[Isolation and characterization of normal mandibular periosteal stem cells from human and macaca mulatta and cross-species single-cell analysis].

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.

Modulating the phenotype and function of bone marrow-derived macrophages via mandible and femur osteoblasts.

Various studies have been investigated the phenotypic and functional distinctions of craniofacial and long bone cells involved in bone regeneration. However, the process of bone tissue regeneration after bone grafting involves complicated interactions between different cell types at the donor-recipient site. Additionally, differences in alterations of the immune microenvironment at the recipient site remained to be explored. Osteoblasts (OBs) and macrophages (MØ) play essential roles in the bone restoration and regeneration processes in the bone and immune systems, respectively. The modulation of MØ on OBs has been extensively explored in the literature, whereas limited research has been conducted on the influence of OBs on the MØ phenotype and function. In the present study, OBs from the mandible and femur (MOBs and FOBs, respectively) promoted cranial defect regeneration in rats, with better outcomes noted in the MOBs-treated group. After MOBs transplantation, a significant inflammatory response was induced, accompanied by an early increase in IL-10 secretion. And then, there was an upregulation in M2-MØ-related cell markers and inflammatory factor expression. Condition media (CM) of OBs mildly inhibited apoptosis in MØ, enhanced their migration and phagocytic functions, and concurrently increased iNOS and Arg1 expression, with MOB-CM demonstrating more pronounced effects compared to FOB-CM. In conclusion, our investigation showed that MOBs and FOBs have the ability to modulate MØ phenotype and function, with MOBs exhibiting a stronger regulatory potential. These findings provide a new direction for improving therapeutic strategies for bone regeneration in autologous bone grafts from the perspective of the immune microenvironment.

Facile manufacturing of fused-deposition modeled composite scaffolds for tissue engineering-an embedding model with plasticity for incorporation of additives.

The fused-deposition modeling (FDM) process is carried out at an elevated temperature, preventing the addition of biological factors, drugs, bioactive compounds, etc, during fabrication. To overcome this disadvantage, a 3D interlinked porous polylactic acid (PLA) scaffold was fabricated by FDM, followed by the embedding of a polycaprolactone (PCL) scaffold into the pores of the PLA at room temperature, yielding a PLA-PCL scaffold. In addition, PLA-PCL scaffolds with nanohydroxyapatite (PLA-PCL-nHAP) and multiwalled carbon nanotubes (PLA-PCL-MWCNT) were also fabricated. Here, the FDM-fabricated PLA scaffold functions as the structural component, whereas the embedded PCL scaffold acts as the functional component, which provides a the ability to functionalize the scaffolds with the desired chemical or biological materials. The embedding process is straightforward, cost effective, and does not require sophistication. A mechanical characterization of the scaffolds suggests that the Young's modulus of the PLA-PCL scaffold (16.02 MPa) was higher than that of the FDM-fabricated PLA (9.98 MPa) scaffold, by virtue of embedded PCL matrix. In addition, finite element analysis showed that the von Mises stress on a mandible with scaffolds was 4.04 MPa, whereas for a mandible with a defect, it was 6.7 MPa, confirming the stress distribution efficiency and mechanical stability of these scaffolds. Furthermore, field emission-scanning electron microscope analysis implied the presence of interlinked porous structures with pore diameters of 50 µm to 300 µm. X-ray diffraction results revealed an increased crystallinity (%) in the embedded models (PLA-PCL, PLA-PCL-nHAP and PLA-PCL-MWCNT), compared to a PLA printed scaffold. Additionally, Raman analysis revealed that the embedding process did not cause chemical alterations in the polymeric chains. In vitro analysis with human osteoblasts demonstrated the osteoconductive nature of the scaffold, which supported mineralization. In brief, the advantage of our model is that it helps to overcome the difficulties of manufacturing a filament with the desired additives for FDM, and offers the ability to incorporate the desired concentrations of heat-labile bioactive molecules during the embedding process at ambient temperatures.

