Facial changes in patients with skeletal class III deformity after bimaxillary surgery: an evaluation based on three-dimensional photographs registered with computed tomography.

The objective of this study was to evaluate facial soft and hard tissue changes, individually and relative to each other, in patients with skeletal class III deformity after bimaxillary surgery using three-dimensional (3D) photos obtained by white light scanning. Thirty patients with skeletal class III deformity who underwent bimaxillary surgery were selected. Each patient underwent white light scanning and spiral computed tomography (CT) within two weeks before (T0) and six months after surgery (T1). The 3D photos were registered with CT soft tissue models for T0 and T1, and the skeletal area unaffected by treatment (cranial base) was used to register T0 and T1. Then, the 3D colour-coded map was analysed to assess both skeletal and soft tissue changes between T0 and T1. Changes in the 3D coordinates of each anatomical landmark were analysed using the Student's t-test. Maxillary advancement by 2-3 mm and mandibular recession by 5-6 mm were observed; the mandible was shortened in the vertical direction. Compared with the preoperative values, the nasal columella was 0.51 mm shorter, the upper lip was 0.71 mm longer, the base of the alar cartilage was 1.38 mm wider, and the nasolabial angle became larger. The ratio of change in the position of soft tissue point Sn to hard tissue point A was 0.73:1, and that of soft tissue point Pg to hard tissue point Pog was 0.86:1. Images obtained by structured white light scanning registered with CT can be used as an alternative to study facial changes after orthognathic surgery.

Soluble Immune-Related Proteins as New Candidate Serum Biomarkers for the Diagnosis and Progression of Lymphangioleiomyomatosis.

Background: The goal of this study was to analyze serum from lymphangioleiomyomatosis (LAM) patients and healthy controls to identify novel biomarkers that could shed light on disease diagnosis and pathogenesis. Methods: From April 2017 to October 2019, qualified serum samples were obtained to explore differences in 59 immune proteins between 67 LAM patients and 49 healthy controls by the Luminex method. Results: We characterized 22 serum immune proteins that were differentially expressed in LAM patients compared with healthy people. Fifty-nine proteins were then classified into eight categories according to their biological function, and the results showed that LAM patients displayed significantly higher levels of growth factors (p = 0.006) and lower levels of costimulatory molecules (p = 0.008). LAG-3 was not only likely to have better predictive value than VEGF-D but also showed a significant difference between patients without elevated VEGF-D and healthy people. IL-18 was positively correlated with lung function and six-minute walk test (6MWT) distance and negatively correlated with St. George's Respiratory Questionnaire (SGRQ) score and pulmonary artery systolic pressure (PASP), which suggested that IL-18 was related to disease severity. PD-1 was significantly different between patients with pneumothorax and/or chylothorax and those without complications. Conclusion: We performed a large-scale serum immune factor analysis of LAM. Our study provides evidence that LAG-3 may be a novel candidate serum biomarker for the diagnosis of LAM. Future independent validation in prospective studies is warranted.

In vivo biocompatibility and degradability of a Zn-Mg-Fe alloy osteosynthesis system.

Zinc is generally considered to be one of the most promising materials to be used in biodegradable implants, and many zinc alloys have been optimized to improve implant biocompatibility, degradation, and mechanical properties. However, long-term degradation leads to the prolonged presence of degradation products, which risks foreign body reactions. Herein, we investigated the in vivo biocompatibility and degradation of a biodegradable Zn-Mg-Fe alloy osteosynthesis system in the frontal bone, mandible, and femur in beagles for 1 year. Results of the routine blood, biochemical, trace element, and histological analyses of multiple organs, peripheral blood CD4/CD8a levels, and serum interleukin 2 and 4 levels showed good biocompatibility of the Zn-Mg-Fe alloy. Zinc content analysis revealed zinc accumulation in adjacent bone tissue, but not in the liver, kidney, and spleen, which was related to the degradation of the Zn-Mg-Fe alloy. The alloy demonstrated a uniform slowing degradation rate in vivo. No degradation differences in the frontal bone, mandible, and femur were observed. The degradation products included zinc oxide [ZnO], zinc hydroxide [Zn(OH)2], hydrozincite [Zn5(OH)6(CO3)2], and hopeite [Zn3(PO4)2·4H2O]. The good biocompatibility and degradation properties of the Zn-Mg-Fe alloy render it a very attractive osteosynthesis system for clinical applications.

