is also dependent on the used surgical technique., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The ability to locally deliver bioactive molecules to distinct regions of the skeleton may provide a novel means by which to improve fracture healing, treat neoplasms or infections, or modulate growth. In this study, we constructed single-sided mineral-coated poly-ε-caprolactone membranes capable of binding and releasing transforming growth factor beta 1 (TGF-β1) and human growth hormone (hGH). After demonstrating biological activity in vitro and characterization of their release, these thin bioabsorbable membranes were surgically implanted using an immature rabbit model. Membranes were circumferentially wrapped under the periosteum, thus placed in direct contact with the proximal metaphysis to assess its bioactivity in vivo. The direct effects on the metaphyseal bone, bone marrow, and overlying periosteum were assessed using radiography and histology. Effects of membrane placement at the tibial growth plate were assessed via physeal heights, tibial growth rates (pulsed fluorochrome labeling), and tibial lengths. Subperiosteal placement of the mineralized membranes induced greater local chondrogenesis in the plain mineral and TGF-β1 samples than the hGH. More exuberant and circumferential ossification was seen in the TGF-β1 treated tibiae. The TGF-β1 membranes also induced hypocellularity of the bone marrow with characteristics of gelatinous degeneration not seen in the other groups. While the proximal tibial growth plates were taller in the hGH treated than TGF-β1, no differences in growth rates or overall tibial lengths were found. In conclusion, these data demonstrate the feasibility of using bioabsorbable mineral coated membranes to deliver biologically active compounds subperiosteally in a sustained fashion to affect cells at the insertion site, bone marrow, and even growth plate., (© 2024 Wiley Periodicals LLC.)

Existing artificial periostea face many challenges, including difficult-to-replicate anisotropy in mechanics and structure, poor tissue adhesion, and neglected synergistic angiogenesis and osteogenesis. Here, inspired by natural wood (NW), a wood-derived elastic artificial periosteum is developed to mimic the structure and functions of natural periosteum, which combines an elastic wood (EW) skeleton, a polydopamine (PDA) binder layer, and layer-by-layer (LBL) biofunctional layers. Specifically, EW derived from NW is utilized as the anisotropic skeleton of artificial periosteum to guide cell directional behaviors, moreover, it also shows a similar elastic modulus and flexibility to natural periosteum. To further enhance its synergistic angiogenesis and osteogenesis, surface LBL biofunctional layers are designed to serve as spatiotemporal release platforms to achieve sequential and long-term release of pamidronate disodium (PDS) and deferoxamine (DFO), which are pre-encapsulated in chitosan (CS) and hyaluronic acid (HA) solutions, respectively. Furthermore, the combined effect of PDA coating and LBL biofunctional layers enables the periosteum to tightly adhere to damaged bone tissue. More importantly, this novel artificial periosteum can boost angiogenesis and bone formation in vitro and in vivo. This study opens up a new path for biomimetic design of artificial periosteum, and provides a feasible clinical strategy for bone repair., (© 2024 Wiley‐VCH GmbH.)

The effective repair of large bone defects remains a major challenge due to its limited self-healing capacity. Inspired by the structure and function of the natural periosteum, an electrospun biomimetic periosteum is constructed to programmatically promote bone regeneration using natural bone healing mechanisms. The biomimetic periosteum is composed of a bilayer with an asymmetric structure in which an aligned electrospun poly(ε-caprolactone)/gelatin/deferoxamine (PCL/GEL/DFO) layer mimics the outer fibrous layer of the periosteum, while a random coaxial electrospun PCL/GEL/aspirin (ASP) shell and PCL/silicon nanoparticles (SiNPs) core layer mimics the inner cambial layer. The bilayer controls the release of ASP, DFO, and SiNPs to precisely regulate the inflammatory, angiogenic, and osteogenic phases of bone repair. The random coaxial inner layer can effectively antioxidize, promoting cell recruitment, proliferation, differentiation, and mineralization, while the aligned outer layer can promote angiogenesis and prevent fibroblast infiltration. In particular, different stages of bone repair are modulated in a rat skull defect model to achieve faster and better bone regeneration. The proposed biomimetic periosteum is expected to be a promising candidate for bone defect healing., (© 2024 Wiley‐VCH GmbH.)

Diabetes-related skin ulcers are of significant clinical concern. Although conventional dressings have been developed, their outcomes have not been adequate, indicating the need to investigate functional dressings for the treatment of diabetic ulcers. Copper selenide nanoparticles (Cu 2 Se NPs) demonstrate outstanding photoresponsiveness, which is critical to the healing process. However, their limited solubility in water restricts their application. To synthesize the ODT-PMMA@Cu 2 Se NP-doped decellularized periosteum‑sodium alginate functional dressing-ODT-PMMA@Cu 2 Se/ECM-S (OP@Cu 2 Se/ECM-S), Cu 2 Se NPs were modified by n-octadecanethiol (ODT) end-functionalized poly (methacrylic acid) (PMAA) ligands homogeneously dispersed in a decellularized periosteum/sodium alginate matrix. This process improved the water solubility and stability. Moreover, under near-infrared irradiation (NIR), ODT-PMMA@Cu 2 Se demonstrated robust photo-responsiveness along with photothermal and photodynamic effects, leading to rapid heating and stimulation of reactive oxygen species (ROS) generation. These two processes work in concert to exhibit excellent antibacterial ability; at 20 μg/mL concentration of Cu 2 Se NPs, the bacterial activities of S. aureus and E. coli were 5.40 % and 0.96 %, respectively. Without the NIR laser irradiation, OP@Cu 2 Se/ECM-S rapidly increased the vascular endothelial growth factor (VEGF) expression, triggered the phosphatidylinositide 3-kinases (PI3K) and protein kinase B (AKT) signaling pathway, affected the expression of bFGF and CD31, and promoted neovascularization, proliferation, and cell migration. In a diabetic mouse wound model, OP@Cu 2 Se/ECM-S exhibited good biocompatibility and promoted epidermal regeneration, collagen deposition, and neovascularization. In a mouse model of subcutaneous abscesses, OP@Cu 2 Se/ECM-S also showed excellent antibacterial activity, in vivo experiments confirmed a decrease in bacterial activity to 1.97 %. Thus, OP@Cu 2 Se/ECM-S is a potentially useful approach for healing diabetic wounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Concentrated growth factor (CGF) is 100% blood-derived, cross-linked fibrin glue with platelets and growth factors. Human CGF clot is transformed into membrane by a compression device, which has been widely used clinically. However, the mechanical properties of the CGF membranes have not been well characterized. The aims of this study were to measure the tensile strength of human CGF membrane and observe its behavior as a scaffold of BMP-2 in ectopic site over the skull. The tensile test of the full length was performed at the speed of 2mm/min. The CGF membrane (5 × 5 × 2 mm 3 ) or the CGF/BMP-2 (1.0 μg) membrane was grafted onto the skull periosteum of nude mice (5-week-old, male), and harvested at 14 days after the graft. The appearance and size of the CGF membranes were almost same for 7 days by soaking at 4 °C in saline. The average values of the tensile strength at 0 day and 7 days were 0.24 MPa and 0.26 MPa, respectively. No significant differences of both the tensile strength and the elastic modulus were found among 0, 1, 3, and 7 days. Supra-periosteal bone induction was found at 14 days in the CGF/BMP-2, while the CGF alone did not induce bone. These results demonstrated that human CGF membrane could become a short-term, sticky fibrin scaffold for BMP-2, and might be preserved as auto-membranes for wound protection after the surgery.

