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BACKGROUND/PURPOSE: Covering the wounds from guided bone regeneration and sinus floor elevation with oral and sinus mucosa is a fundamental criterion for success. This study aimed to verify the regeneration capability of the mucosal connective tissue stromal cells by characterizing their stemness and osteogenic potentials. METHODS: Bone marrow stromal cells (BMSCs), alveolar mucosa cells (AMCs), keratinized gingival cells (KGCs), and sinus mucosal cells (SMCs), were isolated from four Sprague-Dawley rats. The morphology and viability of the cells were investigated under a confocal microscope and by Alamar Blue. Stem cell surface markers were evaluated by flow cytometry. Expressions of pluripotent factors after initial seeding and an early osteogenic gene following 24 h of osteoinduction were evaluated by realtime PCR. Trilineage differentiation capability in long-term inductive cell culture was assessed by Alizarin Red, Alcian Blue, and Oil Red O staining. RESULTS: BMSCs and AMCs were larger cells with smaller aspect ratios relative to KGCs and SMCs, and BMSCs revealed the greatest initial viability but the slowest proliferation. More than 94% of BMSCs, AMCs, and KGCs were double-positive for CD73 and CD90. Compared with BMSCs, AMCs expressed significantly higher Oct4 but reduced Cbfa1 after initial seeding, and AMCs and SMCs expressed significantly higher Cbfa1 following 24 h of osteoinduction. In long-term inductive cell culture, osteogenesis was observed in BMSCs, AMCs, and SMCs, chondrogenesis was observed in BMSCs, AMCs, and KGCs, and adipogenesis was evident in only BMSCs. CONCLUSION: AMCs contain a high percentage of stem/progenitor cells and show differentiation capability toward osteogenic lineage.
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Osteogénesis , Elevación del Piso del Seno Maxilar , Animales , Células de la Médula Ósea , Diferenciación Celular , Células Cultivadas , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND/PURPOSE: Supracrestal ridge augmentation (SRA) is a major challenge for clinicians. This study investigated the efficacy of a 3D-printed (3DP) hydroxyapatite/poly(lactic-co-glycolic acid) (HA/PLGA) scaffold as a potential biologic for SRA. METHODS: Scaffolds that were 5 mm in diameter and 2.5-mm thick with a 1.2-mm diameter through-and-through central hole composed of 90% HA and 10% PLGA were printed using an extrusion-based bioprinter. The HA/PLGA scaffold was fixed with a 1.2-mm titanium mini-implant on the buccal surface of rat mandible (Ti-HPS), and the outcome of SRA were compared with sites treated with a titanium mini-implant alone (control) and a titanium mini-implant covered with deproteinized bovine bone-derived matrix (Ti-DBBM) at 4 and 8 weeks by microcomputed tomography (micro-CT), back-scattered SEM, and histology assessments. RESULTS: The HA/PLGA scaffolds were 2.486 ± 0.082 mm thick with an outer diameter of 4.543 ± 0.057 mm and an inner diameter of 1.089 ± 0.045 mm, and the pore dimensions were 0.48-0.52 mm. There was significantly more mineralized tissue in the Ti-DBBM and Ti-HPS groups than in the control group at both time points. Newly formed bone (NB) was well-integrated with the DBBM and HA/PLGA scaffolds. The framework of the 3DP-HA/PLGA scaffold remained in place, and NB-implant contact (NBIC) was advanced to the middle level in the Ti-HPS group until 8 weeks, whereas dispersion of DBBM with a lower level NBIC was noted in the Ti-DBBM group at both time points. CONCLUSION: The 3DP HA/PLGA scaffold maintains supracrestal space and demonstrates osteoconductivity to facilitate SRA.