Engineering of axially vascularized bone tissue using natural coral scaffold and osteogenic bone marrow mesenchymal stem cell sheets.

INTRODUCTION: Blood supply remains one of the obstacles to large bone tissue engineering. This study aimed to generate vascularized bone tissue by inducing axial vascularization into a construct combining natural coral scaffold and a bone marrow mesenchymal stem cells (BMSCs) sheet. MATERIAL AND METHODS: Isolated BMSCs were cultured to form an osteogenic cell sheet using a continuous culture method. Natural coral scaffolds were prepared into customized shape with a cylinder of 20 mm length, 8 mm in outer diameter and 5 mm in inner diameter. Then, the freed superficial inferior epigastric vessel of rabbits was first wrapped with a cell sheet, and then inserted into the central passage of the scaffold, after being wrapped with another cell sheet, the complexes were implanted subcutaneously into a rabbit groin area. In contrast, the sheet-scaffold construct that implanted into groin subcutaneous area of the other side of the same rabbit with the distal end of the blood vessel was ligated, which was considered as control. New bone and vascularization formation were evaluated at 12 weeks postoperatively. RESULTS: The volume of new bone formation and amount of capillary infiltration in the vascular circulation group were significantly greater than that in the vascular ligation group, which suggested that insertion of axial vessels could significantly promote angiogenesis and osteogenesis of the tissue-engineered bone. CONCLUSIONS: These findings indicate that inserting an arteriovenous bundle into the constructs of mesenchymal stem cell sheet and coral has great potential for clinical applications to repair large bone defects.

Risk factors for right ventricular dysfunction in patients with lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare disease characterized by diffuse cystic lesions of the lung. The present study was designed to evaluate the right ventricular (RV) function in LAM patients via single-beat real-time three-dimensional echocardiography (RT-3DE) and to investigate the factors affecting RV function in LAM patients. According to tricuspid regurgitation velocity (TRV), forty-five female LAM patients [(44.07 ± 10.22) years old] were divided into TRV ≤ 2.8 m/s group (n = 29) and TRV > 2.8 m/s group (n = 16). Relative echocardiography parameters were assessed by conventional transthoracic echocardiography, Doppler tissue imaging (DTI) and RT-3DE, respectively. Pulmonary function tests and the six-minute walk tests (SMWT) were also performed for LAM patients. We found that most of RV functional parameters in LAM patients were worse than that in control patients, although left ventricular dysfunction was not significantly observed. Correlation analysis showed that 3D echocardiographic RV ejection fraction (RVEF) was negatively correlated with pulmonary vascular resistance (PVR), TRV, and the decrease of oxygen saturation (SpO2) post SMWT, and positively correlated with Forced expiratory volume in the first second/forced vital capacity, carbon monoxide diffusion predicted value, SMWT distance, and resting SpO2 in LAM patients. Multivariate stepwise linear regression analysis showed that PVR and SpO2 before SMWT were independent influence factors of RVEF in LAM patients. In this study, we found that RV dysfunction was presented in LAM patients, although left ventricular dysfunction was not significantly obvious. The main influence factors of RVEF were PVR and hypoxia. RT-3DE is a low-cost and noninvasive way to evaluate RV function in LAM patients.

NDRG2 ablation reprograms metastatic cancer cells towards glutamine dependence via the induction of ASCT2.

