OBJECTIVE: To assess the possibility of vertical alveolar ridge augmentation by means of activation of the periosteum.
METHODS: Six adult male Beagle dogs were used for the study. All premolars and first molars were extracted, and one vertical saucer-shaped bony defect was created on each side of the mandible. After 3 months of healing, full-thickness muco-periosteal flaps were elevated, and one distraction device was placed on each side of the mandible. The distraction plate was left submerged, and the activation mechanism connected to the distraction rod was exposed intra-orally. The protocol of periosteal activation (PP: periosteal \'pumping\') was initiated after a latency of 7 days. The alternation of activation and relaxation at the rate of 0.35 mm/12 h during 5 days was followed by the sole activation of 0.35 mm/12 h for 5 days (PP group). Devices were left inactivated on the contralateral control side of the mandible (C group). All animals were euthanized after 8 weeks of consolidation. Samples were analysed histologically and by means of micro-CT.
RESULTS: New mature lamellar bone was formed over the pristine bone in all groups. More intensive signs of bone modelling and remodelling were observed in the PP group compared to the C group. Mean new bone, bone marrow, connective tissue and total volumetric densities were greater in the PP group (p < 0.001, p = 0.001, p = 0.003 and p < 0.001, respectively). No differences were observed in the relative area parameters. Total tissue volume and bone volume were higher in the PP group (p = 0.031 and p = 0.076, respectively), while the bone mineral densities were higher in the C group (p = 0.041 and p = 0.003, respectively). Trabecular number, trabecular thickness and trabecular separation values were similar between the two groups.
CONCLUSIONS: Regeneration of vertical alveolar bone ridge defects may be enhanced by activation of the periosteum, without the application of bone grafting materials.

The vascularized periosteal free flap transposes a non-irradiated soft tissue with neoangiogenesis, bone induction, and osteogenesis qualities. A surgical technique using a humeral periosteal free flap is described for the treatment of recurrent osteoradionecrosis of the lower jaw. The humeral periosteal free flap is a technique associated with low morbidity. The procedure described avoids sacrificing major vessels as seen in other common flap procedures. Hence, this revascularization approach is equivalent to a prevention technique that should be considered early in the development of osteoradionecrosis to avoid further damage and challenging reconstruction.

BACKGROUND: A variety of congenital or acquired conditions can cause craniomaxillofacial bone defects, resulting in a heavy financial burden and psychological stress. Guided bone self-generation with periosteum-preserved has great potential for reconstructing large bone defects.
METHODS: A swine model of guided bone regeneration with occlusive periosteum was established, the rib segment was removed, and the periosteum was sutured to form a closed regeneration chamber. Hematoxylin and eosin staining, Masson\'s staining, and Safranine O-Fast Green staining were done. Nine-time points were chosen for collecting the periosteum and regenerated bone tissue for gene sequencing. The expression level of each secreted frizzled-related protein (SFRP) member and the correlations among them were analyzed.
RESULTS: The process of bone regeneration is almost complete 1 month after surgery, and up to 1 week after surgery is an important interval for initiating the process. The expression of each SFRP family member fluctuated greatly. The highest expression level of all members ranged from 3 days to 3 months after surgery. The expression level of SFRP2 was the highest, and the difference between 2 groups was the largest. Secreted frizzled-related protein 2 and SFRP4 showed a notable positive correlation between the control and model groups. Secreted frizzled-related protein 1, SFRP2, and SFRP4 had a significant spike in fold change at 1 month postoperatively. Secreted frizzled-related protein 1 and SFRP2 had the strongest correlation.
CONCLUSIONS: This study revealed the dynamic expression of the SFRP family in guided bone regeneration with occlusive periosteum in a swine model, providing a possibility to advance the clinical application of bone defect repair.

