Peripheral nerve-derived Sema3A promotes osteogenic differentiation of mesenchymal stem cells through the Wnt/ß-catenin/Nrp1 positive feedback loop.

Sensory nerves play a crucial role in maintaining bone homeostasis by releasing Semaphorin 3A (Sema3A). However, the specific mechanism of Sema3A in regulation of bone marrow mesenchymal stem cells (BMMSCs) during bone remodelling remains unclear. The tibial denervation model was used and the denervated tibia exhibited significantly lower mass as compared to sham operated bones. In vitro, BMMSCs cocultured with dorsal root ganglion cells (DRGs) or stimulated by Sema3A could promote osteogenic differentiation through the Wnt/ß-catenin/Nrp1 positive feedback loop, and the enhancement of osteogenic activity could be inhibited by SM345431 (Sema3A-specific inhibitor). In addition, Sema3A-stimulated BMMSCs or intravenous injection of Sema3A could promote new bone formation in vivo. To sum up, the coregulation of bone remodelling is due to the ageing of BMMSCs and increased osteoclast activity. Furthermore, the sensory neurotransmitter Sema3A promotes osteogenic differentiation of BMMSCs via Wnt/ß-catenin/Nrp1 positive feedback loop, thus promoting osteogenesis in vivo and in vitro.

"Loop-Neurorrhaphy Technique for Preventing Bone Resorption and Preserving Lower Lip Sensation in Mandibular Reconstruction using Vascularized Iliac Bone Flap: A Single-Center Randomized Clinical Trial".

BACKGROUND: The aim of this study was to investigate whether using an innervated vascularized iliac bone flap could effectively prevent bone resorption and maintain sensory function in the lower lip. METHODS: In the innervated group, the deep circumflex iliac artery and recipient vessels were anastomosed, with simultaneous microanastomosis of ilioinguinal nerve, mental nerve and inferior alveolar nerve. Conversely, the control group underwent solely vascular anastomosis. CT was utilized to assess bone quality. Sensory recovery of the lower lip was recorded using two-point discrimination and current perception threshold testing. RESULTS: The study comprised a total of 40 subjects, with each group accounting for 20 participants, equally distributed in terms of gender. Hounsfield unit loss was significantly lower in the innervated group (13.26±8.65 %) as compared to the control group (37.98±8.60 %) (P < 0.001). Moreover, two-point discrimination values were lower in the innervated group (15.11±8.39 mm) when compared to the control group (21.44±7.24 mm) (P = 0.02). The current perception threshold values for the innervated group were 176.19 ± 31.89, 64.21 ± 19.23, and 42.29 ± 18.96 in 2kHz, 250Hz, and 5Hz respectively, while in the control group, the current perception threshold values were 204.47 ± 36.99, 82.26 ± 27.29, and 58.89 ± 25.38 in 2kHz, 250Hz, and 5Hz (P =0.02, 0.02, and 0.03, respectively). CONCLUSIONS: The innervated vascularized iliac bone flap represents a safe and effective novel approach to preserving lower lip sensation and preventing bone resorption through functional mandibular reconstruction.

Distinctive mesenchymal-like neurofibroma stem cells shape NF1 clinical phenotypes controlled by BDNF microenvironment.

BACKGROUND: Neurofibroma type I (NF1) often presents with multiple clinical phenotypes due to mutations of NF1 gene. The aim of this study was to determine the phenotypic and therapeutic relevance of tumor microenvironment in NF1 patients. METHODS: Tumor stem cells (TSCs) from NF1 were isolated and cultured using fluorescence activated cell sorting (FACS) and colony formation experiments. Then, flow cytometry was used to detect the surface markers, osteogenic and adipogenic differentiation were performed as well. Its tumorigenesis ability was confirmed by subcutaneous tumorigenesis in nude mice. Immunohistochemical staining was performed on neurofibroma tissues from the head and trunk with different phenotypes. The expression of BDNF in neurofibroma tissues was detected by Elisa and immunohistochemical staining. Western Blotting was used to detect the expression of p38 MAPK pathway in TSCs. The effect of BDNF neutralizing antibody on the tumorigenesis of TSCs was observed. RESULTS: Herein, we advocate that NF1 contain a new subgroup of mesenchymal-like neurofibroma stem cells (MNSCs). Such colony-forming MNSCs preserved self-renewal, multiple differentiation and tumorigenic capabilities. More interestingly, the MNSCs isolated from neurofibroma tissues of the same patient with different phenotypes presented site-specific capabilities. Moreover, different levels of brain-derived neurotrophic factor (BDNF) in neurofibroma tissues can impact the MNSCs by activating the TrkB/p38 MAPK pathway. Systemic administration of BDNF neutralizing antibodies inhibited MNSCs' characteristics. CONCLUSIONS: We demonstrated that BDNF can modulate MNSCs and thereby controlling different tumor phenotypes between the head and trunk regions. Application of BDNF neutralizing antibodies may inhibit p38 MAPK pathway, therefore providing a promising strategy for managing NF1.