Background: Metastasis is the most common cause of lethal outcome in various types of cancers. Although the cell proliferation related metabolism rewiring has been well characterized, less is known about the association of metabolic changes with tumor metastasis. Herein, we demonstrate that metastatic tumor obtained a mesenchymal phenotype, which is obtained by the loss of tumor suppressor NDRG2 triggered metabolic switch to glutamine metabolism. Methods: mRNA-seq and gene expression profile analysis were performed to define the differential gene expressions in primary MEC1 and metastatic MC3 cells and the downstream pathways of NDRG2. NDRG2 regulation of Fbw7-dependent c-Myc stability were determined by immunoprecipitation and protein half-life assay. Luciferase reporter and ChIP assays were used to determine the roles of Akt and c-Myc in mediating NDRG2-dependent regulation of ASCT2 in in both tumor and NDRG2-knockout MEF cells. Finally, the effect of the NDRG2/Akt/c-Myc/ASCT2 signaling on glutaminolysis and tumor metastasis were evaluated by functional experiments and clinical samples. Results: Based on the gene expression profile analysis, we identified metastatic tumor cells acquired the mesenchymal-like characteristics and displayed the increased dependency on glutamine utilization. Further, the gain of NDRG2 function blocked epithelial-mesenchymal transition (EMT) and glutaminolysis, potentially through suppression of glutamine transporter ASCT2 expression. The ASCT2 restoration reversed NDRG2 inhibitory effect on EMT program and tumor metastasis. Mechanistic study indicates that NDRG2 promoted Fbw7-dependent c-Myc degradation by inhibiting Akt activation, and subsequently decreased c-Myc-mediated ASCT2 transcription, in both tumor and NDRG2-knockout MEF cells. Supporting the biological significance, the reciprocal relationship between NDRG2 and ASCT2 were observed in multiple types of tumor tissues, and associated with tumor malignancy. Conclusions: NDRG2-dependent repression of ASCT2 presumably is the predominant route by which NDRG2 rewires glutaminolysis and blocks metastatic tumor survival. Targeting glutaminolytic pathway may provide a new strategy for the treatment of metastatic tumors.

Identification of driver genes and somatic mutations in cell-free DNA of patients with pulmonary lymphangioleiomyomatosis.

Next-generation sequencing of cell-free circulating DNA (cfDNA) has emerged as promising technique for identifying minimally invasive genomic profiling of tumor cells recently. However, it remains relatively unknown in LAM disease. In our study, paired cfDNA and genomic DNA (gDNA) in blood samples were obtained from 23 LAM patients and seven healthy controls to explore mutations profiles of targeted 70 cancer-related genes. As results, log2-based allele frequencies of mutations in cfDNA were significantly different from those of gDNA. By comparing the mutual mutations identified both in cfDNA and gDNA, a significant correlation was also observed. After removing mutations in gDNA, distinct somatic mutation profiles of cfDNA were observed in LAM patients. Forty of 70 targeted genes had recurrent mutations, of which ATM, BRCA2 and APC showed the highest frequency. Based on the mutation, correlation network constructed of 40 mutated genes, 11 hub genes bearing intensive interactions were highlighted, including BRCA1, BRCA2, RAD50, RB1, NF1, APC, MLH3, ATM, PDGFRA, PALB2 and BLM. Expression of the hub genes showed significant clusters between LAM patients and controls and that RAD50 and BRCA2 had the strongest associations with subject phenotypes. Myogenesis and estrogen response were confirmed to be positively regulated in LAM patients. Collectively, our study provided a landscape of genomic alterations in LAM and discovered several potential driver genes, that is, BRCA2 and RAD50, which shed a substantial light on the clinical application of key molecular markers and potential therapy targets for precision diagnosis and treatment in the future.

Development of biodegradable bioactive glass ceramics by DLP printed containing EPCs/BMSCs for bone tissue engineering of rabbit mandible defects.