Fracture management largely relies on the bone\'s inherent healing capabilities and, when necessary, surgical intervention. Currently, there are limited osteoinductive therapies to promote healing, making targeting skeletal stem/progenitor cells (SSPCs) a promising avenue for therapeutic development. A limiting factor for this approach is our incomplete understanding of the molecular mechanisms governing SSPCs\' behavior. We have recently identified that the Leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6) is expressed in sub-populations of SSPCs, and is required for maintaining bone volume during adulthood and for proper fracture healing. Lgr family members (Lgr4-6) are markers of stem cell niches and play a role in tissue regeneration primarily by binding R-Spondin (Rspo1-4). This interaction promotes canonical Wnt (cWnt) signaling by stabilizing Frizzled receptors. Interestingly, our findings here indicate that Lgr6 may also influence cWnt-independent pathways. Remarkably, Lgr6 expression was enhanced during Bmp-mediated osteogenesis of both human and murine cells. Using biochemical approaches, RNA sequencing, and bioinformatic analysis of published single-cell data, we found that elements of BMP signaling, including its target gene, pSMAD, and gene ontology pathways, are downregulated in the absence of Lgr6. Our findings uncover a molecular interdependency between the Bmp pathway and Lgr6, offering new insights into osteogenesis and potential targets for enhancing fracture healing.

Innovations in surgical techniques have improved the esthetic outcome and predictability of root coverage procedures in recent years. A free gingival graft (FGG) augments the attached gingiva, but the compromised blood supply precludes its use in root coverage. In the surgical technique described in this case report, the FGG kept over a laterally placed periosteal flap enhanced the outcome. A laterally flipped periosteal flap was adapted over the root surface using resorbable sutures. The free graft was secured at the recipient site with cyanoacrylate adhesive, and adaptation was ensured with suspensory sutures. Satisfactory root coverage was appreciated and maintained at 6 months with excellent functional outcomes. Adequate width of the attached gingiva and vestibular depth were also noticed at the recipient site. The patient was highly satisfied with the obtained results, which were maintained until the 1-year postoperative period.

Introduction  Myringoplasty is a common otologic procedure to restore the integrity of the tympanic membrane in cases of traumatic or pathologic perforations. Many grafting materials have been used with different techniques. Objective  In the present work, we evaluate the surgical and audiological outcomes of periosteal graft overlying the mastoid cortex through a retroauricular incision in a pediatric cohort. Methods  A retrospective study was carried out involving all children aged ≤ 16 years who underwent periosteal graft myringoplasty for the treatment of chronic suppurative otitis media with dry central perforation in our hospital from April 2019 to April 2021. All patients were followed up for one year to assess the anatomical success and functional outcomes by comparing the preoperative and postoperative (after six months) results of pure tone audiometry (PTA). Results  The sample was composed of 36 patients; 20 of them were female (55.6%) and 16 were male (44.4%) subjects, with ages ranging from 7 to 16 (mean: 12.7) years. Four patients underwent surgery in both ears (with an interval of 6 to 9 months). Out of 40 surgeries performed, 38 ears have shown anatomical success (95%). A highly significant improvement in hearing was obtained (the mean difference between the pre- and postoperative results of the PTA was of 14.6 ± 3.45 dB ( p  < 0.001). Conclusion  We advocate the use of periosteal graft in the pediatric population as a good alternative for other types of grafts, with comparable and even better functional and anatomical outcomes.