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Durapatita , Andamios del Tejido , Animales , Bovinos , Glicoles , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Impresión Tridimensional , Ratas , Microtomografía por Rayos XRESUMEN
Reconstruction of the periodontal ligament (PDL) to fulfill functional requirement remains a challenge. This study sought to develop a biomimetic microfibrous system capable of withstanding the functional load to assist PDL regeneration. Collagen-based straight and waveform microfibers to guide PDL cell growth were prepared using an extrusion-based bioprinter, and a laminar flow-based bioreactor was used to generate fluidic shear stress. PDL cells were seeded on the respective microfibers with 0 or 6 dynes/cm2 fluidic shear stress for 1-4 h. The viability, morphology, adhesion pattern, and gene expression levels of PDL cells were assessed. The results revealed that upon bioprinting optimization, collagen-based microfibers were successfully fabricated. The straight microfibers were 189.9 ± 11.44 µm wide and the waveform microfibers were 235.9 ± 11.22 µm wide. Under 6 dynes/cm2 shear stress, PDL cells were successfully seeded, and cytoskeleton expansion, adhesion, and viability were greater. Cyclin D, E-cadherin, and periostin were upregulated on the waveform microfibers. In conclusion, 3D-printed collagen-based waveform microfibers preserved PDL cell viability and exhibited an enhanced tendency to promote healing and regeneration under shear stress. This approach is promising for the development of a guiding scaffold for PDL regeneration.
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Colágeno/farmacología , Osteogénesis/efectos de los fármacos , Ligamento Periodontal/efectos de los fármacos , Andamios del Tejido/química , Bioimpresión/métodos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Humanos , Impresión Tridimensional , Estrés MecánicoRESUMEN
OBJECTIVE AND BACKGROUND: To achieve periodontal regeneration, numerous investigations have concentrated on biomolecule supplement and optimization of bone substitute or barrier membrane. This study evaluated the benefit of combining these strategies for periodontal regeneration. METHODS: Biphasic cryogel scaffold (BCS) composed of gelatin (ligament phase) and gelatin with beta-tricalcium phosphate/hydroxyapatite (BH) (bone phase) was designed as tested bone substitute, and both enamel matrix derivatives (EMD) and bone morphogenetic protein-2 (BMP-2) were applied to formulate a biomolecule-aided BCS (BBS). Functionally graded membrane (FGM) was designed as tested barrier membrane by adhering PDGF-encapsulated poly(L-lactide-co-D/L-lactide) nanofibers on the conventional membrane (CM). BBS and FGM were characterized and tested for biocompatibility in vitro. Thirty 4 × 4 × 5 mm3 periodontal intrabony defects were created on 6 Beagle dogs. Each defect was evenly assigned to one of the following treatments including BH-CM, BCS-CM, BBS-CM, BH-FGM, BCS-FGM, and BBS-FGM, for 12 weeks. The therapeutic efficiency was assessed by micro-CT and histology. RESULTS: BCS and FGM sustained the release of biomolecules. The viability of MSCs was maintained in both phases of BCS and was promoted while seeding on the PDGF-encapsulated nanofibers. In CM-covered sites, BBS showed significantly greater osteogenesis (P < .01) and early defect fill (P < .05) relative to BH. FGM significantly promoted osteogenesis (P < .05) in BH-treated sites but showed limited benefit in BBS-treated sites. On denuded roots, cementum deposition was evident in BBS-treated sites. CONCLUSIONS: PDGF-loaded FGM promoted periodontal osteogenesis, and BBS with EMD-BMP-2 had potential for reconstructing alveolar ridge, periodontal ligament, and cementum. FGM and BBS combination provided limited additional benefit.
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Pérdida de Hueso Alveolar , Sustitutos de Huesos , Criogeles , Regeneración Tisular Guiada Periodontal , Nanofibras , Pérdida de Hueso Alveolar/cirugía , Animales , Regeneración Ósea , Cemento Dental , Perros , Ligamento Periodontal , RegeneraciónRESUMEN
BACKGROUND/PURPOSE: It is still challengeable to treat periodontal pockets refractory to mechanical debridement. This study is to evaluate the potential of indocyanine green (ICG)-diode laser-based photothermal therapy (PTT) for solving this dilemma. METHODS: Bone marrow-derived mesenchymal stem cells (BMSCs) and periodontal ligament cells (PDLCs) were incubated with phosphate-buffered saline, chlorhexidine, or ICG, non-irradiated or irradiated with 810-nm diode lasers, and the cell viability was evaluated. Patients with teeth refractory to mechanical periodontal debridement on different quadrants were recruited. At baseline (T0), all examined teeth received scaling and root planing, and those on the test quadrant (PTT group) received ICG-diode laser treatment. The outcome was evaluated using clinical parameters and cytokines in the gingival crevicular fluids at 4-6 weeks (T1) and 6 months (T2). RESULTS: In ICG-treated cultures, the viability of BMSCs and PDLCs was recovered on day 4, and laser irradiation inhibited the metabolic activities of BMSCs. 22 patients with 30 control teeth and 35 PTT-treated teeth were examined. All examined teeth showed modest reductions in probing pocket depth (PPD), clinical attachment loss (CAL), bleeding upon probing (BOP), and plaque score at T1 and T2 and significant reductions in IL-1ß and MMP-8 at T2. Compared with controls, BOP was reduced more prominently, IL-1ß and MMP-8 were significantly lower, and reductions in PPD and CAL were slightly greater in the PTT group at T1 (0.05-0.19 mm). CONCLUSION: ICG-diode laser-based PTT is compatible to periodontium and assists in faster resolution of gingival inflammation in periodontal pockets refractory to mechanical debridement.
