RESUMO
The impact of diabetes mellitus on bone fracture healing is clinically relevant as the patients experience delayed fracture healing. Even though efforts have been made to understand the detrimental effects of type 2 diabetes mellitus (T2DM) on the fracture healing process, the exact mechanisms causing the pathophysiological outcomes remain unclear. The aim of this study was to assess alterations in bone fracture healing (tibial fracture surgery, intramedullary pinning) of diet-induced obese (DIO) mice, and to investigate the in vitro properties of osteochondroprogenitors derived from the diabetic micro-environment. High-resolution contrast-enhanced microfocus X-ray computed tomography (CE-CT) enabled a simultaneous 3D assessment of the amount and spatial distribution of the regenerated soft and hard tissues during fracture healing and revealed that osteogenesis as well as chondrogenesis are altered in DIO mice. Compared to age-matched lean controls, DIO mice presented a decreased bone volume fraction and increased callus volume and adiposity at day 14 post-fracture. Of note, bone turnover was found altered in DIO mice relative to controls, evidenced by decreased blood serum osteocalcin and increased serum CTX levels. The in vitro data revealed that not only the osteogenic and adipogenic differentiation of periosteum-derived cells (PDCs) were altered by hyperglycemic (HG) conditions, but also the chondrogenic differentiation. Elevated PPARγ expression in HG conditions confirmed the observed increase in differentiated adipocytes in vitro. Finally, chondrogenesis-related genes COL2 and COL10 were downregulated for PDCs treated with HG medium, confirming that chondrogenic differentiation is compromised in vitro and suggesting that this may affect callus formation and maturation during the fracture healing process in vivo. Altogether, these results provide novel insights into the alterations of long bone fracture repair and suggest a link between HG-induced dysfunctionality of osteochondroprogenitor differentiation and fracture healing impairment under T2DM conditions.
Assuntos
Diabetes Mellitus Tipo 2 , Consolidação da Fratura , Animais , Calo Ósseo/diagnóstico por imagem , Dieta , Humanos , Camundongos , Camundongos Obesos , Osteogênese , Tomografia , Tomografia Computadorizada por Raios XRESUMO
OBJECTIVES: New strategies for implant surface functionalization in the prevention of peri-implantitis while not compromising osseointegration are currently explored. The aim of this in vivo study was to assess the osseointegration of a titanium-silica composite implant, previously shown to enable controlled release of therapeutic concentrations of chlorhexidine, in the Göttingen mini-pig oral model. MATERIAL AND METHODS: Three implant groups were designed: macroporous titanium implants (Ti-Porous); macroporous titanium implants infiltrated with mesoporous silica (Ti-Porous + SiO2 ); and conventional titanium implants (Ti-control). Mandibular last premolar and first molar teeth were extracted bilaterally and implants were installed. After 1 month healing, the bone in contact with the implant and the bone regeneration in the peri-implant gap was evaluated histomorphometrically. RESULTS: Bone-to-implant contact and peri-implant bone volume for Ti-Porous versus Ti-Porous + SiO2 implants did not differ significantly, but were significantly higher in the Ti-Control group compared with Ti-Porous + SiO2 implants. Functionalization of titanium implants via infiltration of a SiO2 phase into the titanium macropores does not seem to inhibit implant osseointegration. Yet, the importance of the implant macro-design, in particular the screw thread design in a marginal gap implant surgery set-up, was emphasized by the outstanding results of the Ti-Control implant. CONCLUSIONS: Next-generation implants made of macroporous Ti infiltrated with mesoporous SiO2 do not seem to compromise the osseointegration process. Such implant functionalization may be promising for the prevention and treatment of peri-implantitis given the evidenced potential of mesoporous SiO2 for controlled drug release.