Bioactive glass ceramics have excellent biocompatibility and osteoconductivity; and can form direct chemical bonds with human bones; thus, these ceramic are considered as "Smart" materials. In this study, we develop a new type of bioactive glass ceramic (AP40mod) as a scaffold containing Endothelial progenitor cells (EPCs) and Mesenchymal stem cells (BMSCs) to repair critical-sized bone defects in rabbit mandibles. For in vitro experiments: AP40mod was prepared by Dgital light processing (DLP) system and the optimal ratio of EPCs/BMSCs was screened by analyzing cell proliferation and ALP activity, as well as the influence of genes related to osteogenesis and angiogenesis by direct inoculation into scaffolds. The scaffold showed suitable mechanical properties, with a Bending strength 52.7 MPa and a good biological activity. Additionally, when EPCs/BMSCs ratio were combined at a ratio of 2:1 with AP40mod, the ALP activity, osteogenesis and angiogenesis were significantly increased. For in vivo experiments: application of AP40mod/EPCs/BMSCs (after 7 days of in vitro spin culture) to repair and reconstruct critical-sized mandible defect in rabbit showed that all scaffolds were successfully accurately implanted into the defect area. As revealed by macroscopically and CT at the end of 9 months, defects in the AP40mod/EPCs/BMSCs group were nearly completely covered by normal bone and the degradation rate was 29.9% compared to 20.1% in the AP40mod group by the 3D reconstruction. As revealed by HE and Masson staining analyses, newly formed blood vessels, bone marrow and collagen maturity were significantly increased in the AP40mod/EPCs/BMSCs group compared to those in the AP40mod group. We directly inoculated cells on the novel material to screen for the best inoculation ratio. It is concluded that the AP40mod combination of EPCs/BMSCs is a promising approach for repairing and reconstructing large load bearing bone defect.

c-Myc Overexpression Promotes Oral Cancer Cell Proliferation and Migration by Enhancing Glutaminase and Glutamine Synthetase Activity.

BACKGROUND: This study aimed to investigate whether glutaminase (GLS) and glutamine synthetase (GS) are involved in c-Myc-mediated tumor development in oral cancer. METHODS: The correlation between the expressions of c-Myc, GLS, and GS in clinical samples and the clinicopathologic features of oral cancer were examined using immunohistochemistry and quantitative real-time polymerase chain reaction. After overexpressing the c-Myc gene and using an inhibitor of GLS or GS, functional experiments were performed to confirm the effects of c-Myc, GLS and GS on proliferation, cell cycle and migration in KB oral cancer cells. The expressions of E-cadherin and N-cadherin were determined by immunofluorescence assays in KB cells overexpressing c-Myc in the presence of GLS or GS inhibitors. RESULTS: The protein expression of GS was correlated with the Tumor, Lymph Node, and Metastasis (TNM) stage. In addition, c-Myc mRNA levels were positively correlated with GS mRNA levels. Overexpression of c-Myc increased the colonies derived from oral cancer cells and caused more cells to be in S phase compared with the mock-vehicle group. The migratory speed of KB cells was promoted by overexpression of c-Myc compared to the mock-vehicle group. However, these effects were effectively reversed in the presence of GLS or GS inhibitor. Furthermore, c-Myc could inhibit E-cadherin protein expression while promoting N-cadherin expression by enhancing the activity of GLS and GS. CONCLUSIONS: c-Myc overexpression promotes oral cancer cell proliferation and migration by enhancing GLS and GS activity. Our findings are beneficial for the identification of novel molecular targets for the prevention and treatment of oral cancer.

In vitro transdifferentiation of adipose tissue-derived stem cells into salivary gland acinar-like cells.

Current clinical approaches to treat irradiation-induced salivary gland hypofunction are ineffective. We previously reported that adipose-derived stem cell (ADSC)-based therapy ameliorates damaged salivary gland function in mice and that the effects were enhanced when the therapy was co-administrated with platelet-rich fibrin (PRF). We examined the feasibility of ADSC transdifferentiation into salivary gland acinar-like cells (SGALCs) and analyzed the potential of PRF to promote the transdifferentiation process in vitro. Salivary gland cells (SGCs) and ADSCs were indirectly co-cultured using Transwell inserts, and increasing concentrations of PRF-conditioned medium were applied to the co-culture system. The expression of α-amylase and AQP-5 were used to evaluate ADSC transdifferentiation. Notably, on day 7, 14, and 21, expression of both α-amylase and AQP-5 were detected in the co-cultured ADSCs. Additionally, PRF increased α-amylase and AQP-5 levels in ADSCs that were co-cultured for 7 days. These data demonstrate that ADSCs have the potential to transdifferentiate into SGALCs and that PRF can promote the transdifferentiation process. Therefore, these data reveal a possible mechanism to treat irradiation-induced salivary gland hypofunction and have translational medicine implications.

In vivo study of the efficacy, biosafety, and degradation of a zinc alloy osteosynthesis system.