Objective: To investigate the presence of a distinct stem cell populations different from mesenchymal stem cells in the mandibular periosteum of both human and non-human primates (macaca mulatta), to explore its properties during intramembranous osteogenesis and to establish standard protocols for the isolation, culturing and expanding of mandibular periosteal stem cells (PSC) distinguished from other PSCs in other anatomical regions. Methods: Periosteum was harvested from the bone surface during flap bone removal in patients aged 18-24 years undergoing third molar extraction and from the buccal side of the mandibular premolar region of 6-year-old macaca mulatta respectively, and then subjected to single-cell sequencing using the Illumina platform Novaseq 6000 sequencer. Cross-species single-cell transcriptome sequencing results were compared using homologous gene matching. PSC were isolated from primary tissues using two digestion methods with body temperature and low temperature, and their surface markers (CD200, CD31, CD45 and CD90) were identified by cell flow cytometry. The ability of cell proliferation and three-lineage differentiation of PSC expanded to the third generation in vitro in different species were evaluated. Finally, the similarities and differences in osteogenic properties of PSC and bone marrow mesenchymal stem cells (BMSC) were compared. Results: The single-cell sequencing results indicated that 18 clusters of cell populations were identified after homologous gene matching for dimensionality reduction, and manual cellular annotation was conducted for each cluster based on cell marker databases. The comparison of different digestion protocols proved that the low-temperature overnight digestion protocol can stably isolate PSC from the human and m. mulatta mandibular periosteum and the cells exhibited a fibroblast-like morphology. This research confirmed that PSC of human and m. mulatta had similar proliferation capabilities through the cell counting kit-8 assay. Flow cytometry analysis was then used to identify the cells isolated from the periosteum expressed CD200(+), CD31(-), CD45(-), CD90(-). Then, human and m. mulatta PSC were induced into osteogenesis, adipogenesis, and chondrogenesis to demonstrate their corresponding multi-lineage differentiation capabilities. Finally, comparison with BMSC further clarified the oesteogenesis characteristics of PSC. The above experiments proved that the cells isolated from the periosteum were peiosteal cells with characteristics of stem cells evidenced by their cell morphology, proliferation ability, surface markers, and differentiation ability, and that this group of PSC possessed characteristics different from traditional mesenchymal stem cells. Conclusions: In this study, normal mandibular PSC from humans and m. mulatta were stably isolated and identified for the first time, providing a cellular foundation for investigating the mechanism of mandibular intramembranous osteogenesis, exploring ideal non-human primate models and establishing innovative strategies for clinically mandibular injury repair.
目的： 探究人与非人灵长类动物（恒河猴）的下颌骨骨膜中是否存在有别于传统间充质干细胞的干细胞群体及其在膜内成骨过程中的特性，并提供区别于其他解剖区域骨膜干细胞（PSC）的下颌骨PSC的稳定分离、培养、扩增的标准化流程。 方法： 分别从上海交通大学医学院附属第九人民医院的18~24岁行第三磨牙拔除术3例患者翻瓣去骨过程中的骨块表面和3只6岁龄恒河猴下颌骨的磨牙区颊侧获取骨膜，使用Illumina平台Novaseq 6000测序仪进行单细胞测序，并通过同源基因匹配进行跨物种单细胞转录组测序的结果比较。使用37 ℃和低温两种消化方式从原代组织中分离PSC，经流式细胞术分析鉴定其表面标志物（CD200、CD31、CD45和CD90）并通过免疫荧光鉴定组织蛋白酶K（CTSK）与CD200的共定位情况。评估体外扩增至第3代的不同物种PSCs的细胞增殖能力和三系分化能力。比较PSC和骨髓间充质干细胞（BMSC）在成骨方面的特性异同。 结果： 单细胞测序结果提示直系同源基因匹配降维后获得了18个聚类的细胞群，基于各细胞标志物数据库对各聚类进行细胞注释。低温消化方案可以稳定地从人、恒河猴下颌骨骨膜中分离出PSC，细胞呈成纤维细胞状。细胞计数法结果显示人与恒河猴的PSC增殖能力差异无统计学意义，流式细胞术分析鉴定结果显示，从骨膜分离的细胞表面抗原表达CD200+、CD31-、CD45-和 CD90-，免疫荧光提示CTSK与CD200共定位于此细胞中。茜素红染色、油红O染色和阿尔辛蓝染色结果显示，人和恒河猴的PSC均具备成骨、成脂、成软骨的分化能力。与BMSC的成骨能力相比，PSC增殖能力略优，分化过程中，PSC在早期有良好的成骨表现。 结论： 本研究成功稳定分离并鉴定出人和非人灵长类动物（恒河猴）的正常下颌骨PSC，为探索下颌骨膜内成骨的机制、建立理想的非人灵长类动物模型以及下颌骨缺损修复新型策略提供了细胞学基础。.