Assuntos
Próteses e Implantes , Animais , Antibacterianos , Implantes Dentários , Peri-Implantite/prevenção & controle , Dióxido de Silício , Propriedades de Superfície , Suínos , Porco Miniatura , TitânioRESUMO
Obesity is the main cause of type 2 diabetes mellitus (T2DM). Roux-en-Y gastric bypass (RYGB) surgery is an effective treatment for this obesity-related health problem. However, the adverse effects of T2DM on bone tissue persist or even aggravate after this surgical procedure. As studies on the mandibular condyle bone are scarce, the aim of the present study was to assess its compositional characteristics in T2DM and RYGB conditions. Thirty-two male C57BL/6 mice at 8 weeks of age were randomly assigned to receive either a high-fat or low-fat diet. After 14 weeks of high-fat diet intake, seven obese mice were subjected to RYGB surgery. All animals were euthanized at the age of 30 weeks. Mandibular bones were removed and the trabecular condyle region was assessed using Raman spectroscopy. A decreased mineralization was observed for both T2DM and RYGB condyle bones when compared to controls, with elevated carbonate substitutions for the RYGB group. No compositional differences in crystallinity and presence of advanced glycation end products were found between the groups, with the exception of an increased presence of N-carboxymethyl-lysine in RYGB bone compared to their T2DM counterpart. Site-specific measurements revealed a non-uniform bone composition, with increasing mineralization and carbonate substitutions towards the centre of the mandibular condyle. T2DM and RYGB surgery affect the mandibular condyle bone quality, as investigated at compositional level. Assessment of bone structural properties and remodelling should be carried out to further explore the effects of T2DM and RYGB surgery on this skeleton area.
Assuntos
Osso e Ossos/patologia , Diabetes Mellitus Tipo 2/patologia , Derivação Gástrica , Obesidade/patologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição AleatóriaRESUMO
Type 2 diabetes mellitus (T2DM) is a metabolic disorder associated with obesity and hyperglycemia. Roux-en-Y gastric bypass (RYGB) surgery is a common treatment for severely obese patients and T2DM. Both RYGB and T2DM are linked to increased skeletal fragility, though the exact mechanisms are poorly understood. Our aim was to characterize the structural, mechanical and compositional properties of bones from diet-induced obese and RYGB-treated obese (bypass) mice to elucidate which the exact factors are contributing to the increased skeletal fragility. To achieve this, a combinatory approach including microfocus X-ray computed tomography, 3-point bending, finite element modeling and Raman spectroscopy, was used. Compared to aged-matched lean controls, the obese mice displayed decreased cortical thickness, trabecular bone loss, decreased stiffness and increased Young's modulus. For the bypass mice, these alterations were even more pronounced, and additionally they showed low mineral-to-matrix ratio in the cortical endosteal area. Accumulation of the advanced glycation end-product (AGE) pentosidine was found in the cortex of obese and bypass groups and this accumulation was correlated with an increased Young's modulus. In conclusion, we found that the increased fracture risk in T2DM- and post-RYGB bones is mainly driven by accumulation of AGEs and macro-structural alterations, generating biomechanical dysfunctionality.
Assuntos
Osso e Ossos/fisiopatologia , Diabetes Mellitus Tipo 2/cirurgia , Fraturas Ósseas/fisiopatologia , Produtos Finais de Glicação Avançada/metabolismo , Obesidade/cirurgia , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Fenômenos Biomecânicos , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico por imagem , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Fraturas Ósseas/diagnóstico por imagem , Fraturas Ósseas/metabolismo , Derivação Gástrica/efeitos adversos , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Camundongos , Camundongos Obesos , Obesidade/metabolismo , Obesidade/patologia , Esqueleto/fisiopatologia , Tomografia Computadorizada por Raios X , Redução de PesoRESUMO
Biological tissues have a complex and heterogeneous 3D structure, which is only partially revealed by standard histomorphometry in 2D. We here present a novel chemical compound for contrast-enhanced microfocus computed tomography (CE-CT), a Hafnium-based Wells-Dawson polyoxometalate (Hf-POM), which allows simultaneous 3D visualization of mineralized and non-mineralized skeletal tissues, such as mineralized bone and bone marrow vasculature and adipocytes. We validated the novel contrast agent, which has a neutral pH in solution, by detailed comparison with (immuno)histology on murine long bones as blueprint, and showed that Hf-POM-based CE-CT can be used for virtual 3D histology. Furthermore, we quantified the 3D structure of the different skeletal tissues, as well as their spatial relation to each other, during aging and diet-induced obesity. We discovered, based on a single CE-CT dataset per sample, clear differences between the groups in bone structure, vascular network organization, characteristics of the adipose tissue and proximity of the different tissues to each other. These findings highlight the complementarity and added value of Hf-POM-based CE-CT compared to standard histomorphometry. As this novel technology provides a detailed 3D simultaneous representation of the structural organization of mineralized bone and bone marrow vasculature and adipose tissue, it will enable to improve insight in the interactions between these three tissues in several bone pathologies and to evaluate the in vivo performance of biomaterials for skeletal regeneration.