In this study, a comprehensive analysis of a novel zinc alloy osteosynthesis system in a canine mandibular fracture model is presented. The efficacy of the system was compared for PLLA (poly-l-lactic acid) and titanium materials using X-ray radiography, micro-CT tomography, undecalcified bone histomorphometry, and a three-point bending test. Histology, blood normal, blood biochemical, and serum zinc concentration tests were also performed to assess the biosafety of the zinc alloy osteosynthesis system. The degradability of the zinc alloy was evaluated using a micro-CT and scanning electron microscope during the 24-week post operation period. The results showed that zinc alloy possesses good mechanical properties that support fracture healing. Its uniform and slow corrosion leads to adequate degradation behavior in 24 weeks. Additionally, the zinc alloy proved to be biocompatible, indicating that this novel osteosynthesis system is safe for use in the body. The results of the study demonstrate that this zinc alloy-based osteosynthesis system is a promising candidate for a new generation of osteosynthesis systems, with further improvements required in the future.

Cartilaginous extracellular matrix derived from decellularized chondrocyte sheets for the reconstruction of osteochondral defects in rabbits.

Cartilaginous extracellular matrix (ECM) materials derived from decellularized native articular cartilage are widely used in cartilage regeneration. However, it is difficult for endogenous cells to migrate into ECM derived from native cartilage owing to its nonporous structure and dense nature. Moreover, current decellularization approaches frequently lead to architectural breakdown and potential loss of surface composition of ECM. To solve this problem, we aimed to establish a novel biological ECM scaffold from chondrocyte sheets for cartilage regeneration. We cultured chondrocytes harvested from the auricular cartilage of 4-week-old New Zealand rabbits and enabled them to form cell sheets. These sheets were decellularized using sodium dodecyl sulfate (SDS) with three different concentrations, namely, 1%, 5%, and 10%, followed by 1% Triton X-100 and deoxyribonuclease enzyme solution. In vitro microstructural examination and mechanical tests demonstrated that 1% SDS not only removed chondrocytes completely but also maintained the native architecture and composition of ECM, thus avoiding the use of high-concentration SDS. Application of decellularized chondrocyte sheets for osteochondral defects in rabbits resulted in substantial host remodeling and variant regeneration of osteochondral tissues. One percent SDS-treated decellularized chondrocyte sheets contributed to the superior reconstruction of osteochondral defects as compared with 5% and 10% SDS groups, which includes vascularized subchondral bone, articular cartilage with adequate thickness, and integration with host tissues. Furthermore, ECM from 1% SDS significantly increased the migrating potential of bone marrow mesenchymal stem cells (BMSCs) in vitro. RT-PCR and western blot also revealed that ECM increased the expression of SOX-9 in BMSCs, whereas it decreased COL-X expression. In conclusion, our results suggested that the chondrocyte sheets decellularized with 1% SDS preserved the integrity and bioactivity, which favored cell recruitment and enabled osteochondral regeneration in the knee joints of rabbits, thus offering a promising approach for articular cartilage reconstruction without cell transplantation. STATEMENT OF SIGNIFICANCE: Although biological extracellular matrix (ECM) derived from decellularized native cartilage has been widely used in cartilage regeneration, it is difficult for endogenous cells to migrate into ECM owing to its dense nature. Moreover, current decellularization approaches lead to architectural breakdown of ECM. This study established a novel biological ECM from decellularized chondrocyte sheets for cartilage regeneration. Our results suggested that cartilaginous ECM favored cell recruitment and enabled osteochondral regeneration in rabbits, thus offering a promising approach for articular cartilage reconstruction without cell transplantation. SDS 1% adequately decellularized the chondrocytes in cell sheets, whereas it maintained the native architecture and composition of ECM, thereby avoiding the use of high-concentration SDS and providing a new way to acquire cartilaginous ECM.

Effects of incorporation of granule-lyophilised platelet-rich fibrin into polyvinyl alcohol hydrogel on wound healing.