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers. In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45- mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

Superior CKIP-1 sensitivity of orofacial bone-derived mesenchymal stem cells in proliferation and osteogenic differentiation compared to long bone-derived mesenchymal stem cells.

Maxillofacial bone defects caused by multiple factors, including congenital deformations and tumors, have become a research focus in the field of oral medicine. Bone tissue engineering is increasingly regarded as a potential approach for maxillofacial bone repair. Mesenchymal stem cells (MSCs) with different origins display various biological characteristics. The aim of the present study was to investigate the effects of casein kinase­2 interaction protein­1 (CKIP­1) on MSCs, including femoral bone marrow­derived MSCs (BMMSCs) and orofacial bone­derived MSCs (OMSCs), isolated from the femoral and orofacial bones of wild­type (WT) and CKIP­1 knockout (KO) mice. MSCs were isolated using collagenase II and the main biological characteristics, including proliferation, apoptosis and osteogenic differentiation, were investigated. Subcutaneous transplantation of MSCs in mice was also performed to assess ectopic bone formation. MTT and clone formation assay results indicated that cell proliferation in the KO group was increased compared with the WT group, and OMSCs exhibited significantly increased levels of proliferation compared with BMMSCs. However, the proportion of apoptotic cells was not significantly different between CKIP­1 KO OMSCs and BMMSCs. Furthermore, it was revealed that osteogenic differentiation was increased in CKIP­1 KO MSCs compared with WT MSCs, particularly in OMSCs. Consistent with the in vitro results, enhanced ectopic bone formation was observed in CKIP­1 KO mice compared with WT mice, particularly in OMSCs compared with BMMSCs. In conclusion, the present results indicated that OMSCs may have a superior sensitivity to CKIP­1 in promoting osteogenesis compared with BMMSCs; therefore, CKIP­1 KO in OMSCs may serve as an efficient strategy for maxillofacial bone repair.

RNA-sequencing reveals positional memory of multipotent mesenchymal stromal cells from oral and maxillofacial tissue transcriptomes.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) can be isolated from numerous tissues and are attractive candidates for therapeutic clinical applications due to their immunomodulatory and pro-regenerative capacity. Although the minimum criteria for defining MSCs have been defined, their characteristics are known to vary depending on their tissue of origin. RESULTS: We isolated and characterized human MSCs from three different bones (ilium (I-MSCs), maxilla (Mx-MSCs) and mandible (Md-MSCs)) and proceeded with next generation RNA-sequencing. Furthermore, to investigate the gene expression profiles among other cell types, we obtained RNA-seq data of human embryonic stem cells (ESCs) and several types of MSCs (periodontal ligament-derived MSCs, bone marrow-derived MSCs, and ESCs-derived MSCs) from the Sequence Reads Archive and analyzed the transcriptome profile. We found that MSCs derived from tissues of the maxillofacial region, such as the jaw bone and periodontal ligament, were HOX-negative, while those derived from other tissues were HOX-positive. We also identified that MSX1, LHX8, and BARX1, an essential regulator of craniofacial development, were strongly expressed in maxillofacial tissue-derived MSCs. Although MSCs may be divided into two distinct groups, the cells originated from over the neck or not, on the basis of differences in gene expression profile, the expression patterns of all CD antigen genes were similar among different type of MSCs, except for ESCs. CONCLUSIONS: Our findings suggest that MSCs from different anatomical locations, despite meeting general characterization criteria, have remarkable differences in gene expression and positional memory. Although stromal cells from different anatomical sources are generally categorized as MSCs, their differentiation potential and biological functions vary. We suggested that MSCs may retain an original tissue memory about the developmental process, including gene expression profiles. This could have an important impact when choosing an appropriate cell source for regenerative therapy using MSCs.

Vessel-derived angiocrine IGF1 promotes Meckel's cartilage proliferation to drive jaw growth during embryogenesis.