The touchstone for bone replacing or anchoring trauma implants, besides resorption, includes functional ankylosis at a fixation point and replacement by viable functional neo-bone tissues. These parameters redefined the concept of "resorbability" as "bioresorbability." Interference screws are the most commonly used resorbable anchoring implants for anterior cruciate ligament (ACL) reconstruction (surgery). Over the years, the bioresorbable screw fixation armamentarium has amplified countless choices, but instability and postimplantation complications have raised concerns about its reliability and efficacy. Owing to this interest, in this work, bioactive glass fiber-reinforced plastic (BGFP) composites with (BGFP nb 5) and without (BGFP5) niobicoxide composing multiplexed network modifiers are reported as bioresorbable bone-anchoring substitutes. These synergistically designed composites have a fabricated structure of continuous, unidirectional BG fibers reinforced in an epoxy resin matrix using "melt-drawing and microfabrication" technology. The BGFP microarchitecture is comprised of multiplexed oxide components that influence bioactive response in a distinctive lophelia atoll-like apatite formation. Furthermore, it assists in the proliferation, adherence, and migration of bone marrow-derived mesenchymal stem cells. It also exhibits superior physicochemical characteristics such as surface roughness, hydrophilic exposure, distinctive flexural strength, and bioresorption. Thus, it induces restorative bone osseointegration and osteoconduction and actuates periosteum function. In addition, the BGFP influences the reduction of DH5-α Escherichia coli in suspension culture, demonstrating potential antibacterial efficacy. In conclusion, the BGFP composite therapeutic efficacy demonstrates distinctive material characteristics aiding in bone regeneration and restoration that could serve as a pioneer in orthopedic regenerative medicine.

Purpose: Intra-articular administration of tranexamic acid (TXA) in orthopaedic arthroplasty and arthroscopic procedures has become increasingly common over the past decade. However, several recent reports have shown that TXA has the potential to be cytotoxic to cartilage, tendon and synovium. Our aim was to review the literature for evidence of toxic effects from TXA exposure to intra-articular tissue., Methods: A scoping review methodology was used to search for studies assessing the toxic effects of TXA exposure to intra-articular tissues. MEDLINE, EMBASE, SCOPUS and The Cochrane Library were searched. Relevant information was extracted and synthesis of the retrieved data followed a basic content analytical approach., Results: A total of 15 laboratory studies were retrieved. No clinical studies reporting a toxic effect of TXA on intra-articular tissue were identified in our search. Studies were analysed according to species of origin, tissue of origin and study setting (in vitro, ex vivo, or in vivo). There was increasing cytotoxicity to chondrocytes, tenocytes, synoviocytes and periosteum-derived cells with TXA concentrations beyond 20 mg/ml. Monolayer cell cultures appear more susceptible to TXA exposure, than three-dimensional and explant culture models. In vivo studies have not demonstrated a major toxic effect., Conclusions: Current evidence suggests a dose-dependent toxic effect on cartilage, tendon, and synovial tissue. Concentrations of 20 mg/ml or less are expected to be safe. There is a significant body of evidence to suggest the need for caution with intraarticular administration of TXA. There is a need for further human clinical trials in order to clarify the long-term safety of TXA topical application.

Background: Although complications associated with pazopanib, a multitarget tyrosine kinase inhibitor, are known, periosteal reaction as a side effect has never been reported., Observation: We present a case involving a male pediatric patient with desmoid tumors treated for 6 months with pazopanib who presented with pain and periosteal reaction in the ilium and scapula. Three months after termination of pazopanib therapy, the periosteal reaction in the scapula resolved and that in the ilium improved., Conclusion: Children receiving pazopanib presenting with focal pain should be examined for the periosteal reaction; this knowledge may facilitate correct diagnosis of symptoms as a drug-associated finding.

Filler injection demand is increasing worldwide, but no ideal filler with safety and longevity currently exists. Sodium alginate (SA) is the sodium salt of alginic acid, which is a polymeric polysaccharide obtained by linear polymerization of two types of uronic acid, d-mannuronic acid (M) and l-guluronic acid (G). This study aimed to evaluate the therapeutic value of SA. Nine SA types with different M/G ratios and viscosities were tested and compared with a commercially available sodium hyaluronate (SH) filler. Three injection modes (onto the periosteum, intradermally, or subcutaneously) were used in six rats for each substance, and the animals were sacrificed at 4 or 24 weeks. Changes in the diameter and volume were measured macroscopically and by computed tomography, and histopathological evaluations were performed. SA with a low M/G ratio generally maintained skin uplift. The bulge gradually decreased over time but slightly increased at 4 weeks in some samples. No capsule formation was observed around SA. However, granulomatous reactions, including macrophage recruitment, were observed 4 weeks after SA implantation, although fewer macrophages and granulomatous reactions were observed at 24 weeks. The long-term volumizing effects and degree of granulomatous reactions differed depending on the M/G ratio and viscosity. By contrast, SH showed capsule formation but with minimal granulomatous reactions. The beneficial and adverse effects of SA as a filler differed according to the viscosity or M/G ratio, suggesting a better long-term volumizing effect than SH with relatively low immunogenicity.

A 7-year-old boy presented with diffuse bone pain. FDG PET/CT was performed to find the possible underlying malignant cause of hypercalcemia. The images demonstrated multiple foci of abnormal FDG activity at the sites of periosteal reaction. In addition, calcium deposit was noted in the basal ganglia, stomach, and the colon. History taking revealed that the patient had routinely taken an over-the-counter "supplement" that contains a high dose of vitamin D. One week after calcitonin therapy and stopping the supplement, the patient became symptom free. This case suggests that hypervitaminosis D might cause hypermetabolic periosteal reaction on FDG PET/CT imaging.

Background: The periosteum is a highly vascularized, collagen-rich tissue that plays a crucial role in directing bone repair. This is orchestrated primarily by its resident progenitor cell population. Indeed, preservation of periosteum integrity is critical for bone healing. Cells extracted from the periosteum retain their osteochondrogenic properties and as such are a promising basis for tissue engineering strategies for the repair of bone defects. However, the culture expansion conditions and the way in which the cells are reintroduced to the defect site are critical aspects of successful translation. Indeed, expansion in human serum and implantation on biomimetic materials has previously been shown to improve in vivo bone formation., Aim: This study aimed to develop a protocol to allow for the expansion of human periosteum derived cells (hPDCs) in a biomimetic periosteal-like environment., Methods: The expansion conditions were defined through the investigation of the bioactive cues involved in augmenting hPDC proliferative and multipotency characteristics, based on transcriptomic analysis of cells cultured in human serum., Results: Master regulators of transcriptional networks were identified, and an optimized periosteum-derived growth factor cocktail (PD-GFC; containing β-estradiol, FGF2, TNFα, TGFβ, IGF-1 and PDGF-BB) was generated. Expansion of hPDCs in PD-GFC resulted in serum mimicry with regard to the cell morphology, proliferative capacity and chondrogenic differentiation. When incorporated into a three-dimensional collagen type 1 matrix and cultured in PD-GFC, the hPDCs migrated to the surface that represented the matrix topography of the periosteum cambium layer. Furthermore, gene expression analysis revealed a down-regulated WNT and TGFβ signature and an up-regulation of CREB, which may indicate the hPDCs are recreating their progenitor cell signature., Conclusion: This study highlights the first stage in the development of a biomimetic periosteum, which may have applications in bone repair., (Copyright © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Nuclear factor kappa B (NF-κB) regulates inflammatory gene expression and represents a likely target for novel disease treatment approaches, including skeletal disorders. Several plant-derived sesquiterpene lactones can inhibit the activation of NF-κB. Parthenolide (PTL) is an abundant sesquiterpene lactone, found in Mexican Indian Asteraceae family plants, with reported anti-inflammatory activity, through the inhibition of a common step in the NF-κB activation pathway. This study examined the effects of PTL on the enhanced, in vitro, osteogenic phenotypes of human periosteum-derived cells (hPDCs), mediated by the inflammatory cytokine tumor necrosis factor (TNF)-α. PTL had no significant effects on hPDC viability or osteoblastic activities, whereas TNF-α had positive effects on the in vitro osteoblastic differentiation of hPDCs. c-Jun N-terminal kinase (JNK) signaling played an important role in the enhanced osteoblastic differentiation of TNF-α-treated hPDCs. Treatment with 1 µM PTL did not affect TNF-α-treated hPDCs; however, 5 and 10 µM PTL treatment decreased the histochemical detection and activity of alkaline phosphatase (ALP), alizarin red-positive mineralization, and the expression of ALP and osteocalcin mRNA. JNK phosphorylation decreased significantly in TNF-α-treated hPDCs pretreated with PTL. These results suggested that PTL exerts negative effects on the increased osteoblastic differentiation of TNF-α-treated hPDCs by inhibiting JNK signaling.