Identification of Functional Modules and Key Pathways Associated with Innervation in Graft Bone-CGRP Regulates the Differentiation of Bone Marrow Mesenchymal Stem Cells via p38 MAPK and Wnt6/ß-Catenin.

Bone resorption occurs after bone grafting, however, contemporaneous reconstruction of the innervation of the bone graft is a potential treatment to maintain the bone mass of the graft. The innervation of bone is an emerging research topic. To understand the potential molecular mechanisms of bone innervation after bone grafting, we collected normal iliac bone tissue as well as bone grafts with or without innervation from nine patients 1 year after surgery and performed RNA sequencing. We identified differentially expressed genes) from these samples and used the gene ontology and Kyoto Encyclopedia of Genes and Genomes databases for functional enrichment and signaling pathway analysis. In parallel, we established protein-protein interaction networks to screen functional modules. Based on bioinformatic results, we validated in vitro the osteogenic differentiation potential of rat bone marrow mesenchymal stem cells (BMMSCs) after calcitonin gene-related peptide (CGRP) stimulation and the expression of p38 MAPK and Wnt6/ß-catenin pathways during osteogenesis. Our transcriptome analysis of bone grafts reveals functional modules and signaling pathways of innervation which play a vital role in the structural and functional integration of the bone graft. Simultaneously, we demonstrate that CGRP regulates the differentiation of BMMSCs through p38 MAPK and Wnt6/ß-catenin.

[Evaluation of the effect of simultaneous neuralized iliac bone flap on the preservation of lower lip and chin sensation during mandibular reconstruction].

PURPOSE: To evaluate the effect of reconstructing inferior alveolar nerve and preserving the sensation of lower lip and chin in repairing mandibular defect by simultaneous neuralized iliac bone flap. METHODS: Patients with continuous mandibular defects requiring reconstruction were randomly assigned to the innervated(IN) group and the control(CO) group by random number table. In the IN group, the deep circumflex iliac artery and recipient vessels were anastomosed microscopically during mandible reconstruction, and the ilioinguinal nerve(IN), mental nerve(MN) and inferior alveolar nerve(IAN) were anastomosed at the same time. In the CO group, only vascular anastomosis was performed without nerve reconstruction. During the operation, the nerve electrical activity after nerve anastomosis was detected by nerve monitor, and the sensory recovery of lower lip was recorded by two-point discrimination(TPD), current perception threshold (CPT) and Touch test sensory evaluator(TTSE) test. SPSS 26.0 software package was used for data analysis. RESULTS: According to the inclusion and exclusion criteria, a total of 20 patients were included, with 10 patients in each group. All the flaps survived in both groups, and no serious complications such as flap crisis occurred, and no obvious complications occurred in the donor site. The results of TPD test, CPT test and TTSE test all indicated that the degree of postoperative hypoesthesia in the IN group was less(P＜0.05). CONCLUSIONS: Simultaneous nerve anastomosis vascularized iliac bone flap can effectively preserve the feeling of lower lip and improve the postoperative quality of life of patients. It is a safe and effective technique.

Frontiers of Hydroxyapatite Composites in Bionic Bone Tissue Engineering.