Craniofacial development is a complex morphogenic process that requires highly orchestrated interactions between multiple cell types. Blood vessel-derived angiocrine factors are known to promote proliferation of chondrocytes in Meckel's cartilage to drive jaw outgrowth, however the specific factors controlling this process remain unknown. Here, we use in vitro and ex vivo cell and tissue culture, as well as genetic mouse models, to identify IGF1 as a novel angiocrine factor directing Meckel's cartilage growth during embryonic development. We show that IGF1 is secreted by blood vessels and that deficient IGF1 signalling underlies mandibular hypoplasia in Wnt1-Cre; Vegfafl/fl mice that exhibit vascular and associated jaw defects. Furthermore, conditional removal of IGF1 from blood vessels causes craniofacial defects including a shortened mandible, and reduced proliferation of Meckel's cartilage chondrocytes. This demonstrates a crucial angiocrine role for IGF1 during craniofacial cartilage growth, and identifies IGF1 as a putative therapeutic for jaw and/or cartilage growth disorders.

Comparison of Proliferation and Osteogenic Differentiation Potential of Rat Mandibular and Femoral Bone Marrow Mesenchymal Stem Cells In Vitro.

This study was conducted to compare the in vitro proliferation and osteogenic differentiation potential of mesenchymal stem cells (MSCs) derived from mandibular (M-MSCs) or femur (F-MSCs) tissues of rats. M-MSC and F-MSC cultures were isolated and established from the same rat. Cultures were observed for morphological changes by microscope and growth characteristics by CCK-8 and cloning assays. Cell adhesion ability on a culture plate and titanium sheet was detected by staining with toluidine blue and Hoechst 33258, respectively. The levels of Ca, P, and ALP (serially) during osteogenic differentiation were evaluated. Cultures were analyzed for mineralization potential with alizarin red and ALP staining methods and for differentiation markers with RT-PCR (ALP, Runx2, and OCN). M-MSCs and F-MSCs were successfully isolated from the same rat with uncontaminated culture, which showed significant differences in morphology. The proliferation rate of M-MSCs was higher than F-MSCs in primary culture, but significantly lower after passage. More colonies are formed from F-MSCs than from M-MSCs. M-MSCs showed a significantly higher mineralization and osteogenic differentiation potential, which might be of significance for use in bone/dental tissue engineering. In vitro, cell passage will decrease the proliferation ability of M-MSCs. The higher mineralization and osteogenic differentiation potential of M-MSCs could make them an approachable stem cell source for further application in stem cell-based clinical therapies.

Efficacy of three-dimensionally printed polycaprolactone/beta tricalcium phosphate scaffold on mandibular reconstruction.

It has been demonstrated that development of three-dimensional printing technology has supported the researchers and surgeons to apply the bone tissue engineering to the oromandibular reconstruction. In this study, poly caprolactone/beta tricalcium phosphate (PCL/ß-TCP) scaffolds were fabricated by multi-head deposition system. The feasibility of the three-dimensionally (3D) -printed PCL/ß-TCP scaffolds for mandibular reconstruction was examined on critical-sized defect of canine mandible. The scaffold contained the heterogeneous pore sizes for more effective bone ingrowth and additional wing structures for more stable fixation. They were implanted into the mandibular critical-sized defect of which periosteum was bicortically resected. With eight 1-year-old male beagle dogs, experimental groups were divided into 4 groups (n = 4 defects per group, respectively). (a) no further treatment (control), (b) PCL/ß-TCP scaffold alone (PCL/TCP), (c) PCL/ß-TCP scaffold with recombinant human bone morphogenetic protein-2 (rhBMP-2) (PCL/TCP/BMP2) and (d) PCL/ß-TCP scaffold with autogenous bone particles (PCL/TCP/ABP). In micro-computed tomography, PCL/TCP/BMP2 and PCL/TCP/ ABP groups showed significant higher bone volume in comparison to Control and PCL/TCP groups (P < 0.05). In histomorphometric analysis, a trend towards more bone formation was observed in PCL/TCP/BMP2 and PCL/TCP/ABP groups, but the results lacked statistical significance (P = 0.052). Within the limitations of the present study, 3D-printed PCL/ß-TCP scaffolds showed acceptable potential for oromandibular reconstruction.