Assuntos
Meios de Contraste/química , Esqueleto/citologia , Tomografia Computadorizada por Raios X/métodos , Compostos de Tungstênio/química , Adipócitos/citologia , Animais , Células da Medula Óssea/citologia , Osso Esponjoso/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise Espectral RamanRESUMO
More insight into the biological fundamentals of leukocyte platelet-rich fibrin (L-PRF) guided healing is necessary to recommend its application, in particular in deficient bone sites that need to support implants. This study investigated the short-term bone healing effect of L-PRF treatment in cylindrical non-critical sized bone defects with 3 mm diameter and 6 mm depth in tibiae of 18 adult male New Zealand White rabbits. After a randomization process, 96 bone defects were prepared and half of them were filled with a L-PRF membrane, while untreated defects in the opposite tibia served as control group. The rabbits were euthanized after 7, 14 or 28 days of healing. The bone healing of the cortical and medullary areas was investigated by micro-CT, while the expression of molecular markers (RUNX2, VEGFA, COL1A2 and BMP2) was assessed by qRT-PCR. Treatment with L-PRF did not affect the micro-structural bone characteristics of the repaired bone tissue, except for a decrease in the trabecular connectivity at the cortical level after 14 days of healing. At this time, RUNX2 and VEGFA mRNA levels were significantly lower in the treated defects. L-PRF membranes thus had a temporary negative influence on the bone microarchitecture (Tb.Pf) and on the RUNX2 and VEGFA expression during early bone healing. Overall, L-PRF treatment did not enhance bone regeneration in these non-critical size defects after 28 days.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Leucócitos , Fibrina Rica em Plaquetas , Tíbia/lesões , Cicatrização/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Coelhos , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Microtomografia por Raio-XRESUMO
AIM: Osseointegration of titanium implants is predictable, but can be improved via surface functionalization. MATERIALS AND METHODS: One hundred and twenty implants were installed in parietal bone of 12 domestic pigs and left to heal for 1 or 3 months. Five groups were defined according surface treatments: immersion in water (H2 O), 10% polyphosphoric acid (PPA10), 1% phosphorylated pullulan (PPL1), 10% phosphorylated pullulan (PPL10) or 10% phosphorylated pullulan + 1 µg bone morphogenetic protein-2 (PPL10 BMP). As primary outcome, implant osseointegration was evaluated by quantitative histology, namely peri-implant bone formation (B/T in %) and bone-to-implant contact (BIC in %) for each healing period. The Wilcoxon signed-rank test and Mann-Whitney U-test with α = 0.05 were performed. RESULTS: PPL10 and PPA10 groups showed significantly higher B/T and BIC results than the control (H2 O) group at 1-month (p < .05). No significant difference was found between PPL1 and H2 O or between PPL10 BMP and H2 O, irrespective of healing time (1 or 3 months) or investigated parameter (B/T and BIC; p > .05). After 3 months, no experimental group showed a significant difference compared to the control group (H2 O) for both investigated parameters (B/T and BIC; p > .05). CONCLUSION: Functionalizing titanium implants with inorganic or organic phosphate-containing polymers at 10 wt% concentration may stimulate peri-implant bone formation and implant osseointegration at early healing times.