Dressings are commonly used to treat skin wounds. In this study, we aimed to develop a new scaffold composed of a polyvinyl alcohol (PVA) hydrogel containing granule-lyophilised platelet-rich fibrin (G-L-PRF) as a dressing. G-L-PRF was prepared by freeze-drying and was then incorporated into PVA hydrogel by freezing-thawing. Notably, the mechanical strength and degradation rate of the scaffold were found to be related to G-L-PRF concentrations, reaching 6.451 × 10-2 MPa and 17-22%, respectively, at a concentration of 1%. However, the strength decreased and the degradation was accelerated when the G-L-PRF concentration was over 1%. The elastic properties and biocompatibility of the scaffold were independent of G-L-PRF concentration, and both showed excellent elasticity and biocompatibility. The release of vascular endothelial growth factor and platelet-derived growth factor-AB was no significant time dependent. Additionally, application of 1% G-L-PRF/PVA to acute full-thickness dorsal skin wounds accelerated wound closure at days 7 and 9. Healing also increased on day 11. Histological and immunohistochemical analyses showed that the scaffold enhanced granulation tissue, maturity, collagen deposition, and new vessel formation. These results demonstrated that the prepared G-L-PRF/PVA scaffolds accelerated wound healing in acute full-thickness skin wounds, suggesting potential applications as an ideal wound dressing.

The use of allogenic adipose-derived stem cells in combination with platelet-rich fibrin for the treatment of cartilage defects in rabbit ear.

The current study aims to assess the efficacy of allogenic adipose-derived stem cells (ADSCs) together with platelet-rich fibrin (PRF) for the treatment of rabbit ear cartilage defects. For this study, 12 New Zealand white rabbits were randomly allocated into 4 groups. Two full-thickness cartilage defects were created in the rabbit ears. Group 1 was left untreated; Group 2 was treated with allogenic ADSCs, Group 3 was treated with PRF; Group 4 was treated with allogenic ADSCs and PRF. Macroscopic observation, hematoxylin and eosin staining, and Alcian blue staining after 3 months suggested that the allogenic ADSCs/PRF significantly accelerated cartilage regeneration compared to other groups and this was associated with increased expression of collagen II relative to the other groups. Expression of genes associated with immune response such as cluster of differentiation 4 and 8 (CD4, CD8), and interleukin 2 and 4 (IL-2, IL-4) displayed no significant statistical difference compared to Group 1. In conclusion, these results suggest that allogenic ADSCs in combination with PRF can accelerate regeneration in full-thickness cartilage defects in the rabbit ear model without causing a significant immune response. The results suggest that allogenic ADSCs with PRF could successfully be used for cartilage regeneration.

[Three-dimensional study of facial soft tissue changes in patients with skeletal Class â ¢ malocclusion before and after orthognathic surgery].

Objective: To investigate the changes of facial soft tissue before and after orthognathic surgery in patients with skeletal Class Ⅲ malocclusion. Methods: Between August 2016 and April 2017, 30 patients with skeletal Class Ⅲ malocclusion who underwent maxillary LeFort Ⅰ osteotomy and sagittal split mandible osteotomy were selected as study subjects. Among them, 11 were male and 19 were female with an average age of 22.6 years (range, 18-35 years). Full head CT scan and facial soft tissue three-dimensional image scan were performed within 2 weeks before surgery and at 6 months after surgery. A three-dimensional facial image model was established using Artec Studio 11.0 and CMF Proplan 3.0 software to analyze the facial soft tissue changes before and after surgery. The soft tissue anatomical landmarks in each area of the face were measured and compared before and after surgery. Results: The area of facial soft tissue change after surgery was the maxillary nose and the lower jaw area, and the two sides did not exceed the vertical boundary of the outer canthus. After surgery, the horizontal points of bilateral alar bases and bilateral cheeks changed significantly ( P<0.05). The sagittal points of subnasale, pronasale, bilateral alar bases, upper lip margin significantly forwarded ( P<0.05); the sagittal points of the bilateral cheilions, lower lip margin, midpoint of chin-lip groove, pogonion, and menton significantly backwarded ( P<0.05). The vertical points of the upper lip margin, bilateral cheilions, lower lip margin, bilateral cheeks, and bilateral inner canthus points significantly descended ( P<0.05), and the vertical point of the menton significantly elevated ( P<0.05). After surgery, the nasal column was significantly shortened, the upper lip got longer and the alar base widened when compared with those before surgery ( P<0.05). Conclusion: The overall change of face after double jaw surgery is shorter and fuller, and the mandible of facial soft tissue change is larger than that of maxillary, which suggests that the postoperative facial changes should be taken into account in the surgical design.