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Periodontitis Crónica/radioterapia , Verde de Indocianina/uso terapéutico , Láseres de Semiconductores , Bolsa Periodontal/radioterapia , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Femenino , Líquido del Surco Gingival/metabolismo , Humanos , Interleucina-1beta/metabolismo , Masculino , Metaloproteinasa 8 de la Matriz/metabolismo , Persona de Mediana Edad , Índice Periodontal , Taiwán , Resultado del TratamientoRESUMEN
BACKGROUND: Diabetes mellitus deteriorates the destruction and impairs the healing of periodontal wounds and craniofacial defects. This study is to evaluate the potential of self-assembled adipose-derived stem cell spheroids (ADsp) in microbial transglutaminase cross-linked gelatin hydrogel (mTG) for treating diabetic periodontal wounds and craniofacial defects. METHODS: Human adipose-derived stem cells (ADSCs) were isolated by lipoaspiration, pluripotent genes and trilineage differentiation were examined, and the maintenance of ADsp properties in mTG was verified. Oral mucosal wounds and calvarial osseous defects were created in diabetic rats. Gross observation, histologic evaluation, and immunohistochemistry for proliferating cells and keratinization were conducted in the mucosal wounds within 4-28 days. Micro-CT imaging, histologic evaluation, and immunohistochemistry for proliferating cells and osteogenic differentiation were conducted in the osseous defects at 7 and 28 days. RESULTS: ADSCs expressed pluripotent genes and were capable of trilineage differentiation. ADsp retained morphology and stemness in mTG. In diabetic mucosal wounds, wound closure, epithelization, and keratinization were accelerated in those with ADsp and ADsp-mTG. In diabetic osseous defects, osteogenic differentiation markers were evidently expressed, cell proliferation was promoted from day 7, and bone formation was significantly promoted at day 28 in those with osteogenically pretreated ADsp-mTG. CONCLUSIONS: ADsp-mTG accelerated diabetic oral mucosal wound healing, and osteogenically pretreated ADsp-mTG promoted diabetic osseous defect regeneration, proving that ADsp-mTG facilitated diabetic periodontal wound healing and craniofacial osseous defect regeneration.
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Diabetes Mellitus Experimental , Hidrogeles , Ratas , Humanos , Animales , Hidrogeles/química , Gelatina , Transglutaminasas/genética , Osteogénesis , Tejido Adiposo , Células MadreRESUMEN
BACKGROUND: Alveolar mucosa could be a promising source of mesenchymal stem cells (MSCs) for regeneration therapeutics because it exhibits faster healing potential and can be easily collected with minimal periodontal disturbance. This study aimed to evaluate the potential of alveolar mucosal cell (AMC) spheroids for promoting extraction socket healing and calvarial osseous defect regeneration. METHODS: AMCs were isolated from Sprague-Dawley rats. Antigenic and MSC surface marker expressions and trilineage differentiation capability were assessed. AMCs were then osteogenically stimulated (OAs) or unstimulated (UAs), self-aggregated to form spheroids, and encapsulated in gelatin hydrogel to fill rat extraction sockets or combined with freeze-dried bone graft (FDBG) to fill rat calvarial osseous defects. The outcome was assessed by gross observation, micro-CT imaging, and immunohistochemistry. RESULTS: AMCs highly expressed MSC surface markers, showed weak antigenicity, and were capable of trilineage differentiation at Passage 3. In the extraction sockets, wound closure, socket fill, keratinization, and proliferative activities were accelerated in those with AMC spheroids treatment. Socket fill and maturation were further promoted by OA spheroids. In the calvarial osseous defects, the mineralized tissue ratio was promoted with AMC spheroids/FDBG treatment, and bone sialoprotein expression and cell proliferation were more evident with OA spheroids/FDBG treatment. CONCLUSION: AMCs exhibited MSC properties with weak antigenicity. AMC spheroids promoted extraction socket healing, AMC spheroids/FDBG promoted calvarial osseous defect regeneration, and the outcomes were further enhanced by osteogenically stimulation of AMCs.