Background: Osteoporosis is a metabolic bone disorder that leads to low bone mass and microstructural deterioration of bone tissue and increases bone fractures. Resveratrol, a natural polyphenol compound, has pleiotropic effects including anti-oxidative, anti-aging, and anti-cancer effects. Resveratrol also has roles in increasing osteogenesis and in upregulating mitochondrial biogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is still unclear that resveratrol can enhance osteogenic differentiation or mitochondrial biogenesis of periosteum-derived MSCs (PO-MSCs), which play key roles in bone tissue maintenance and fracture healing. Thus, in order to test a possible preventive or therapeutic effect of resveratrol on osteoporosis, this study investigated the effects of resveratrol treatments on osteogenic differentiation and mitochondrial biogenesis of PO-MSCs., Methods: The optimal doses of resveratrol treatment on PO-MSCs were determined by cell proliferation and viability assays. Osteogenic differentiation of PO-MSCs under resveratrol treatment was assessed by alkaline phosphatase activities (ALP, an early biomarker of osteogenesis) as well as by extracellular calcium deposit levels (a late biomarker). Mitochondrial biogenesis during osteogenic differentiation of PO-MSCs was measured by quantifying both mitochondrial mass and mitochondrial DNA (mtDNA) contents., Results: Resveratrol treatments above 10 μM seem to have negative effects on cell proliferation and viability of PO-MSCs. Resveratrol treatment (at 5 μM) on PO-MSCs during osteogenic differentiation increased both ALP activities and calcium deposits compared to untreated control groups, demonstrating an enhancing effect of resveratrol on osteogenesis. In addition, resveratrol treatment (at 5 μM) during osteogenic differentiation of PO-MSCs increased both mitochondrial mass and mtDNA copy numbers, indicating that resveratrol can bolster mitochondrial biogenesis in the process of PO-MSC osteogenic differentiation., Conclusion: Taken together, the findings of this study describe the roles of resveratrol in promoting osteogenesis and mitochondrial biogenesis of human PO-MSCs suggesting a possible application of resveratrol as a supplement for osteoporosis and/or osteoporotic fractures.

Periosteum as an important component in the construct of bone is mainly responsible for providing nourishment and regulating osteogenic differentiation. When bone defect happens, the functionality of periosteum will also be influenced, furthermore, it will finally hamper the process of bone regeneration. However, fabrication of an artificial periosteum with the capabilities in accelerating angiogenesis and osteogenesis in the defect area is still a challenge for researchers. In this study, we fabricated an organic-inorganic hybrid biomimetic periosteum by electrospinning, which can induce mineralization in situ and control the ions release for long-term in local area. Further, this system exhibited potential capabilities in promoting in vitro, which means the potentiality in accelerating bone regeneration in vivo. Calcium phosphate nanoparticles (CaPs) were fabricated by emulsion method, then CaPs were further incorporated with gelatin-methacryloyl (GelMA) by electrospinning fibers to construct the hybrid hydrogel fibers. The fibers exhibited satisfactory morphology and mechanical properties, additionally, controlled ions release could be observed for over 10 days. Further, significant mineralization was proved on the surface of hybrid fibers after 7 days and 14 days' co-incubation with simulated body fluid (SBF). Then, favorable biocompatibility of the hybrid fibers was approved by co-cultured with MC3T3-E1 cells. Finally, the hybrid fibers exhibited potential capabilities in promoting angiogenesis and osteogenesis by co-culture with HUVECs and MC3T3-E1 cells. This biomimetic organic-inorganic hybrid hydrogel electrospinning periosteum provided a promising strategy to develop periosteum biomaterials with angiogenesis and osteogenesis capabilities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Recombinant human bone morphogenetic protein-2 (rhBMP-2) and bioceramic are the widely used bioactive factors in treatment of bone defects, but these easily cause side effects because of uncontrollable local concentration. In this study, rhBMP-2 was grafted on the surface of mesoporous bioglass nanoparticles (MBGNs) with an amide bond and then photo-cross-linked together with methacrylate gelatin (GelMA); in this way, a GelMA/MBGNs-rhBMP-2 hydrogel membrane was fabricated to release rhBMP-2 in a controllable program during the early bone regeneration period and then release calcium and silicon ions to keep promoting osteogenesis instead of rhBMP-2 in a long term. In this way, rhBMP-2 can keep releasing for 4 weeks and then the ions keep releasing after 4 weeks; this process is matched to early and late osteogenesis procedures. In vitro study demonstrated that the early release of rhBMP-2 can effectively promote local cell osteogenic differentiation in a short period, and then, the inorganic ions can promote cell adhesion not only in the early stage but also keep promoting osteogenic differentiation for a long period. Finally, the GelMA/MBGNs-rhBMP-2 hydrogel shows a superior capacity in long-term osteogenesis and promoting bone tissue regeneration in rat calvarial critical size defect. This GelMA/MBGNs-rhBMP-2 hydrogel demonstrated a promising strategy for the controllable and safer use of bioactive factors such as rhBMP-2 in artificial periosteum to accelerate bone repairing.

Background: Pain and blood loss after total knee arthroplasty (TKA) are unsolved clinical problems. Some studies reported that periarticular cocktail injection can effectively reduce pain and blood loss. However, there was no gold standard about the cocktail ingredient and injection location. More osteotomy and less soft tissue release in TKA with mild deformity; besides, plenty of nerves and blood vessels are contained in the periosteums and bone marrow. In this study, we aimed to detect the clinical results of subperiosteal cocktail application in TKA., Methods: Two groups were included according to the different injection location in our study. In group 1, cocktails were injected into the muscles, tendons, suprapatellar bursa, and subpatellar bursa surrounding knee joint. In group 2, cocktail injection was performed under the periosteum of the distal femur and proximal tibia. Our primary outcomes were visual analogue scale (VAS) and hemoglobin (Hb), and the secondary outcomes were wound healing, infection, deep vein thrombosis (DVT), operation time, and hospitalization., Results: At the first operative day, the mean (standard deviation) VAS score in a state of static was lower in group 2 compared with group 1 (0.98 ± 0.27 in group 1 and 0.86 ± 0.60 in group 2, p < 0.05). In the state of flexion and extension, the mean (standard deviation) VAS was 1.61 ± 0.66 in group 1 and 1.10 ± 0.57 in group 2 (p < 0.05). The mean (standard deviation) blood loss was higher in group 1 than in group 2 at the first postoperative day (440.19 (167.68) ml in group 1 and 333.67 (205.99) ml in group 2, p < 0.05). At the third day after surgery, the mean (standard deviation) blood loss was 686.44 (140.29) ml in group 1 and 609.19 (260.30) ml in group 2, and there was significant difference between these two groups (p < 0.05)., Conclusions: We concluded that subperiosteal cocktail injection can significantly reduce pain and blood loss compared with periarticular cocktail injection after TKA.