Bone defects caused by various factors may cause morphological and functional disorders that can seriously affect patient's quality of life. Autologous bone grafting is morbid, involves numerous complications, and provides limited volume at donor site. Hence, tissue-engineered bone is a better alternative for repair of bone defects and for promoting a patient's functional recovery. Besides good biocompatibility, scaffolding materials represented by hydroxyapatite (HA) composites in tissue-engineered bone also have strong ability to guide bone regeneration. The development of manufacturing technology and advances in material science have made HA composite scaffolding more closely related to the composition and mechanical properties of natural bone. The surface morphology and pore diameter of the scaffold material are more important for cell proliferation, differentiation, and nutrient exchange. The degradation rate of the composite scaffold should match the rate of osteogenesis, and the loading of cells/cytokine is beneficial to promote the formation of new bone. In conclusion, there is no doubt that a breakthrough has been made in composition, mechanical properties, and degradation of HA composites. Biomimetic tissue-engineered bone based on vascularization and innervation show a promising future.

Novel loop neurorrhaphy technique to preserve lower lip sensate in mandibular reconstruction using an innervated vascularized iliac bone flap.

BACKGROUND: This study aimed to introduce a novel loop neurorrhaphy technique using an innervated vascularized iliac bone flap (VIBF) with vascularized ilioinguinal nerve (IIN) to reconstruct the inferior alveolar nerve (IAN) and preserve lower lip sensation simultaneously with mandibular reconstruction. METHODS: This study prospectively included patients who underwent mandibular reconstruction using VIBF from May 2018 to April 2020. Subjects were allocated into two groups: (1) Group I; innervated VIBF with loop neurorrhaphy (IIN doubly anastomosed with IAN and mental nerve), (2) Group II (control); conventional VIBF. Evaluation was done with operative time, intraoperative indocyanine green (ICG), lower lip sensory assessment (two-point discrimination [TPD] test and current perception threshold [CPT]), and drooling. RESULTS: Twelve patients were included; 6 in each group, (7 males and 5 females), age ranging from 18 to 57 years (average: 36.75 years). In all cases, intraoperative perfusion of IIN was confirmed by ICG. Group I showed a statistically significant more flap harvesting time compared with group II (mean difference, 5.67 min; P = 0.0091). There was a significant difference in sensory recovery favoring group I (P < 0.05). The TPD results in group I showed an average of 9.8 ± 6.9 mm and 6.2 ± 5.7 mm on operated and non-operated sides, while Group II showed a poor sensory recovery, and the TPD showed an average of 24.6 ± 6.7 mm and 8.4 ± 2.3 mm on operated and non-operated sides. The CPT results showed a significant difference between both groups. In Group I, the extent of drooling was 3.16 ± 0.75, while in Group II, the score was 1.6 ± 0.81, revealing a significant difference favoring Group I. CONCLUSIONS: Concurrent mandibular reconstruction using VIBF and loop neurorrhaphy with vascularized IIN to reconstruct IAN successfully restore lower jaw form and preserve lip sensation.

[Simultaneous innervation revitalizes bone marrow mesenchymal stem cells from postoperative iliac grafts].

PURPOSE：To compare the osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) from innervated iliac graft bone flap and traditional one for reconstructing mandibular defects. METHODS：Graft bone marrow samples were harvested 1 year after free vascularized iliac reconstruction of mandibular defects, with or without innervation through simultaneous nerve anastomosis. BMMSCs were isolated and cultured in vitro. Colony forming units-fibrosis observation, Brdu incorporation assay, population doubling, Alizarin red staining for in vitro calcified nodule formation and in vivo assay of subcutaneous osteogenesis in nude mice were used to detect BMMSCs proliferation, self-renewal and osteogenic differentiation capabilities, respectively. SPSS 24.0 software package was used for statistical analysis. RESULTS：Colony formation, proliferation, population doubling and osteogenic differentiation abilities of BMMSCs from innervated group were significantly higher than those from non-innervated group（P<0.05）. CONCLUSIONS：Simultaneous innervation of free vascularized iliac during reconstruction of mandibular defects may maintain self-renewal and osteogenic differentiation potentials of BMMSCs in graft bones, thereby maintaining bone homeostasis and reducing postoperative graft bone resorption.