Assuntos
Materiais Revestidos Biocompatíveis/farmacologia , Implantação Dentária Endóssea/métodos , Implantes Dentários , Osseointegração/fisiologia , Titânio/farmacologia , Animais , Proteína Morfogenética Óssea 2/farmacologia , Interface Osso-Implante , Planejamento de Prótese Dentária , Glucanos/farmacologia , Implantes Experimentais , Modelos Animais , Ácidos Fosfóricos/farmacologia , Polímeros/farmacologia , Crânio/cirurgia , Propriedades de Superfície , Retalhos Cirúrgicos , SuínosRESUMO
The spread of antibiotic resistance and the challenges associated with antiseptics such as chlorhexidine have necessitated a search for new antibacterial agents against oral bacterial pathogens. As a result of failing traditional approaches, drug repurposing has emerged as a novel paradigm to find new antibacterial agents. In this study, we examined the effects of the FDA-approved anticancer agent toremifene against the oral bacteria Porphyromonas gingivalis and Streptococcus mutans We found that the drug was able to inhibit the growth of both pathogens, as well as prevent biofilm formation, at concentrations ranging from 12.5 to 25 µM. Moreover, toremifene was shown to eradicate preformed biofilms at concentrations ranging from 25 to 50 µM. In addition, we found that toremifene prevents P. gingivalis and S. mutans biofilm formation on titanium surfaces. A time-kill study indicated that toremifene is bactericidal against S. mutans Macromolecular synthesis assays revealed that treatment with toremifene does not cause preferential inhibition of DNA, RNA, or protein synthesis pathways, indicating membrane-damaging activity. Biophysical studies using fluorescent probes and fluorescence microscopy further confirmed the membrane-damaging mode of action. Taken together, our results suggest that the anticancer agent toremifene is a suitable candidate for further investigation for the development of new treatment strategies for oral bacterial infections.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos Hormonais/farmacologia , Biofilmes/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Porphyromonas gingivalis/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacos , Toremifeno/farmacologia , Biofilmes/crescimento & desenvolvimento , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Placa Dentária/tratamento farmacológico , Placa Dentária/microbiologia , Reposicionamento de Medicamentos , Farmacorresistência Bacteriana Múltipla/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Periodontite/tratamento farmacológico , Periodontite/microbiologia , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/ultraestrutura , Streptococcus mutans/metabolismo , Streptococcus mutans/ultraestrutura , Titânio/análiseRESUMO
Porphyromonas gingivalis is a major pathogen involved in oral diseases such as periodontitis and peri-implantitis. Management of these diseases typically includes mechanical debridement of the colonized surfaces followed by application of antiseptics or antibiotics. Disadvantages associated with the use of antiseptics and the growing worldwide problem of antibiotic resistance have necessitated the search for alternative agents. In this study, the antibacterial and antibiofilm properties of AM404, an active metabolite of paracetamol, were tested against P. gingivalis and other bacterial pathogens. The activity of AM404 was tested against 10 bacteria, including both oral and nonoral human pathogens. The minimal inhibitory concentration (MIC) of AM404 was determined by measuring optical density (OD) values. The minimum biofilm inhibitory concentration (MBIC) was detected by crystal violet staining. The activity of structural analogs of AM404 was tested by MIC determinations. The effect of AM404 on P. gingivalis biofilms formed on titanium disks as a model for dental implants was evaluated by colony forming unit counting. Potential cytotoxicity of AM404 towards HEK-293 (human embryonic kidney cells), HepG2 (human hepatoma cells), IEC-6 (rat intestinal cells), and Panc-1 cells (pancreatic cancer cells) was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. To get more insight in the mode of action of AM404, we used the fluorescent dyes N-phenyl-1-napthylamine and SYTOX green to investigate outer and inner membrane damage of P. gingivalis induced by AM404, respectively. Of all tested pathogens, AM404 only inhibited growth and biofilm formation of P. gingivalis. Moreover, it showed potent activity against P. gingivalis biofilms formed on titanium surfaces. A structure-activity analysis demonstrated that the unsaturated carbon chain is essential for its antibacterial activity. Importantly, AM404 was not toxic towards the tested mammalian cells up to concentrations approaching 4× the MIC. Membrane damage assays using fluorescent probes N-phenyl-1-napthylamine and SYTOX green revealed that membrane permeabilization presumably is the primary antibacterial mode of action of AM404. Collectively, our results suggest that AM404 has the potential to be used for the development of new drugs specifically targeting P. gingivalis-related infections.