The periosteum is a membrane that surrounds bones, providing essential cellular and biological components for fracture healing and bone repair. Tissue engineered scaffolds able to function as periosteum substitutes can significantly improve bone regeneration in severely injured tissues. Efforts to develop more bioactive and tunable periosteal substitutes are required to improve the success of this tissue engineering approach. In this work, a chemical modification was performed in chitosan, a polysaccharide with osteoconductive properties, by introducing phosphate groups to its structure. The phosphorylated polymer (Chp) was used to produce chitosan-xanthan-based scaffolds for periosteal tissue engineering. Porous and mechanically reinforced matrices were obtained with addition of the surfactant Kolliphor® P188 and the silicone rubber Silpuran® 2130A/B. Scaffolds properties, such as large pore sizes (850-1097 μm), micro-roughness and thickness (0.7-3.5 mm in culture medium), as well as low thrombogenicity compared to standard implantable materials, extended degradation time and negligible cytotoxicity, enable their application as periosteum substitutes. Moreover, the higher adsorption of bone morphogenetic protein mimic (cytochrome C) by Chp-based formulations suggests improved osteoinductivity of these materials, indicating that, when used in vivo, the material would be able to concentrate native BMPs and induce osteogenesis. The scaffolds produced were not toxic to adipose tissue-derived stem cells, however, cell adhesion and proliferation on the scaffolds surfaces can be still further improved. The mineralization observed on the surface of all formulations indicates that the materials studied have promising characteristics for the application in bone regeneration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: The aim of this research is to investigate the effects of concentrated growth factor (CGF) on the proliferation, osteogenic differentiation, and angiogenic potential of rabbit periosteum-derived cells (PDCs) in vitro., Methods: PDCs were isolated from the femoral and tibial periosteum of rabbits and cultured with or without CGF membranes or CGF conditioned media. Scanning electron microscopy (SEM) was used for the structural characterization. Cell Counting Kit-8 assay was used to measure cell proliferation. Alkaline phosphatase (ALP) activity of PDCs was also measured. Immunohistochemistry was used to detect the expression of CD34. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qPCR), and Western blot were used to evaluate the secretion and expression levels of osteogenic differentiation markers (bone morphogenetic protein-2, type I collagen, osteocalcin) and angiogenesis markers (vascular endothelial growth factor, basic fibroblast growth factor) in supernatants and PDCs at days 3, 7, 14, and 21., Results: The SEM analysis showed a dense three-dimensional fibrin network in CGF, and CGF membranes were covered by PDCs with elongated or polygonal morphological features. Compared with the control group, CGF significantly promoted the proliferation of PDCs during the experimental period (p < 0.05). Immunohistochemistry revealed that PDCs were dispersedly distributed among the CGF substrates, and CD34-positive cells were also present. Moreover, CGF significantly increased the ALP activity and upregulated the expression and secretion of osteogenic differentiation and angiogenesis markers in PDCs at days 3, 7, 14, and 21 (p < 0.05)., Conclusion: CGF can increase the proliferation and promote the osteogenic differentiation and angiogenic potential of PDCs in vitro. These results indicate that CGF can be used as a new therapeutic means for biotechnological and clinical applications.

Background: Nitrogen-containing bisphosphonates (BIS) are potent therapeutics in osteoporosis, but their use may result in osteonecrotic side-effects in the maxillofacial region. Periosteal microcirculatory reactions may contribute to the development of bone-healing complications, particularly in osteoporotic bones, where ischemia-reperfusion (IR) events often develop during orthopaedic/trauma interventions. The effect of BIS on the inflammatory reactions of appendicular long bones has not yet been evaluated; thus, we aimed to examine the influence of chronic zoledronate (ZOL) administration on the periosteal microcirculatory consequences of hindlimb IR in osteopenic rats., Materials and Methods: Twelve-week-old female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated, and ZOL (80 μg/kg iv, weekly) or a vehicle was administered for 8 weeks, 4 weeks after the operation. At the end of the pre-treatment protocols, 60-min limb ischemia was induced, followed by 180-min reperfusion. Leukocyte-endothelial interactions were quantitated in tibial periosteal postcapillary venules by intravital fluorescence videomicroscopy. CD11b expression of circulating polymorphonuclear leukocytes (PMN, flow cytometry) and plasma TNF-alpha levels (ELISA) were also determined. Two-way RM ANOVA followed by the Holm-Sidak and Dunn tests was used to assess differences within and between groups, respectively., Results: Limb IR induced significant increases in PMN rolling and firm adhesion in sham-operated and OVX rats, which were exacerbated temporarily in the first 60 min of reperfusion by a ZOL treatment regimen. Postischemic TNF-alpha values showed a similar level of postischemic elevations in all groups, whereas CD11b expression only increased in rats not treated with ZOL., Conclusions: The present data do not show substantial postischemic periosteal microcirculatory complications after chronic ZOL treatment either in sham-operated or OVX rats. The unaltered extent of limb IR-induced local periosteal microcirculatory reactions in the presence of reduced CD11b adhesion molecule expression on circulating PMNs, however, may be attributable to local endothelial injury/activation caused by ZOL.

Strength training induces not only muscle growth but also increased bone strength, a change that is expected to be associated with increased bone blood flow. However, the effects of exercise on contractile properties of bone microvascultaure have not been investigated. Once-a-week strength training with electrical muscle stimulation was applied unilaterally to tibialis anterior muscle of guinea pigs, while muscle force was measured from both legs to compare their muscle strength and endurance. After 10 weeks of training, changes in the arteriolar diameters of isolated periosteum taken from both trained and non-trained legs were measured using a video tracking system. Electrical field stimulation evoked a phasic constriction followed by a sustained dilatation in periosteal arterioles of trained legs, while triggering only vasoconstriction in the arterioles of non-trained legs. In trained leg arterioles, phentolamine, an α-adrenoceptor antagonist, inhibited both the constriction and dilatation. Prazosin, an α 1 -adrenoceptor antagonist, inhibited only the constriction, while yohimbine, α 2 -adrenoceptor antagonist, or l-nitro arginine (L-NA), a nitric oxide (NO) synthase inhibitor, inhibited the dilatation. In non-trained leg arterioles, phentolamine or prazosin largely suppressed the constriction, but failed to unmask any dilatation. Consistently, noradrenaline (NAd)-induced arteriolar constriction was enhanced and prolonged by L-NA in trained but not non-trained side arterioles. Thus, NAd released from sympathetic nerves appears to activate endothelial α 2 -adrenoceptors to release NO resulting in the sustained dilatation of periosteum arterioles from trained leg. The altered sympathetic vasomotor function would facilitate the blood supply to the bone and may contribute to the exercise-induced bone strength gain., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In 2004, we developed autologous periosteal sheets for the treatment of periodontal bone defects. This regenerative therapy has successfully regenerated periodontal bone and augmented alveolar ridge for implant placement. However, the necessity for 6-week culture is a limitation. Here, we examined the applicability of a human platelet-rich fibrin extract (PRFext) as an alternative to fetal bovine serum (FBS) for the explant culture of periosteal sheets in a novel culture medium (MSC-PCM) originally developed for maintaining mesenchymal stem cells. Small periosteum tissue segments were expanded in MSC-PCM + 2% PRFext for 4 weeks, and the resulting periosteal sheets were compared with those prepared by the conventional method using Medium199 + 10% FBS for their growth rate, cell multilayer formation, alkaline phosphatase (ALP) activity, and surface antigen expression (CD73, CD90, and CD105). Periosteal sheets grew faster in the novel culture medium than in the conventional medium. However, assessment of cell shape and ALP activity revealed that the periosteal cells growing in the novel medium were relatively immature. These findings suggest that the novel culture medium featuring PRFext offers advantages by shortening the culture period and excluding possible risks associated with xeno-factors without negatively altering the activity of periosteal sheets.