Intraglandular transplantation of adipose-derived stem cells combined with platelet-rich fibrin extract for the treatment of irradiation-induced salivary gland damage.

The aim of the present study was to determine the effect of adipose-derived mesenchymal stem cells (ADSCs) combined with heterologous platelet-rich fibrin extract (PRFe) on irradiation-induced salivary gland (SG) damage. ADSCs were isolated from C3H mice, whereas PRFe was obtained from New Zealand rabbits. Twelve weeks post irradiation, the ADSCs or PRFe or their combination were transplanted into the submandibular glands of C3H mice with irradiation-induced damage. The salivary flow rate (SFR) was determined and histopathological analysis was performed at 12 weeks post transplantation. Immunofluorescence, microvessel density measurements and transmission electron microscopy were performed to assess α-amylase (AMY) production, apoptosis and microstructural changes in the cells. The administration of ADSCs combined with PRFe increased the SFR at 12 weeks post transplantation, whereas ADSCs alone or PRFe alone failed to do so. The ADSCs+PRFe-treated, irradiated SGs had fewer damaged and atrophied acinar cells, higher AMY levels and an increased microvessel density compared with the untreated irradiated SGs. Moreover, SG tissue from the ADSCs+PRFe group also showed decreased apoptotic and increased proliferative activity compared to that from the irradiated group. In conclusion, ADSCs or PRFe alone did not restore permanent, irradiation-induced damage of SG tissue when used alone, but when used together, they provided effective treatment outcomes.

Intraglandular Transplantation of Adipose-Derived Stem Cells for the Alleviation of Irradiation-Induced Parotid Gland Damage in Miniature Pigs.

PURPOSE: In a previous study, the authors verified the protective efficacy of adipose-derived stem cells (ADSCs) on the prevention of salivary gland (SG) damage induced by irradiation in mice. As a critical step before implementation in clinical practice, the present study investigated the protective effect of ADSCs in a miniature pig SG model, because miniature pigs share many characteristics with humans. MATERIALS AND METHODS: Third-passage autologous ADSCs at a concentration of 4 × 106 cells/mL were transplanted by intraglandular injection into parotid glands (PGs) immediately after local irradiation at a single dose of 20 Gy. The injection process was repeated twice a week for 6 consecutive weeks. At 12 weeks after irradiation, functional and histologic evaluations were performed by measuring salivary flow rate (SFR) and hematoxylin and eosin and periodic acid-Schiff staining. Immunohistochemical and transmission electron microscopic examinations also were conducted to evaluate amylase (AMY) production, microvessel density (MVD), and microstructural changes. RESULTS: The irradiated PGs showed remarkable decreases in SFR, AMY production, and MVD. However, transplantation of ADSCs alleviated irradiated PG morphology and function by preserving more functional acinar cells and increasing SFR and AMY production. In addition, greater MVD was observed in the ADSC-treated group than in the irradiated group. CONCLUSIONS: These results indicated that intraglandular transplantation of autologous ADSCs is an effective method to protect PGs against damage from irradiation in miniature pigs, which might have clinic application in the future.

Scaffold-Based Delivery of Bone Marrow Mesenchymal Stem Cell Sheet Fragments Enhances New Bone Formation In Vivo.