RESUMO
The current literature was reviewed to determine the impact of autologous platelet concentrates (APCs) on endodontic healing. All types of clinical study designs concerning any kind of endodontic treatment involving the application of APCs were included. Two independent reviewers searched three databases (PubMed, Embase, and Cochrane Library) for studies, complemented by hand search, until 16/1/2016. From the 423 identified records, 48 articles met the inclusion criteria. Selected randomized controlled clinical trials (RCTs) underwent Cochrane Collaboration's risk-of-bias assessment and data extraction. Only two RCTs showed low risk of bias. There was considerable heterogeneity between the RCTs with regard to the type of therapy, type of APCs, assessment method, and study quality, and therefore the data could not be analyzed quantitatively. The included case reports/series and non-randomized comparative studies underwent qualitative analysis with the revised Methodological Index for Non-Randomized Studies (MINORS) and data extraction. The two comparative non-randomized studies scored qualitatively high, though the MINORS-scores of the case series and reports were dispersed. APCs were involved in five endodontic treatment modalities, namely apexification, regenerative endodontic procedures, pulpotomy, apical surgery, and treatment of endo-perio/perio-endo lesions. APCs seem to accelerate postoperative bone healing, augment the patients' postoperative quality of life, aid further root development, and support maintenance/regaining of pulp vitality. No adverse events were reported. APCs in endodontic treatments seem to contribute to the healing of soft and hard tissues, though there is a lack of long-term high quality clinical trials and standardized treatment protocols.
Assuntos
Doenças da Polpa Dentária/terapia , Transfusão de Plaquetas , Plasma Rico em Plaquetas , Cicatrização , Terapia Combinada , Humanos , Transfusão de Plaquetas/efeitos adversos , Transfusão de Plaquetas/métodos , Viés de Publicação , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do TratamentoRESUMO
Within the bone marrow, the endosteal niche plays a crucial role in B-cell differentiation. Because spaceflight is associated with osteoporosis, we investigated whether changes in bone microstructure induced by a ground-based model of spaceflight, hind limb unloading (HU), could affect B lymphopoiesis. To this end, we analyzed both bone parameters and the frequency of early hematopoietic precursors and cells of the B lineage after 3, 6, 13, and 21 d of HU. We found that limb disuse leads to a decrease in both bone microstructure and the frequency of B-cell progenitors in the bone marrow. Although multipotent hematopoietic progenitors were not affected by HU, a decrease in B lymphopoiesis was observed as of the common lymphoid progenitor (CLP) stage with a major block at the progenitor B (pro-B) to precursor B (pre-B) cell transition (5- to 10-fold decrease). The modifications in B lymphopoiesis were similar to those observed in aged mice and, as with aging, decreased B-cell generation in HU mice was associated with reduced expression of B-cell transcription factors, early B-cell factor (EBF) and Pax5, and an alteration in STAT5-mediated IL-7 signaling. These findings demonstrate that mechanical unloading of hind limbs results in a decrease in early B-cell differentiation resembling age-related modifications in B lymphopoiesis.