Introduction: Aminocaproic acid is approved as an anti-fibrinolytic for use in joint replacement and spinal fusion surgeries to limit perioperative blood loss. Previous animal studies have demonstrated a pro-osteogenic effect of aminocaproic acid in spine fusion models. Here, we tested if aminocaproic acid enhances appendicular bone healing and we sought to uncover the effect of aminocaproic acid on osteoprogenitor cells (OPCs) during bone regeneration., Methods: We employed a well-established murine femur fracture model in adult C57BL/6J mice after receiving two peri-operative injections of aminocaproic acid. Routine histological assays, biomechanical testing and micro-CT analyses were utilized to assess callus volume, and strength, progenitor cell proliferation, differentiation, and remodeling in vivo. Two disparate ectopic transplantation models were used to study the effect of the growth factor milieu within the early fracture hematoma on osteoprogenitor cell fate decisions., Results: Aminocaproic acid treated femur fractures healed with a significantly smaller cartilaginous callus, and this effect was also observed in the ectopic transplantation assays. We hypothesized that aminocaproic acid treatment resulted in a stabilization of the early fracture hematoma, leading to a change in the growth factor milieu created by the early hematoma. Gene and protein expression analysis confirmed that aminocaproic acid treatment resulted in an increase in Wnt and BMP signaling and a decrease in TGF-β-signaling, resulting in a shift from chondrogenic to osteogenic differentiation in this model of endochondral bone formation., Conclusion: These experiments demonstrate for the first time that inhibition of the plasminogen activator during fracture healing using aminocaproic acid leads to a change in cell fate decision of periosteal osteoprogenitor cells, with a predominance of osteogenic differentiation, resulting in a larger and stronger bony callus. These findings may offer a promising new use of aminocaproic acid, which is already FDA-approved and offers a very safe risk profile., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: To evaluate whether the subperiosteal injection of simvastatin (SIM) with a novel in situ gel-forming system, SrHA/Alg (strontium hydroxyapatite/alginate), can stimulate vertical bone augmentation in a rat calvarial model., Material and Methods: The SrHA/Alg solution was synthesized and combined with different doses of SIM (0.01, 0.02, 0.1, and 0.2 mg) to form the following groups: (1) SrHA/Alg only, (2) SrHA/Alg/0.01, (3) SrHA/Alg/0.02, (4) SrHA/Alg/0.1, and (5) SrHA/Alg/0.2. The SIM release pattern was analyzed, and rat primary periosteum-derived cell (PDC) responses were investigated. Twenty male Wistar rats were enrolled in the calvarial subperiosteal injection experiment with each animal receiving a 200-μl single subperiosteal injection of SrHA/Alg with different amounts of SIM (0, 0.01, 0.02, and 0.1 mg) incorporated (n = 5). The 0.2 mg dose group was not tested in vivo due to the severe toxicity found in vitro. The new bone formation was assessed histologically and radiologically at 8 weeks., Results: The slow release of SIM was confirmed, and PDC viability decreased in the SrHA/Alg/0.2 group. Alkaline phosphatase positive areas and mineralization areas were significantly greater in the SrHA/Alg/0.01 and SrHA/Alg/0.02 groups (p < .05). The mRNA expression level of Runx2 significantly increased in the SrHA/Alg/SIM-0.02 group by day 7 (p < .05) and significantly higher levels of VEGF were found in the SrHA/Alg/0.01 and SrHA/Alg/0.02 groups at different time points (p < .05). In vivo, no prominent clinical sign of inflammation was observed, and the most significant bone gain was shown in the SrHA/Alg/0.02 group (p < .05). The osteoclast formation within the newly formed bone area was reduced in the SrHA/Alg/0.1 group (p < .05)., Conclusions: When combined with SrHA/Alg system, the 0.02 mg SIM seemed to be the optimal dose to stimulate subperiosteal bone formation without inducing inflammation. This combination may hold potential therapeutic benefits for clinical bone augmentation in a minimally invasive manner., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Most recent advances in tissue engineering in the fields of oral surgery and dentistry have aimed to restore hard and soft tissues. Further improvement of these therapies may involve more biological approaches and the use of dental tissue stem cells in combination with inorganic/organic scaffolds. In this study, we analyzed the osteoconductivity of two different inorganic scaffolds based on poly (lactic-co-glycolic) acid alone (PLGA-Fisiograft) or in combination with hydroxyapatite (PLGA/HA-Alos) in comparison with an organic material based on equine collagen (PARASORB Sombrero) both in vitro and in vivo. We developed a simple in vitro model in which periosteum-derived stem cells were grown in contact with chips of these scaffolds to mimic bone mineralization. The viability of cells and material osteoconductivity were evaluated by osteogenic gene expression and histological analyses at different time points. In addition, the capacity of scaffolds to improve bone healing in sinus lift was examined. Our results demonstrated that the osteoconductivity of PLGA/HA-Alos and the efficacy of scaffolds in promoting bone healing in the sinus lift were increased. Thus, new clinical approaches in sinus lift follow-up should be considered to elucidate the clinical potential of these two PLGA-based materials in dentistry., Competing Interests: The authors declare no conflict of interest.

AbstractThe etiologic agent of yaws, Treponema pallidum subsp. pertenue , causes a multistage infection transmitted by nonsexual contact with the exudates from active lesions. Bone lesions in the form of osteoperiostitis are common and occur in numerous bones simultaneously in early stages. Although a multinational eradication campaign with mass administration of intramuscular benzathine benzylpenicillin in the 1950s greatly reduced its global incidence, a resurgence of yaws has occurred since around 2000 in western and central Africa and the Pacific Islands. The finding that a single oral dose of azithromycin (30 mg/kg) was as effective as benzathine benzylpenicillin prompted renewed interest by World Health Organization in 2012 toward eradication of this infection by 2020. We previously reported the excellent response to benzathine benzylpenicillin therapy for yaws osteoperiostitis. Herein, we document a confirmed case of yaws with osteoperiostitis successfully treated with single-dose azithromycin and discuss the pathology of yaws periostitis and comment on the implications of this in light of the new campaign toward yaws eradication.

Background: Poor bone quality, increased fracture risks, and impaired bone healing are orthopedic comorbidities of type 1 diabetes (T1DM). Standard osteogenic growth factor treatments are inadequate in fully rescuing retarded healing of traumatic T1DM long bone injuries where both periosteal and bone marrow niches are disrupted. We test the hypotheses that osteogenesis of bone marrow-derived stromal cells (BMSCs) and periosteum-derived cells (PDCs), two critical skeletal progenitors in long bone healing, are both impaired in T1DM and that they respond differentially to osteogenic bone morphogenetic proteins (BMPs) and/or insulin-like growth factor-1 (IGF-1) rescue., Methods: BMSCs and PDCs were isolated from Biobreeding Diabetes Prone/Worcester rats acquiring T1DM and normal Wistar rats. Proliferation, osteogenesis, and adipogenesis of the diabetic progenitors were compared with normal controls. Responses of diabetic progenitors to osteogenesis rescue by rhBMP-2/7 heterodimer (45 or 300 ng/ml) and/or rhIGF-1 (15 or 100 ng/ml) in normal and high glucose cultures were examined by alizarin red staining and qPCR., Results: Diabetic BMSCs and PDCs proliferated slower and underwent poorer osteogenesis than nondiabetic controls, and these impairments were exacerbated in high glucose cultures. Osteogenesis of diabetic PDCs was rescued by rhBMP-2/7 or rhBMP-2/7 + rhIGF-1 in both normal and high glucose cultures in a dose-dependent manner. Diabetic BMSCs, however, only responded to 300 ng/nl rhBMP-2/7 with/without 100 ng/ml rhIGF-1 in normal but not high glucose osteogenic culture. IGF-1 alone was insufficient in rescuing the osteogenesis of either diabetic progenitor. Supplementing rhBMP-2/7 in high glucose osteogenic culture significantly enhanced gene expressions of type 1 collagen (Col 1), osteocalcin (OCN), and glucose transporter 1 (GLUT1) while suppressing that of adipogenic marker peroxisome proliferator-activated receptor gamma (PPARγ) in diabetic PDCs. The same treatment in high glucose culture only resulted in a moderate increase in Col 1, but no significant changes in OCN or GLUT1 expressions in diabetic BMSCs., Conclusions: This study demonstrates more effective osteogenesis rescue of diabetic PDCs than BMSCs by rhBMP-2/7 with/without rhIGF-1 in a hyperglycemia environment, underscoring the necessity to tailor biochemical therapeutics to specific skeletal progenitor niches. Our data also suggest potential benefits of combining growth factor treatment with blood glucose management to optimize orthopedic therapeutic outcomes for T1DM patients.