Tuning of Titanium Microfiber Scaffold with UV-Photofunctionalization for Enhanced Osteoblast Affinity and Function.

Titanium (Ti) is an osteoconductive material that is routinely used as a bulk implant to fix and restore bones and teeth. This study explored the effective use of Ti as a bone engineering scaffold. Challenges to overcome were: (1) difficult liquid/cell infiltration into Ti microfiber scaffolds due to the hydrophobic nature of Ti; and (2) difficult cell attachment on thin and curved Ti microfibers. A recent discovery of UV-photofunctionalization of Ti prompted us to examine its effect on Ti microfiber scaffolds. Scaffolds in disk form were made by weaving grade 4 pure Ti microfibers (125 µm diameter) and half of them were acid-etched to roughen the surface. Some of the scaffolds with original or acid-etched surfaces were further treated by UV light before cell culture. Ti microfiber scaffolds, regardless of the surface type, were hydrophobic and did not allow glycerol/water liquid to infiltrate, whereas, after UV treatment, the scaffolds became hydrophilic and immediately absorbed the liquid. Osteogenic cells from two different origins, derived from the femoral and mandibular bone marrow of rats, were cultured on the scaffolds. The number of cells attached to scaffolds during the early stage of culture within 24 h was 3-10 times greater when the scaffolds were treated with UV. The development of cytoplasmic projections and cytoskeletal, as well as the expression of focal adhesion protein, were exclusively observed on UV-treated scaffolds. Osteoblastic functional phenotypes, such as alkaline phosphatase activity and calcium mineralization, were 2-15 times greater on UV-treated scaffolds, with more pronounced enhancement on acid-etched scaffolds compared to that on the original scaffolds. These effects of UV treatment were associated with a significant reduction in atomic carbon on the Ti microfiber surfaces. In conclusion, UV treatment of Ti microfiber scaffolds tunes their physicochemical properties and effectively enhances the attachment and function of osteoblasts, proposing a new strategy for bone engineering.

Human Dental Pulp Stem Cells in Rat Mandibular Bone Defects.

This study aimed to evaluate the use of human dental pulp stem cells (hDPSCs) in non-critical-sized mandibular bone defects in rats. hDPSCs from permanent teeth were isolated and engrafted in mandibular bone defects in rats for 7, 14, and 28 days; bone defects without cells formed the control group. Samples were evaluated by scanning electron microscopy (SEM), light microscopy (hematoxylin and eosin staining), and the regeneration area was measured by the Image J program. Before surgery procedures, the human dental pulp cells were characterized as dental pulp stem cells: fusiform morphology, plastic-adherent; expression of CD105, CD73, and CD90; lack of expression of CD45 and CD34, and differentiated into osteoblasts, adipocytes, and chondroblasts. The results indicated that within 7 days the control group presented a pronounced bone formation when compared with the treated group (p < 0.05). After 14 days, the treated group showed an increase in bone formation, but with no statistical difference among the groups (p > 0.05). In the final evaluated period there was no difference between the control group and the treated group (p > 0.05). There was a significant difference between 7 and 14 days (p < 0.05) and between 7 and 28 days (p < 0.05) in the treated group. In conclusion, there is no evidence that the use of hDPSCs in the conditions of this study could improve bone formation in non-critical-sized mandibular bone defects.

Skeletal Stem Cell-Schwann Cell Circuitry in Mandibular Repair.