Assuntos
Linfócitos B/citologia , Elevação dos Membros Posteriores/fisiologia , Linfopoese/fisiologia , Voo Espacial , Corticosteroides/metabolismo , Envelhecimento , Animais , Células da Medula Óssea/citologia , Remodelação Óssea , Diferenciação Celular , Linhagem da Célula , Citocinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Imunoglobulinas/metabolismo , Interleucina-7/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fator de Transcrição PAX5/metabolismo , Fator de Transcrição STAT5/metabolismo , Células-Tronco , Fatores de Tempo , Transativadores/metabolismo , Microtomografia por Raio-XRESUMO
The present study aimed at elucidating the effect of local pH in the extracellular microenvironment of tissue-engineered (TE) constructs on bone cell functions pertinent to new tissue formation. To this aim, we evaluated the osteogenicity process associated with bone constructs prepared from human Bone marrow-derived mesenchymal stem cells (hBMSC) combined with 45S5 bioactive glass (BG), a material that induces alkalinization of the external medium. The pH measured in cell-containing BG constructs was around 8.0, that is, 0.5 U more alkaline than that in two other cell-containing materials (hydroxyapatite/tricalcium phosphate [HA/TCP] and coral) constructs tested. When implanted ectopically in mice, there was no de novo bone tissue in the BG cell-containing constructs, in contrast to results obtained with either HA/TCP or coral ceramics, which consistently promoted the formation of ectopic bone. In addition, the implanted 50:50 composites of both HA/TCP:BG and coral:BG constructs, which displayed a pH of around 7.8, promoted 20-30-fold less amount of bone tissue. Interestingly, hBMSC viability in BG constructs was not affected compared with the other two types of material constructs tested both in vitro and in vivo. Osteogenic differentiation (specifically, the alkaline phosphatase [ALP] activity and gene expression of RUNX2, ALP, and BSP) was not affected when hBMSC were maintained in moderate alkaline pH (≤7.90) external milieu in vitro, but was dramatically inhibited at higher pH values. The formation of mineralized nodules in the extracellular matrix of hBMSC was fully inhibited at alkaline (>7.54) pH values. Most importantly, there is a pH range (specifically, 7.9-8.27) at which hBMSC proliferation was not affected, but the osteogenic differentiation of these cells was inhibited. Altogether, these findings provided evidence that excessive alkalinization in the microenvironment of TE constructs (resulting, for example, from material degradation) affects adversely the osteogenic differentiation of osteoprogenitor cells.
Assuntos
Microambiente Celular , Células-Tronco Mesenquimais/citologia , Osteogênese , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Adolescente , Adulto , Animais , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Microambiente Celular/efeitos dos fármacos , Meios de Cultura , Feminino , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Implantes Experimentais , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos Nus , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Osteogênese/efeitos dos fármacos , Tela Subcutânea/efeitos dos fármacosRESUMO
Skeletal unloading provokes bone loss. These bone alterations have been shown to be associated with impairment of osteoblastic activity. In the present study, we evaluated the effect of skeletal unloading on bone marrow progenitor cells, for exploration of the underlying mechanism. Wistar rats were randomized to be either hindlimb unloaded for 9 days or to act as controls. Micro-CT was used to detect tibial trabecular architecture changes in response to skeletal unloading. Microgravity conditions for 9 days resulted in a decreased number and an increased spacing of the bone trabeculae in the proximal tibia. The proliferative capacity of the femoral bone marrow samples was assessed (fibroblast-colony-forming assay). By using qPCR, the expression of selected markers of vascularization (Vegfa; Hif1a; Angpt1), energy metabolism (Prkaa2; Mtor), bone formation (Runx2; Alp; Bglap; Bmp2; Bmp4; Bmp7) and bone resorption (Acp5; Tnfsf11; Tnfrsf11b) in these bone marrow suspensions was measured. We demonstrated a striking decrease in the number of fibroblastic progenitors in response to hindlimb unloading. This deficit in proliferation was shown to be accompanied by altered hindlimb perfusion and cellular energy homeostasis. Ex vivo culture assays of the bone marrow-derived progenitor cells screened for osteogenic (Runx2; Alp; Bglap) and adipogenic (Pparg; Fabp4) differentiation alterations in response to microgravity. Induced progenitor cells from unloaded rats showed a delay in osteogenic differentiation and impaired adipogenic differentiation compared to control. The data of this multi-level approach demonstrate that skeletal unloading significantly affects the bone tissue and its metabolism at the progenitor stage. The molecular expressions of the bone marrow population support a role of cellular metabolic stresses in skeletal alterations induced by inactivity.