Apart from its nutritive functions, the periosteum critically affects bone regeneration via its stem/osteoprogenitor cell content. Normal healing after bone fractures, trauma-orthopedic interventions and invasive dental procedures is critically linked to the reestablishment of the periosteal microcirculation, but the reconstruction, replacement or repair of lost tissues may also be performed with autologous periosteum. Besides the initiation of cell differentiation during bone repair and remodeling processes, the periosteum together with the endosteum plays significant roles in the pathogenesis of both hormone-related and trauma-induced osteoporotic alterations in the bone metabolism. Nevertheless, the axial bones, and in particular the jawbones, and the appendicular bones display differences not only in their blood supply and fracture healing characteristics, but also in respect of the development of osteoporosis and their reactions to treatment modalities (i.e. bisphosphonates). These reactions may also be linked to the differences in periosteal microcirculatory reactions. The present overview summarizes the relevant data of microcirculatory studies focusing on the periosteal reactions in different anatomical locations together with the optimal background methodologies, study models and the most significant observations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Intermittent administration of parathyroid hormone (PTH) is used to stimulate bone formation in patients with osteoporosis. A reduction in the degree of matrix mineralisation has been reported during treatment, which may reflect either production of undermineralised matrix or a greater proportion of new matrix within the bone samples assessed. To explore these alternatives, high resolution synchrotron-based Fourier Transform Infrared Microspectroscopy (sFTIRM) coupled with calcein labelling was used in a region of non-remodelling cortical bone to determine bone composition during anabolic PTH treatment compared with region-matched samples from controls. 8week old male C57BL/6 mice were treated with vehicle or 50μg/kg PTH, 5 times/week for 4weeks (n=7-9/group). Histomorphometry confirmed greater trabecular and periosteal bone formation and 3-point bending tests confirmed greater femoral strength in PTH-treated mice. Dual calcein labels were used to match bone regions by time-since-mineralisation (bone age) and composition was measured by sFTIRM in six 15μm 2 regions at increasing depth perpendicular to the most immature bone on the medial periosteal edge; this allowed in situ measurement of progressive changes in bone matrix during its maturation. The sFTIRM method was validated in vehicle-treated bones where the expected progressive increases in mineral:matrix ratio and collagen crosslink type ratio were detected with increasing bone maturity. We also observed a gradual increase in carbonate content that strongly correlated with an increase in longitudinal stretch of the collagen triple helix (amide I:amide II ratio). PTH treatment did not alter the progressive changes in any of these parameters from the periosteal edge through to the more mature bone. These data provide new information about how the bone matrix matures in situ and confirm that bone deposited during PTH treatment undergoes normal collagen maturation and normal mineral accrual., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mature osteoblasts have three fates: as osteocytes, quiescent lining cells, or osteoblasts that undergo apoptosis. However, whether intermittent parathyroid hormone (PTH) can modulate the fate of mature osteoblasts in vivo is uncertain. We performed a lineage-tracing study using an inducible gene system. Dmp1-CreERt2 mice were crossed with Rosa26R reporter mice to obtain targeted mature osteoblasts and their descendants, lining cells or osteocytes, which were detected using X-gal staining. Rosa26R:Dmp1-CreERt2(+) mice were injected with 0.25 mg 4-OH-tamoxifen (4-OHTam) on postnatal days 5, 7, 9, 16, and 23. In a previous study, at 22 days after the last 4-OHTam, most LacZ+ cells on the periosteal surface were inactive lining cells. On day 25 (D25), the mice were challenged with an injection of human PTH (1-34, 80 μg/kg) or vehicle daily for 10 (D36) or 20 days (D46). We evaluated the number and thickness of LacZ+ osteoblast descendants in the calvaria and tibia. In the vehicle group, the number and thickness of LacZ+ osteoblast descendants at both D36 and D46 significantly decreased compared to D25, which was attenuated in the PTH group. In line with these results, PTH inhibited the decrease in the number of LacZ+/osteocalcin-positive cells compared to vehicle at both D36 and D46. As well, the serum levels of sclerostin decreased, as did the protein expression of sclerostin in the cortical bone. These results suggest that intermittent PTH treatment can increase the number of periosteal osteoblasts by preventing mature osteoblasts from transforming into lining cells in vivo.

Current concepts of facial aging propose that volume losses of bone and fat in specific compartmentalized areas are the primary and the most important causes of the characteristic changes seen in the aging face. The authors propose that the use of Radiesse in the supra-periosteal space of the temple, zygomatic arch, anterior cheek, pyriform aperture, and pre-jowl sulcus most effectively and most efficiently corrects these volume losses, and therefore best helps to restore a youthful appearance. Placement of filler directly on bone produces a lifting effect on all of the overlying tissues as a single unit which the authors term "composite volumization". , , J Drugs Dermatol. 2016;15(9):1136-1141.

Voriconazole-induced periostitis (VIP) is a rare but increasingly encountered entity since Food and Drug Administration (FDA) approval of the second generation antifungal medication in 2002. Literature reports most commonly include transplant recipients on immunosuppressive therapy simultaneously requiring antifungal therapy. Nontransplant patients receiving long-term voriconazole have an equal risk of developing the disease, but may experience a delay in diagnosis due to a lack of familiarity with the process outside of the post-transplant and/or immunosuppressed population. We present a case of VIP in a nontransplant, immunocompetent patient on suppressive antifungal therapy for prior abdominal aortic stent graft fungal infection. Radiologist review of current medications and recognition of periostitis on multiple imaging modalities may hasten the diagnosis and lead to earlier treatment and resolution of symptoms.

Ethnopharmacological Relevance: Hancornia speciosa Gomes, commonly known as Mangabeira, is a Brazilian native fruit tree belonging to the Apocynaceae family. In folk medicine, the latex obtained from Mangabeira's trunk has been used as an adjunct therapy for bone fractures. Few pharmacological studies on the Hancornia speciosa latex have been developed and despite its popular use for bone healing there is no data about its biological effect on bone., Aim of the Study: The present study aimed to investigate the osteogenic potential of Hancornia speciosa latex in rat calvaria, as well as its phytochemical profile., Materials and Methods: A neutral gel composition containing 5% latex was topical applied to a critical size bone defect and over intact calvaria of rats. Areas of newly formed bone on the borders of the defect and of calvaria periosteum were quantified, as well as the percentage of BrdU-positive cells and total cells in the periosteum at different periods of time after latex application. The cytotoxicity of the latex aqueous phase was evaluated in rat calvarial cells in vitro by MTT assay and its phytochemical profile was investigated by ESI-MS/MS., Results: The area of newly formed bone on the borders of the calvaria defect was larger in rats that received latex at 15 and 30 days of healing. After 3 days of latex application over the intact calvaria, the periosteum area was increased and newly formed bone was observed after 5 and 11 days. There was also an increase in periosteum cell proliferation and population followed latex application on calvaria (p<0.05). The latex aqueous phase limited rat calvarial cell viability in vitro in concentrations larger than 0.6mg/mL. Chlorogenic acid and naringenin-7-O-glucoside were identified in the latex aqueous phase, along with catechin and procyanidin compounds., Conclusion: There was a stimulus for periosteum cell proliferation and bone formation when Hancornia speciosa latex was topically applied on rat calvaria. In addition, chlorogenic acid and naringenin-7-O-glucoside present in Hancornia speciosa latex may contribute to its effects on bone formation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-β1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (μMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p < 0.001), which in combination with BMP6 synergistically enhanced cartilage-like tissue formation that displayed in vitro mineralization capacity at day 14 (p < 0.001). Gene expression of μMasses cultured for 7 days with a medium formulation supplemented with 100 ng/mL of BMP2 and BMP6 and a low concentration of GDF5, TGF-β1, and FGF2 showed increased expression of Sox9 (1.7-fold) and the matrix molecules aggrecan (7-fold increase) and COL2A1 (40-fold increase) compared to nonstimulated control μMasses. The DoE analysis indicated that in GF combinations, BMP2 was the strongest effector for chondrogenic differentiation of hPDCs. When transplanted ectopically in nude mice, the in vitro-differentiated μMasses showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This study indicates the power of using the DoE approach for the creation of new medium formulations for skeletal tissue engineering approaches.