OBJECTIVE: Following a mild traumatic brain injury (mTBI), the most prevalent and profoundly debilitating occurrence is the emergence of an acute and persistent post-traumatic headache (PTH), for which there are presently no approved treatments. A crucial gap in knowledge exists regarding the consequences of an mTBI, which could serve as a foundation for the development of therapeutic approaches. The activation of trigeminal sensory nerve terminals that innervate the calvarial periosteum (CP)-a densely innervated tissue layer covering the calvarial skull-has been implicated in both migraines and PTHs. We have previously shown that trigeminal oxytocin receptors (OTRs) may provide a therapeutic target for PTHs. This study examined the expression of oxytocin receptors on trigeminal nerves innervating the periosteum and whether these receptors might serve as a therapeutic target for PTHs using a direct application of oxytocin to the periosteum in a rodent model of PTH.
METHODS: We used retrograde tracing and immunohistochemistry to determine if trigeminal ganglion (TG) neurons innervating the periosteum expressed OTRs and/or CGRPs. To model the impact of local inflammation that occurs following an mTBI, we applied chemical inflammatory mediators directly to the CP and assessed for changes in immediate-early gene expression as an indication of neuronal activation. We also determined whether mTBI would lead to expression changes to OTR levels. To determine whether these OTRs could be a viable therapeutic target, we assessed the impact of oxytocin injections into the CP in a mouse model of PTH-induced periorbital allodynia.
RESULTS: The results of these experiments demonstrate the following: (1) the cell bodies of CP afferents reside in the TG and express both OTRs and CGRPs; (2) inflammatory chemical stimulation of the periosteum leads to rapid activation of TG neurons (phospho-ERK (p-ERK) expression), (3) mTBI-induced inflammation increased OTR expression compared to the sham group; and (4) administration of oxytocin into the periosteum on day 2 and day 40 blocked cutaneous allodynia for up to one hour post-administration for both acute and persistence phases in the PTH model-an effect that was preventable by the administration of an OTR antagonist.
CONCLUSIONS: Taken together, our observations suggest that periosteal trigeminal afferents contribute to post-TBI craniofacial pain, and that periosteum tissue can be used as a potential local target for therapeutics such as oxytocin.

The aim of this study is to validate a minimally invasive surgical procedure to harvest palate periosteum as a source of tissue for mesenchymal stromal/stem cells. We performed a standardized procedure to harvest the palate periosteum in ten subjects, which consisted of a 3 mm disposable punch and a Molt periosteal elevator to harvest a small full-thickness fragment of soft tissue at the hard palate area, between the upper bicuspids, 3 to 4 mm apical to the cement enamel junction. The one-third inner portion was fragmented, and following standard cell culture procedures, the adherent cells were cultured for three passages, after obtaining 70-90% confluence. Cell morphology analysis, flow cytometry analysis, and viability and osteogenic differentiation assays were performed. In all 10 cases, uneventful healing was observed, with no need for analgesic intake. The evaluation of cell morphology showed elongated spindle-shaped cells distributed in woven patterns. A high viability range was verified as well as an immunophenotype compatible with mesenchymal stem cell lineage. The differentiation assay showed the potential of the cells to differentiate into the osteogenic lineage. These results demonstrate that the minimally invasive proposed surgical technique is capable of supplying enough periosteum source tissue for stem cell culture and bone tissue engineering.

Congenital pseudarthrosis of the forearm poses a considerable challenge because of its rarity. The objective of this report is to introduce a novel surgical technique for its treatment. Here, we document a case of congenital pseudarthrosis of the radius in a 3-year-old boy diagnosed with type-1 neurofibromatosis. The surgical treatment involved the excision of approximately 9 cm of native radial periosteum and a bifocal radius osteotomy, which was supplemented with a vascularized tibial periosteal transplant to facilitate bone healing. Anastomosis between the anterior tibial vessels and radial vessels was performed. No immediate or late postoperative complications were observed. After 3 weeks, a robust callus formation was observed, and during a follow-up examination 3 years and 4 months later, a wide range of active forearm rotation was noted. This report suggests that vascularized periosteal flaps show promise as a viable treatment option for congenital pseudarthrosis of the forearm. They offer an alternative to vascularized fibular grafts or single-bone forearm constructs.