Assuntos
Células da Medula Óssea/citologia , Elevação dos Membros Posteriores , Ossos da Perna/fisiologia , Osteogênese , Células-Tronco/citologia , Animais , Medula Óssea/irrigação sanguínea , Medula Óssea/fisiologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Metabolismo Energético , Feminino , Regulação da Expressão Gênica , Membro Posterior/irrigação sanguínea , Membro Posterior/fisiologia , Ossos da Perna/irrigação sanguínea , Neovascularização Fisiológica , Osteoblastos/citologia , Ratos , Ratos Wistar , Células-Tronco/metabolismoRESUMO
Local tissue ischemia is a prime cause responsible for the massive cell death in tissue-engineered (TE) constructs observed postimplantation. To assess the impact of ischemia on the death of implanted human multipotent stromal cells (hMSCs), which have great potential for repairing damaged tissues, we hereby investigated the in vivo temporal and spatial fate of human Luc-GFP-labeled MSCs within fibrin gel/coral scaffolds subcutaneously implanted in nude mice. In vivo bioluminescence imaging monitoring and histological analyses of the constructs tested confirmed the irremediable death of hMSCs over 30 days postimplantation. The kinetics of expression of three hypoxic/ischemic markers (HIF-1α, LDH-A, and BNIP3) was also monitored. Our results provided evidence that hMSCs located within the core of implanted constructs died faster and predominantly and strongly expressed the aforementioned ischemic markers. In contrast, cells located in the outer regions of TE constructs were reperfused by neovascularization and were still viable (as evidenced by their ex-vivo proliferative potential) at day 15 postimplantation. These results support the explanation that in the central part of the constructs tested, death of hMSCs was due to ischemia, whereas in the periphery of these constructs, cell death was due to another mechanism that needs to be elucidated.
Assuntos
Hipóxia Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Engenharia Tecidual/métodosRESUMO
Optimization of implant osseointegration in patients with reduced bone healing potential is a challenge remaining in implant dentistry. Identification of the genes that are modulated during implant osseointegration in normal versus osteopenic bone is needed to successfully address these pertinent clinical needs. The present study aimed to assess the initial and early molecular events following titanium implant installation in normal and compromised bone in a rat tibia model. Peri-implant tissue from a well-defined tissue regeneration compartment was analyzed at 2 and 7 days post-surgery for the expression of select markers of inflammation, angiogenesis, bone resorption and bone formation. Impaired bone was induced by hindlimb unloading and validated using µCT. The essential step of angiogenesis preceding bone regeneration was evidenced for the peri-implant setting in healthy bone. Compromised bone significantly affected the angiogenesis-osteogenesis coupling in the initial phase (2 days post-surgery), with altered expressions of Vegfa and Epas1 coinciding with downregulated expressions of Col1a1, Bmp2, Bmp4, Alpl and Bglap. At 7 days post-implantation, differences between normal and compromised peri-implant bone were no longer observed. This in vivo molecular evidence of delayed implant osseointegration in compromised bone reassert modern strategies in implant development, such as surface modifications and bioengineered approaches, to improve implant osseointegration in compromised conditions.
Assuntos
Osseointegração/fisiologia , Titânio/química , Animais , Feminino , Membro Posterior/diagnóstico por imagem , Próteses e Implantes , Ratos , Ratos Wistar , Tíbia/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
Shortening of the healing time before loading risks impeding successful titanium implant anchorage into compromised bone. A thorough understanding at the genetic scale of the early phases of bone regeneration at the implant interface is required before the development of strategies to enhance implant osseointegration. In this study a new in vivo implant model to explore the mechanism by which titanium implant osseointegration is affected by the host bone properties is presented. An implant was conceptualized enabling standardized harvesting of peri-implant tissue for quantitative molecular analysis while preserving the mimicking of the clinical setting. The implant is partly indented to provide a well-defined healing compartment from where tissue differentiation and de novo bone formation can be investigated and partly screw-threaded to provide a good implant anchorage into the bone. The feasibility of the implant design was assessed in osteopenic bone conditions, evoked by simulated weightlessness. Wistar rats were either hindlimb unloaded by tail suspension (HU) for 9 days or acted as controls (CTL). The status of compromised bone tissue through 9-days HU was confirmed by micro-X-ray computed tomography. The implant was installed in the proximal tibial bone 7 days after the onset of HU or CTL. Two days postimplantation, the peri-implant regenerating tissue responses were recorded by measuring expression of inflammatory, angiogenic, and bone resorption parameters (hypoxia-inducible factor 1, alpha subunit; vascular endothelial growth factor A; angiopoietin 1; endothelial PAS domain protein 1; fibroblast growth factor 2; tumor necrosis factor; interleukin 11; acid phosphatase 5, tartrate resistant; tumor necrosis factor (ligand) superfamily, member 11/RANKL). We successfully demonstrated that HU-associated bone conditions evoked a significant alteration of expression of the angiogenic markers in the peri-implant regenerative tissue during initial implant osseointegration, whereas the expression levels of the inflammatory and bone resorption parameters remained unchanged. We concluded that this in vivo implant model provides a well-designed and controlled method to examine molecular responses in implant osseointegration to impaired bone conditions. This model may serve to explore the application of anabolic strategies in peri-implant osteogenesis.