As part of our ongoing efforts to understand underlying mechanisms contributing to radiation-associated bone fragility and to identify possible treatments, we evaluated the longitudinal effects of parathyroid hormone (PTH) treatment on bone quality in a murine model of limited field irradiation. We hypothesized PTH would mitigate radiation-induced changes in the chemical composition and structure of bone, as measured by microscope-based Raman spectroscopy. We further hypothesized that collagen crosslinking would be especially responsive to PTH treatment. Raman spectroscopy was performed on retrieved tibiae (6-7/group/time point) to quantify metrics associated with bone quality, including: mineral-to-matrix ratio, carbonate-to-phosphate ratio, mineral crystallinity, collagen crosslink (trivalent:divalent) ratio, and the mineral and matrix depolarization ratios. Irradiation disrupted the molecular structure and orientation of bone collagen, as evidenced by a higher collagen crosslink ratio and lower matrix depolarization ratio (vs. non-irradiated control bones), persisting until 12weeks post-irradiation. Radiation transiently affected the mineral phase, as evidenced by increased mineral crystallinity and mineral-to-matrix ratio at 4weeks compared to controls. Radiation decreased bone mineral depolarization ratios through 12weeks, indicating increased mineral alignment. PTH treatment partially attenuated radiation-induced increases in collagen crosslink ratio, but did not restore collagen or mineral alignment. These post-radiation matrix changes are consistent with our previous studies of radiation damage to bone, and suggest that the initial radiation damage to bone matrix has extensive effects on the quality of tissue deposited thereafter. In addition to maintaining bone quality, preventing initial radiation damage to the bone matrix (i.e. crosslink ratio, matrix orientation) may be critical to preventing late-onset fragility fractures., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Biodegradable poly([D,L]-lactide-co-glycolide) (PLGA) nanofibrous membrane embedded with two drug-to-polymer weight ratios, namely 1:3 and 1:6, which comprised PLGA 180 mg, lidocaine 20 mg, vancomycin 20 mg, and ceftazidime 20 mg, and PLGA 360 mg, lidocaine 20 mg, vancomycin 20 mg, and ceftazidime 20 mg, respectively, was produced as an artificial periosteum in the treatment of segmental femoral fractures. The nanofibrous membrane's drug release behavior was assessed in vitro using high-performance liquid chromatography and the disk-diffusion method. A femoral segmental fracture model with intramedullary Kirschner-wire fixation was established for the in vivo rabbit activity study. Twenty-four rabbits were divided into two groups. Twelve rabbits in group A underwent femoral fracture fixation only, and 12 rabbits in group B underwent femoral fracture fixation and were administered the drug-loaded nanofibers. Radiographs obtained at 2, 6, and 12 weeks postoperatively were used to assess the bone unions. The total activity counts in animal behavior cages were also examined to evaluate the clinical performance of the rabbits. After the animals were euthanized, both femoral shafts were harvested and assessed for their torque strengths and toughness. The daily in vitro release curve for lidocaine showed that the nanofibers eluted effective levels of lidocaine for longer than 3 weeks. The bioactivity studies of vancomycin and ceftazidime showed that both antibiotics had effective and sustained bactericidal capacities for over 30 days. The findings from the in vivo animal activity study suggested that the rabbits with the artificial drug-eluting periosteum exhibited statistically increased levels of activity and better clinical performance outcomes compared with the rabbits without the artificial periosteum. In conclusion, this artificial drug-eluting periosteum may eventually be used for the treatment of open fractures.

The treatment of bone defects still presents complex problems, although various techniques have been developed. The periosteum is considered a good source of osteogenic precursor cells for new bone formation. It can be collected easily in the clinical setting and is less invasive to the donor site. However, the murine skull periosteum has a poor cellular component, and growth is very slow, making it important to identify a culture method for efficient growth. In the present study, we used three-dimensional cell migration with atelocollagen and gelatin media and found that both were effective for promoting the proliferation of periosteum-derived cells. Moreover, atelocollagen medium is expected to provide an added benefit as a scaffold structure in the ambient temperature of the human body. The selection of a proper surface marker for osteogenesis is imperative for bone regeneration. CD90 is a mesenchymal stem cell marker. Periosteum-derived cells sorted with CD90 showed higher proliferative capacity and osteogenic potential than that of unsorted periosteum-derived cells in vivo and in vitro. Thus, periosteum-derived cells sorted with CD90 are expected to be a good source for bone regeneration. Significance: Periosteum-derived cells showed higher proliferative capacity and osteogenic potential. Periosteum can be collected easily in the clinical setting and is less invasive to the donor site. Thus, periosteum-derived cells can be expected to be a good source for bone regeneration., (©AlphaMed Press.)

The sympathetic nervous system controls bone remodeling by regulating bone formation and resorption. How nerves and bone cells influence each other remains elusive. Here we modulated the content or activity of the neuropeptide Vasoactive Intestinal Peptide to investigate nerve-bone cell interplays in the mandible periosteum by assessing factors involved in nerve and bone behaviors. Young adult rats were chemically sympathectomized or treated with Vasoactive Intestinal Peptide or Vasoactive Intestinal Peptide10-28, a receptor antagonist. Sympathectomy depleted the osteogenic layer of the periosteum in neurotrophic proNerve Growth Factor and neurorepulsive semaphorin3a; sensory Calcitonin-Gene Related Peptide-positive fibers invaded this layer physiologically devoid of sensory fibers. In the periosteum non-osteogenic layer, sympathectomy activated mast cells to release mature Nerve Growth Factor while Calcitonin-Gene Related Peptide-positive fibers increased. Vasoactive Intestinal Peptide treatment reversed sympathectomy effects. Treating intact animals with Vasoactive Intestinal Peptide increased proNerve Growth Factor expression and stabilized mast cells. Vasoactive Intestinal Peptide10-28 treatment mimicked sympathectomy effects. Our data suggest that sympathetic Vasoactive Intestinal Peptide modulate the interactions between nervous fibers and bone cells by tuning expressions by osteogenic cells of factors responsible for mandible periosteum maintenance while osteogenic cells keep nervous fibers at a distance from the bone surface.

Due to the indispensable role of periosteum in bone defect healing and regeneration, a promising method to enhance osteogenesis of bone grafts by using an engineered biomimetic periosteum would be highly beneficial. The stromal microenvironment of periosteum is composed of various highly organized extracellular matrix (ECM) fibers, so an aligned natural ECM sheet, derived from the human dermal fibroblast cell sheet, may be advantageous when applied for artificial periosteum fabrication. Human mesenchymal stem cells (hMSCs) have been used to replace the osteoprogenitor cell population in native periosteum due to hMSCs' great osteogenic potential and fast in vitro expansion capacity. The objective of this work is to investigate if the natural ECM sheet and the substrate alignment can promote in vitro osteogenesis of hMSCs. The conventional cell culture substrates collagen I-coated polydimethylsiloxane (PDMS) and tissue culture plastic (TCP) were used as controls. It was found that the ECM sheet significantly increased alkaline phosphatase activity and calcium deposition. The enhanced osteogenic potential was further confirmed by increased bone-specific gene expression. The ECM sheet can bind significantly higher amounts of growth factors including ANG-1, TGF-β1, bFGF, and VEGF, as well as calcium phosphate nanoparticles, which contributed to high osteogenesis of the hMSCs on ECM sheet. However, the alignment of the substrates did not show significant influence on osteogenic activity and growth factor binding. These results demonstrated the great potential of hMSC-seeded ECM sheet as a biomimetic periosteum to improve critical sized bone regeneration.