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Functionally graded membranes (FGM) with regenerative signals and nanofibrous topography mimicking the native extracellular matrix have been shown to improve the outcome of alveolar ridge regeneration (ARR). This study developed a novel FGM with doxycycline-enamel matrix derivative (EMD) nanofibrous composites deposition to coordinate anti-inflammation and differentiation signals, thus facilitating ARR. Doxycycline-loaded PDLLA nanofibers (PD), EMD-loaded chitosan nanospheres (CE), and CE-embedded PD (CE-PD) were fabricated by electrospinning, deposited on the surfaces of barrier membrane to develop a FGM, and the efficacy was validated by delivering the FGM to regenerate experimental alveolar ridge defects in rats. Results revealed that PD had potent antibacterial capability, and CE-PD allowed sustained release of EMD to promote osteogenesis in vitro. In the alveolar ridge defects, FGM with PD on the outer surface downregulated MMP-8, and wound dehiscence was further reduced with Cbfa1 upregulation in those treated by FGM with CE-PD on the inner surface at 1 week. FGM with CE-PD revealed significantly greater new bone formation and defect fill at 4 weeks. In conclusion, FGM with PD reduced early tissue breakdown and with CE-PD nanofibrous composites accelerated wound healing and facilitated osteogenesis, and thus could be an advantageous strategy for ARR.
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Quitosano , Nanofibras , Nanosferas , Proceso Alveolar , Animales , Regeneración Ósea , Doxiciclina/farmacología , RatasRESUMEN
BACKGROUND: Three-dimensional (3D) printing has become an available technology to fabricate customized tissue engineering scaffolds with delicate architecture. This exploratory study aimed to evaluate the potential of a 3D-printed hydroxyapatite-based scaffold as a biomaterial for obtaining guided bone regeneration (GBR) in vivo. METHODS: Scaffolds composed of 90% hydroxyapatite and 10% poly(lactic-co-glycolic acid) were printed using a microextrusion process to fit 4 mm diameter and 0.5 mm thick through-and-through osseous defects on the mandibular ramus of rats, with unfilled defects serving as controls. Specimens were analyzed for regeneration-associated gene expression on day 7, and micro-computed tomography (micro-CT) and histology assessments were carried out on day 28. RESULTS: The scaffolds were 3.56 ± 0.43 mm (x-axis) and 4.02 ± 0.44 mm (y-axis) in diameter and 0.542 ± 0.035 mm thick (z-axis), with a mean pore size of 0.420 ± 0.028 × 0.328 ± 0.005 mm2 . Most scaffolds fit the defects well. Type I collagen, VEGF, and Cbfa1 were upregulated in the scaffold-treated defects by day 7. By day 28, de novo osteogenesis and scaffold-tissue integration were evident in the scaffold-treated defects, and entire mineralized tissue, as well as newly formed bone, was significantly promoted, as seen in the micro-CT and histologic analyses. CONCLUSION: The 3D-printed hydroxyapatite-based scaffold showed acceptable dimensional stability and demonstrated favorable osteoregenerative capability that fulfilled the need for GBR.
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Durapatita , Impresión Tridimensional , Animales , Regeneración Ósea , Osteogénesis , Ratas , Andamios del Tejido , Microtomografía por Rayos XRESUMEN
BACKGROUND: Infection control is a major determinant of guided tissue regeneration (GTR). This study aims to develop an antibiotic-loaded membrane to assist periodontal repair. METHODS: Poly(D,L-lactic acid) (PDLLA) nanofibers encapsulating amoxicillin (PDLLA-AMX) were fabricated using the electrospinning technique, and their structures, drug encapsulation efficiency, and release characteristics were assessed. The viability and behaviors of periodontal ligament (PDL) cells on nanofibers, and antibacterial capabilities of nanofibers were evaluated in vitro. Early therapeutic efficiency of the antibiotic-loaded membranes was investigated in rats with ligature-induced experimental periodontitis, and the outcomes were evaluated by gene expression, microcomputed tomography imaging, and histology within 7 days of membrane placement. RESULTS: AMX was successfully encapsulated in the PDLLA nanofibers and released in a sustained manner. After initial attachment was achieved, cells stretched out along with the directions of nanofibers. The viability and expression of migration-associated gene of PDL cells were significantly improved, and the growth of Streptococcus sanguinis and Porphyromonas gingivalis was significantly reduced in the PDLLA-AMX group compared with the controls. On PDLLA-AMX-treated sites, wound dehiscence and sulcular inflammation were reduced. Collagen fiber matrix deposition was accelerated with upregulated type I collagen and interleukin-1ß, and downregulated matrix metalloproteinase-8, whereas periodontal bone level and the expressions of vascular endothelial growth factor and core-binding factor subunit alpha-1 were equivalent to conventional membrane treatment. CONCLUSIONS: PDLLA-AMX nanofibers inhibited bacterial growth and promoted the viability and mobility of PDL cells after initial cell attachment. Membranes with PDLLA-AMX nanofibers reduced inflammation and accelerated periodontal repair at an early stage, providing good prospects for the further development of GTR membranes.