Assuntos
Implantes Experimentais , Modelos Animais , Osseointegração/efeitos dos fármacos , Tíbia/efeitos dos fármacos , Titânio/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Elevação dos Membros Posteriores , Ratos , Ratos Wistar , Tíbia/diagnóstico por imagem , Cicatrização/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
BACKGROUND: Critical factors for the establishment of osseointegration are the implant surface microtopography and the local mechanical environment. The present study evaluated the bone response around a turned (T) and a roughened (R) implant for either an unloaded or a well-controlled loaded situation. METHODS: Bone chambers were installed in the tibia of 20 rabbits. In each of the chambers, two identical displacement-controlled loading experiments were performed: 30 microm for 400 cycles at 1 Hz, three times a week for 9 weeks versus 0-microm implant displacement. A linear mixed model and a logistic mixed model with alpha = 5% were set to study the significant effect of the surface texture on the peri-implant bone response in the unloaded (T-0 microm versus R-0 microm) and the loaded (T-30 microm versus R-30 microm) mode. RESULTS: Results indicated no microtopographic dependence of the bone response further away from the implant in unloaded and loaded conditions. For a load-free implant, osseointegration seemed to occur with a higher incidence at a roughened compared to a turned implant surface. In the presence of loading, the topographic dependency of the osteogenic activity at the interface was overruled by the loading-related bone response, revealing no significant differences in osseointegration incidence between T and R. CONCLUSION: A predominant effect of the interfacial mechanical environment over the implant surface characteristics on the differentiating cell population is suggested.
Assuntos
Implantes Dentários , Planejamento de Prótese Dentária , Osseointegração/fisiologia , Ligas , Animais , Fenômenos Biomecânicos , Medula Óssea/patologia , Matriz Óssea/patologia , Tecido Conjuntivo/patologia , Ligas Dentárias/química , Materiais Dentários/química , Feminino , Osteoblastos/patologia , Porosidade , Coelhos , Distribuição Aleatória , Estresse Mecânico , Propriedades de Superfície , Tíbia/patologia , Tíbia/cirurgia , Titânio/químicaRESUMO
UNLABELLED: Both implant surface characteristics and mechanical loading are known to affect implant osseointegration. Their interaction and the underlying mechanisms by which they affect peri-implant healing processes are still unknown. The aim of this study is therefore to investigate the influence of a turned versus a rough (Plus), Dentsply Friadent) implant surface on peri-implant bone formation in case of unloaded or loaded implant healing. MATERIAL AND METHODS: Bone formation was evaluated around screw-shaped implants under four experimental conditions using a repeated sampling bone chamber methodology: (1) unloaded turned implant (CU), (2) unloaded implant with a rough surface (TU), (3) loaded turned implant (CL), and (4) loaded implant with a rough surface (TL). Peri-implant tissue samples were paraffin embedded after implant removal and examined histologically and histomorphometrically. A mixed model was used for statistical analysis. RESULTS: The surface of bone tissue relative to the total tissue area (bone area fraction) was not affected by the experimental conditions. The areas of bone trabeculae relative to the bone area (bone fraction) were significantly higher for TL compared with CU and TU. The bone fraction in the vicinity (100 microm zone) of the implant (BFZ) was significantly the highest around the loaded roughened implants (TL). CONCLUSION: Implant loading did not affect bone formation in the absence of surface roughness, and implant surface roughness had no effect in the absence of loading. However, a bone-stimulating effect in the implant's vicinity was assigned to the rough surface when the implant was loaded.