Anodizing could be used for bio-functionalization of the surfaces of titanium alloys. In this study, we use anodizing for creating nanotubes on the surface of porous titanium alloy bone substitutes manufactured using selective laser melting. Different sets of anodizing parameters (voltage: 10 or 20V anodizing time: 30min to 3h) are used for anodizing porous titanium structures that were later heat treated at 500°C. The nanotopographical features are examined using electron microscopy while the bioactivity of anodized surfaces is measured using immersion tests in the simulated body fluid (SBF). Moreover, the effects of anodizing and heat treatment on the performance of one representative anodized porous titanium structures are evaluated using in vitro cell culture assays using human periosteum-derived cells (hPDCs). It has been shown that while anodizing with different anodizing parameters results in very different nanotopographical features, i.e. nanotubes in the range of 20 to 55nm, anodized surfaces have limited apatite-forming ability regardless of the applied anodizing parameters. The results of in vitro cell culture show that both anodizing, and thus generation of regular nanotopographical feature, and heat treatment improve the cell culture response of porous titanium. In particular, cell proliferation measured using metabolic activity and DNA content was improved for anodized and heat treated as well as for anodized but not heat-treated specimens. Heat treatment additionally improved the cell attachment of porous titanium surfaces and upregulated expression of osteogenic markers. Anodized but not heat-treated specimens showed some limited signs of upregulated expression of osteogenic markers. In conclusion, while varying the anodizing parameters creates different nanotube structure, it does not improve apatite-forming ability of porous titanium. However, both anodizing and heat treatment at 500°C improve the cell culture response of porous titanium., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The expression of transforming growth factor-beta 1 (TGF-β1) inside the callus cells of diabetic rats and the impact of insulin therapy on its expression and biomechanics was investigated. The rats were randomly divided as follows: an insulin therapy group (IT), a diabetic model group (DM), and a non-diabetic control group (NC). Bone specimens from each group were extracted at different times for immunohistochemical observation of the expression of TGF-β1. Concurrently, the destruction torque and torsional stiffness were detected at different times. One to four weeks after fracture, TGF-β1 was widely expressed in fractured callus cells and periosteal proliferating cells, while the expression inside diabetic cells was significantly reduced. The expression of TGF-β1 decreased over the first 68 weeks, and the mature bone cells never expressed TGF-β1. The destruction torque (Nm) detected in the 6th week revealed that there was a statistically significant difference between the DM, NC, and IT groups (P < 0.01). In conclusion, TGF-β1 expression was significantly reduced inside the callus cells of diabetic rats. Insulin therapy increased TGF-β1 expression inside the callus cells of diabetic rats and improved the biomechanical characteristics of the callus.

Our aim was to evaluate the effect of local simvastatin on the formation of new bone using a new design of periosteal distractor. The distractors were placed between the periosteum and bone at the inferior border of the mandible of 20 New Zealand rabbits. In the first group (n=10) simvastatin was applied locally to the distraction zone. The other 10 rabbits served as controls. The formation of new bone was evaluated with digital direct radiography, computed tomography (CT), and histomorphometric analyses. New bone formed in all rabbits, but more formed in the experimental group according to histomorphometric variables. However, other measurements did not differ significantly between the groups. The new design of the periosteal distraction device was successful in causing new bone to form. Local simvastatin made no significant contribution to the procedure., (Copyright © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Although bone pain caused by cancer chemotherapy is a well-recognized and significant problem, with approximately 1 in 10 childhood cancer patients being reported to experience isolated bone pain along with other skeletal complications, the underlying mechanisms are poorly understood and there is no specific treatment. In this study, effects of methotrexate (MTX) treatment on pain in the hind legs and the extent of sensory innervation of the tibial bone were examined through a 20-day time course in young rats after 5 daily 0.75 mg/kg MTX injections. MTX treatment increased von-Frey filament stimulation-induced mechanical allodynia and palpation nocifensive score in the tibia. MTX-treated rats showed trends in reduced loading (numbers of stands) on hind limbs after palpation, commencing early during treatment and 2 weeks after the end of treatment despite no signs of ongoing pain during normal locomotion. Immunohistochemical analyses showed an increase in innervation of calcitonin gene-related peptide (CGRP)-positive sensory nerve fibres in tibial periosteum on days preceding and overlapping with those rats with touch-evoked pain responses and the bone repair phase. These data suggest that methotrexate chemotherapy triggers touch-evoked pain involving enhanced sensory nerve innervation of the bone., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

The preservation of the bone-forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell-based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2-primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2-expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2-primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell-based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects., (© 2014 AlphaMed Press.)

The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid-alkali (AcAl), alkali-acid-heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown., Methods: Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools., Results: One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-μg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin-leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides., Conclusions: These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.

Bone loss is still a major problem in orthopedics. The purpose of this experimental study is to evaluate the safety and regenerative potential of a new scaffold based on a bio-ceramization process for bone regeneration in long diaphyseal defects in a sheep model. The scaffold was obtained by transformation of wood pieces into porous biomorphic silicon carbide (BioSiC®). The process enabled the maintenance of the original wood microstructure, thus exhibiting hierarchically organized porosity and high mechanical strength. To improve cell adhesion and osseointegration, the external surface of the hollow cylinder was made more bioactive by electrodeposition of a uniform layer of collagen fibers that were mineralized with biomimetic hydroxyapatite, whereas the internal part was filled with a bio-hybrid HA/collagen composite. The final scaffold was then implanted in the metatarsus of 15 crossbred (Merinos-Sarda) adult sheep, divided into 3 groups: scaffold alone, scaffold with platelet-rich plasma (PRP) augmentation, and scaffold with bone marrow stromal cells (BMSCs) added during implantation. Radiological analysis was performed at 4, 8, 12 weeks, and 4 months, when animals were sacrificed for the final radiological, histological, and histomorphometric evaluation. In all tested treatments, these analyses highlighted the presence of newly formed bone at the bone scaffolds' interface. Although a lack of substantial effect of PRP was demonstrated, the scaffold+BMSC augmentation showed the highest value of bone-to-implant contact and new bone growth inside the scaffold. The findings of this study suggest the potential of bio-ceramization processes applied to vegetable hierarchical structures for the production of wood-derived bone scaffolds, and document a suitable augmentation procedure in enhancing bone regeneration, particularly when combined with BMSCs.

Aneurysmal bone cyst (ABC), once considered a reactive lesion, has been proven to be a neoplasia characterized by rearrangements of the USP6-gene. Aggressive local growth and recurrences are common and therapeutic options may be limited due to the vicinity of crucial structures. We describe a case of a locally aggressive, multinucleated giant cell-containing lesion of the forearm of a 21-year old woman, treated with denosumab for recurrent, surgically uncontrollable disease. Under the influence of this RANKL inhibitor, the tumor showed a marked reduction of the content of the osteoclastic giant cells and an extensive metaplastic osteoid production leading to the bony containment, mostly located intracortically in the proximal radius. The diagnosis of a periosteal ABC was confirmed by FISH demonstrating USP6 gene rearrangement on the initial biopsy. Function conserving surgery could be performed, enabling reconstruction of the affected bone. Inhibition of RANKL with denosumab may offer therapeutic option for patients not only with giant cell tumors but also with ABCs.

Prostaglandin E1 (PGE1) is the drug of choice for providing ductal patency in cyanotic congenital heart disease (CCHD) for a short period of time until essential surgical management. Occasionally, prolonged use of PGE1 is required when the surgical procedure is delayed due to certain clinical conditions. Prolonged use of PGE1 may lead to bone and tissue changes such as pretibial and soft tissue swelling of the extremities and reversible cortical proliferation of the bones. Venous stasis as an additional risk factor can result in generalized periosteal reaction that initially can cause the separation of the periosteum from the cortex. We report an infant with CCHD who developed severe tissue swelling and generalized periosteal reaction due to coexistence of prolonged use of PGE1 and venous stasis. To the best of our knowledge, this is the first case report with both of these risk factors.