Regenerative paradigms exhibit nerve dependency, including regeneration of the mouse digit tip and salamander limb. Denervation impairs regeneration and produces morphological aberrancy in these contexts, but the direct effect of innervation on the stem and progenitor cells enacting these processes is unknown. We devised a model to examine nerve dependency of the mouse skeletal stem cell (mSSC), the progenitor responsible for skeletal development and repair. We show that after inferior alveolar denervation, mandibular bone repair is compromised because of functional defects in mSSCs. We present mSSC reliance on paracrine factors secreted by Schwann cells as the underlying mechanism, with partial rescue of the denervated phenotype by Schwann cell transplantation and by Schwann-derived growth factors. This work sheds light on the nerve dependency of mSSCs and has implications for clinical treatment of mandibular defects.

Effects of nicotine exposure on murine mandibular development.

Nicotine is known to affect cell proliferation and differentiation, two processes vital to proper development of the mandible. The mandible, the lower jaw in mammals and fish, plays a crucial role in craniofacial development. Malformation of the jaw can precipitate a plethora of complications including disrupting development of the upper jaw, the palate, and or impeding airway function. The purpose of this study was to test the hypothesis that in utero nicotine exposure alters the development of the murine mandible in a dose dependent manner. To test this hypothesis, wild type C57BL6 mice were used to produce in utero nicotine exposed litters by adding nicotine to the drinking water of pregnant dams at concentrations of 0 µg/ml (control), 50 µg/ml (low), 100 µg/ml (medium), 200 µg/ml (high) throughout pregnancy to birth of litters mimicking clinically relevant nicotine exposures. Resultant pups revealed no significant differences in body weight however, cephalometric investigation revealed several dimensions affected by nicotine exposure including mandibular ramus height, mandibular body height, and molar length. Histological investigation of molars revealed an increase in proliferation and a decrease in apoptosis with nicotine exposure. These results demonstrate the direct effects of nicotine on the developing mandible outside the context of tobacco use, indicating that nicotine use including tobacco alternatives, cessation methods, and electronic nicotine delivering products may disrupt normal growth and development of the craniofacial complex.

High Prevalence of BRAF V600E Mutations in Langerhans Cell Histiocytosis of Head and Neck in Chinese Patients.

Langerhans cell histiocytosis (LCH) is characterized by clonal proliferation of Langerhans cells and has been classified as a hematolymphoid tumor. BRAF V600E mutation was found to be frequent in LCH; however, it has also been reported that Asia patients with LCH tend to show a lower rate of BRAF V600E mutation. In this study, we found LCH from the head and neck region often involved bone especially the posterior of the mandible and presented a high prevalence of BRAF V600E mutation in Chinese patients. Our findings also showed immunohistochemical detection correlated very well to DNA sequencing of BRAF alterations, which may be useful in the diagnosis of LCH, especially in cases with a low proportion of Langerhans cells, and BRAF inhibitors might be a treatment option for patients with LCH harboring BRAF V600E mutation.

Comparative characterization of mesenchymal stem cells from human dental pulp and adipose tissue for bone regeneration potential.

Bone tissue engineering is an area of regenerative medicine that attempts to repair bone defects. Seed cells such as dental pulp stem cells (DPSCs) and adipose tissue-derived stem cells (ADSCs) are two of the most well-characterized cells for bone regeneration because their use involves few ethical constraints and they have the ability to differentiate into multiple cell types, secreting growth factors and depositing mineral. However, bone regeneration ability of these cells remains unclear. This study aimed to compare the bone formation capacity of DPSCs and ADSCs in vitro and in vivo. Studies revealed that DPSCs had enhanced colony-forming ability, higher proliferative ability, stronger migration ability and higher expression of angiogenesis-related genes. They also secreted more vascular endothelial growth factor compared to ADSCs. In contrast, ADSCs grew more slowly compared to DPSCs but exhibited greater osteogenic differentiation potential, higher expression of osteoblast marker genes, and greater mineral deposition. Furthermore, after DPSCs and ADSCs were implanted into a mandibular defect of a rat for 6 weeks, ADSCs showed visible bone tissue as early as week 1 and promoted faster and greater bone regeneration compared to the DPSC group. These results suggest that ADSCs might be more useful than DPSCs for bone regeneration.