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Nanofibras , Periodontitis , Amoxicilina/farmacología , Animales , Periodontitis/tratamiento farmacológico , Ratas , Factor A de Crecimiento Endotelial Vascular , Microtomografía por Rayos XRESUMEN
This study aimed to evaluated the potential of core-shell poly(D,l-lactide-co-glycolide)-chitosan (PLGA-chitosan) nanospheres encapsulating simvastatin (SIM) and doxycycline (DOX) for promoting periodontal and large-sized osseous defects. SIM, and/or DOX were encapsulated in PLGA-chitosan nanospheres using double emulsion technique and were delivered to sites of experimental periodontitis and large-sized mandibular osseous defects of rats for 1-4 weeks. The resultant nanospheres were ~ 200 nm diameter with distinct core-shell structure and released SIM and DOX sustainably for 28 days. DOX and SIM-DOX nanospheres significantly inhibited P. gingivalis and S. sanguinis. In experimental periodontitis sites, SIM-DOX nanospheres significantly down-regulated IL-1b and MMP-8 and significantly reduced bone loss. In mandibular osseous defects, VEGF was up-regulated, and osteogenesis was significantly augmented with SIM nanospheres treatment. In conclusion, core-shell PLGA-chitosan nanospheres released SIM and DOX sustainably. SIM-DOX and SIM nanospheres could be considered to promote the repair of infected periodontal sites and non-infected osseous defects respectively.
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BACKGROUND: Particulate anorganic bovine bone matrix (ABBM) is extensively used for alveolar ridge preservation (ARP). This study evaluates the potential of ABBM/gelatin cryogel scaffold of regular (250 to 1,000 µm) and small (50 to 100 µm) particles for ARP. METHODS: ABBM was either condensed in a gypsum ring to simulate filling of ABBM in a defect or incorporated into a gelatin cryogel scaffold to disperse ABBM. Condensed regular-sized or small-sized ABBM (rABBM or sABBM) (± bone morphogenetic protein [BMP]-2) were subcutaneously implanted in rats to evaluate biocompatibility and osteogenic potential. Experimental extraction sockets were surgically created on the maxillary ridges of rats and were unfilled (control), filled with rABBM/gelatin, or sABBM/gelatin cryogel scaffold. The socket fill and dimensional changes of the ridge were evaluated by microcomputed tomography imaging and histology. RESULTS: Condensed sABBM showed acceptable biocompatibility but significantly lower interparticle distance (IPD) and porosity (P < 0.001) compared with rABBM, whereas rABBM/gelatin and sABBM/gelatin cryogel scaffold showed similar IPD and porosity. Osteogenesis took place in rABBM/gelatin and sABBM/gelatin-treated extraction sockets showed osteogenesis at 8 weeks and had increased ridge width and reduced ridge discrepancy compared with the control sites. sABBM/gelatin scaffold significantly increased socket fill and reduced ridge discrepancy at 4 weeks, increased ridge width at 8 weeks, and reduced buccal ridge height resorption at both 4 and 8 weeks (P < 0.05 for all). CONCLUSIONS: Osteoconductivity was suppressed in condensed sABBM, even after adding BMP-2. By dispersing sABBM in a gelatin cryogel scaffold, sABBM/gelatin showed a greater potential in promoting socket fill, reducing buccal ridge atrophy, and providing equivalent ridge stability compared with rABBM/gelatin.