PURPOSE: Stem cell therapy is becoming a potent strategy to shorten the consolidation time and reduce potential complications during distraction osteogenesis (DO). However, the conventional local injection or scaffold-based delivery of bone marrow mesenchymal stem cell (BMSC) suspension deprives the cells of their endogenous extracellular matrix, which might dampen cell differentiation and tissue regeneration after implantation. Therefore, in our study, a BMSC sheet was established and was then minced into fragments and loaded onto a hydroxyapatite (HA) scaffold for grafting. MATERIALS AND METHODS: The purified and characterized BMSCs were grown into a cell sheet, and bone formation and mineralization capacity, as well as the cell sheet composition, were analyzed. Afterward, the in vivo osteogenic ability of cell sheet fragments (CSFs) was evaluated in immunocompromised mouse and rabbit models of DO. RESULTS: The BMSC sheet exhibited higher alkaline phosphatase activity than osteogenic cell suspension cultures. Alkaline phosphatase activity and mineral particles in the cell sheet increased further after osteogenic induction. Moreover, calcium and phosphorus were present only in the osteogenic cell sheet, along with the common elements carbon, oxygen, chlorine, sodium, and sulfur, as indicated by x-ray photoelectron spectroscopy analysis. In a mouse model, the CSF-HA complex was injected subcutaneously. Micro-computed tomography analysis showed that the osteogenic CSF-HA complex led to a considerably higher bone volume than the BMSC-HA or CSF-HA complex. The osteogenic CSF-HA specimens showed increased angiogenesis and deposition of type I collagen compared with the non-osteogenic CSF-HA or BMSC-HA specimens. Moreover, the osteogenic CSF-HA markedly improved bone consolidation and increased bone mass in DO rabbits. CONCLUSIONS: Collectively, the incorporation of osteogenic BMSC sheets into HA particles greatly promoted bone regeneration, which offers therapeutic alternatives for DO.

The influence of the sensory neurotransmitter calcitonin gene-related peptide on bone marrow mesenchymal stem cells from ovariectomized rats.

In order to explore the effects of calcitonin gene-related peptide (CGRP) on bone mesenchymal stem cells (BMSCs) from ovariectomized (OVX) rats, an OVX rat model was used. An ELISA was performed to examine the changes in CGRP level in the plasma and skeleton. The BMSCs from the sham rats were designated group A. The BMSCs from the OVX rats (groups B, C, D and E) were treated with different concentrations of CGRP (10-6, 10-8, 10-10 and 0 M) in vitro. The proliferation and osteogenic and adipogenic differentiation potential of the BMSCs were evaluated. BMSCs sheets and Bio-Oss® mixtures were transplanted into nude mice to observe the effects of CGRP on bone formation in vivo. The level of CGRP was decreased by almost 27 and 17 % in the plasma and bone, respectively, in OVX rats compared with sham rats (p < 0.05). Treatment with CGRP increased the proliferation and mineralization of BMSCs, and significantly decreased the lipid accumulation of BMSCs in a dose-dependent manner. The expression of Runx2 and Osterix was upregulated, but the expression of peroxisome proliferator-activated receptor γ was significantly downregulated in groups B, C and D compared with group E (p < 0.05). Micro computed tomography showed no difference between the images of the planted mixtures. Hematoxylin and eosin stain revealed the formation of slightly more hard bone-like structures in groups B and C. These results suggested that CGRP played a role in adjusting bone mass and strength by promoting the proliferation and osteogenic differentiation of BMSCs, as well as significantly suppressing the adipogenic differentiation of BMSCs.

Macrophages Undergo M1-to-M2 Transition in Adipose Tissue Regeneration in a Rat Tissue Engineering Model.

Macrophages are involved in the full processes of tissue healing or regeneration and play an important role in the regeneration of a variety of tissues. Although recent evidence suggests the role of different macrophage phenotypes in adipose tissue expansion, metabolism, and remodeling, the spectrum of macrophage phenotype in the adipose tissue engineering field remains unknown. The present study established a rat model of adipose tissue regeneration using a tissue engineering chamber. Macrophage phenotypes were assessed during the regenerative process in the model. Neo-adipose tissue was generated 6 weeks after implantation. Macrophages were obvious in the chamber constructs 3 days after implantation, peaked at day 7, and significantly decreased thereafter. At day 3, macrophages were predominantly M1 macrophages (CCR7+), and there were few M2 macrophages (CD206+). At day 7, the percentage of M2 macrophages significantly increased and remained stable at day 14. M2 macrophages became the predominant macrophage population at 42 days. Enzyme-linked immunosorbent assay demonstrated transition of cytokines from pro-inflammatory to anti-inflammatory, which was consistent with the transition of macrophage phenotype from M1 to M2. These results showed distinct transition of macrophage phenotypes from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 in adipose tissue regeneration in our tissue engineering model. This study provides new insight into macrophage phenotype transition in the regeneration of adipose tissue.