Histologic and Radiographic Characteristics of Bone Filler Under Bisphosphonates.

BACKGROUND: Dental implants and bone augmentation are well-established procedures used for oral rehabilitation. There is an increasing interest in biological mediators used topically for prevention of bone resorption maybe enhancement of osseointegration of dental implants. The purpose of the manuscript is to describe preliminarily the effect of bisphosphonates on the ossification pattern of bone grafts in a rat model. MATERIAL AND METHODS: Twenty Wistar-derived male rats were divided into 2 groups study and control. Bone substitute was added to mandibular defects and was covered by a resorbable collagen membrane. In the study group, the membrane was soaked with bisphosphonates suspension. In the control group, the membrane was soaked with saline solution. Radiographic and histomorphometric evaluation were performed. RESULTS: Radiographically, it was found that bone density was significantly higher in the study group. Histomorphometric analysis revealed a trend of higher bone volume fraction along with reduced bone substitute volume fraction in the study group, and increased number of osteoclasts and blood vessels in the control group. CONCLUSIONS: Within the limitations of our study it was found that there is a trend of increasing bone quantity and radiographic bone density by application of bisphosphonates.

Activity and morphologic changes in the mandible after mandibular osteotomy.

INTRODUCTION: Orthognathic surgery accelerates orthodontic tooth movement, and tooth movement accelerates with demineralized bone and accelerated bone remodeling. The purpose of this study was to ascertain whether orthognathic surgery induces accelerated bone remodeling. The research design included a human model and an animal model. METHODS: The levels of serum tartrate resistant acid phosphatase-5b (TRAP) and bone alkaline phosphatase (BALP) were measured in 15 patients after sagittal split ramus osteotomy. For the animal study, 18 rabbits were divided into 6 groups: a control group and 5 surgery groups. The rabbits in the surgery groups had osteotomies in the molar regions of the mandible. Changes in bone mass of the anterior mandibles were examined by microcomputed tomography, and changes in osteoblast and osteoclast numbers were analyzed by real-time polymerase chain reaction, hematoxylin and eosin staining, TRAP staining, and alkaline phosphatase staining. RESULTS: In the 15 patients, TRAP-5b increased from 1 to 8 weeks postoperatively, and BALP increased significantly in 2 weeks postoperatively. In the rabbits, the levels of mRNA expression of TRAP were increased at 3 weeks, and matrix metalloproteinase 9 was increased at 4 and 8 weeks, whereas mRNA expression of BALP and bone morphogenetic protein 2 were increased at 4 weeks. Bone loss was detected from 1 week postoperatively and reached the maximum at 3 weeks; and bone mass and mechanical structure did not recoverer to preoperative levels until 8 weeks postoperatively. CONCLUSIONS: These findings show active bone remodeling induced by osteotomy.

Long-lived murine osteocytes are embodied by craniofacial skeleton in young and old animals whereas they decrease in number in postcranial skeletons at older ages.

BACKGROUND: Osteocytes are engaged in life-enduring processes such as bone remodelling, fracture healing or osseointegration of implants. Over age, ossification processes and regenerative capacity can greatly differ in mandible and femur. OBJECTIVE: Mesenchymal stem cells from cranial and postcranial bones are of different embryologic origin. This may be the reason why the regenerative capacity differs between cranial and postcranial bones in old patients. It was hypothesised that different ageing patterns, reflected by osteocyte density, lacunar density and osteoid formation, exist between murine mandibles and femurs. MATERIAL AND METHODS: Mandible and femur of young (4 months) and old (34-36 months old) male C57Bl/6 mice were histologically investigated to determine the number of lacunae occupied with osteocytes. Osteoid formation was revealed by Masson-Goldner staining, and the spatial distribution of BMP-2 synthesis was examined. RESULTS: Over lifetime, the number of lacunae occupied with osteocytes only showed a modest decrease in mandibular bone (old 85.63%/young 91.12%) while greatly diverging in the femur (old 55.99%/young 93.28%). In equal measure, old femur exhibited less osteoid formation and decreased BMP-2 expression. CONCLUSION: Tissue-specific conduct of bone ageing is moulded by osteocytic activities, which was found to vary between postcranial and craniofacial skeleton. The latter harbours long-lived osteocytes also in old animals which assures lifelong bone integrity. Preliminary concurring findings from a human cadaver, also presented in this contribution, provided a rationale for recommending the translatability to humans.

Evaluation of the bisphosphonate effect on stem cells derived from jaw bone and long bone rabbit models: A pilot study.

BACKGROUND AND OBJECTIVE: Bisphosphonates have been widely used and the number of patients experiencing medication-related osteonecrosis of the jaw (MRONJ) has been increasing. This study was designed to evaluate the effect of zoledronate on stem cells derived from different tissues. DESIGN: Stem cells derived from four different tissues were compared using rabbit models (JPO: periosteum from the jaw bone (mandible), JBM: bone marrow from the jaw bone, LPO: periosteum from long bone (tibia), and LBM: bone marrow from long bone). Stem cells were grown in the presence of zoledronate at final concentrations ranging from 10-6M to 10-10M. Morphology was viewed under an inverted microscope, and the analysis of cell proliferation was performed using a Cell Counting Kit-8 (CCK-8) on days 1, 2, 4, and 7. RESULTS: The CCK-8 results for LBM showed that the increase of CCK-8 values was correlated with a longer incubation time. Compared to the untreated control, growth in the presence of zoledronate at 10-10M and 10-8M resulted in decreased CCK-8 values for LBM on day 7 (P<0.05). The CCK-8 results for JBM, LPO, and JPO on days 1, 2, 4, and 7 showed that the presence of zoledronate did not produce statistically significant changes compared with the untreated control. CONCLUSION: Zoledronate in the tested concentrations from JBM, LPO, and JPO did not produce noticeable alterations in the viability of mesenchymal stem cells. This in vitro experiment suggests that the occurrence of MRONJ solely in the oral cavity is not due to differences in the cellular proliferation of stem cells in the response to zoledronate.

Effect of Irrigation Time of Antiseptic Solutions on Bone Cell Viability and Growth Factor Release.

Antiseptic solutions are commonly utilized to treat local infection in the oral and maxillofacial region. However, surrounding vital bone is also exposed to antiseptic agents during irrigation and may have a potential negative impact on bone survival. The aim of the present study was therefore to investigate the effect of rinsing time with various antiseptic solutions on bone cell viability, as well as their subsequent release of growth factors important for bone regeneration. The bone samples collected from porcine mandible were rinsed in the following commonly utilized antiseptic solutions; povidone-iodine (0.5%), chlorhexidine digluconate (CHX, 0.2%), hydrogen peroxide (1%), and sodium hypochlorite (0.25%) for 1, 5, 10, 20, 30, or 60 minutes and assessed for cell viability and release of growth factors including vascular endothelial growth factor, transforming growth factor beta 1, bone morphogenetic protein 2, receptor activator of nuclear factor kappa-B ligand, and interleukin-1 beta by enzyme-linked immunosorbent assay. It was found in all the tested groups that the long exposure of any of the tested antiseptic solutions drastically promoted higher cell death. Sodium hypochlorite demonstrated the significantly highest cell death and at all time points. Interestingly, bone cell viability was highest in the CHX group post short-term rinsing of 1, 5, or 10 minutes when compared with the other 4 tested groups. A similar trend was also observed in subsequent growth factor release. The present study demonstrated that of the 4 tested antiseptic solutions, short-term CHX rinsing (ideally within 1 minute) favored bone cell viability and growth factor release. Clinical protocols should be adapted accordingly.