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1.
Neurosci Lett ; 763: 136179, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34416344

RESUMO

Neural differentiation of mesenchymal stromal cells has been widely studied. However, a comparative characterization of ultrastructural changes during neural differentiation has not been performed. In this study, we conducted scanning electron microscopy and transmission electron microscopy analysis to show the morphological changes in mesenchymal stromal cells upon induction of neural differentiation. In addition, transmission electron microscopy results demonstrated ultrastructural differences between human cranial bone marrow mesenchymal stromal cells and iliac crest bone marrow mesenchymal stromal cells. We propose that enriched microvesicles in cranial bone marrow mesenchymal stromal cells may be responsible for the increased efficiency of neural differentiation.


Assuntos
Células-Tronco Mesenquimais/ultraestrutura , Neurogênese , Crânio/citologia , Medula Óssea/irrigação sanguínea , Células Cultivadas , Humanos , Ílio/citologia , Células-Tronco Mesenquimais/fisiologia , Microscopia Eletrônica de Transmissão , Microvasos/anatomia & histologia , Cultura Primária de Células
2.
J Bone Joint Surg Am ; 103(17): 1628-1636, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33844657

RESUMO

BACKGROUND: Connective tissue progenitors (CTPs) resident in native tissues serve as biological building blocks in tissue repair and remodeling processes. Methods for analysis and reporting on CTP quantity and quality are essential for defining optimal cell sources and donor characteristics and the impact of cell processing methods for cell therapy applications. The present study examines the influence of donor characteristics and cell concentration (nucleated cells/mL) on CTP prevalence (CTPs/million nucleated cells) and CTP concentration (CTPs/mL) in bone marrow aspirates (BMAs). METHODS: Iliac crest bone marrow was aspirated from 436 patients during elective total knee or hip arthroplasty. Bone marrow-derived nucleated cells were plated at a density of 1.19 × 105 cells/cm2. Colony-forming unit analysis was performed on day 6. RESULTS: Large variation was seen between donors. Age (p < 0.05) and cell concentration (p < 0.001) significantly influenced CTP prevalence and CTP concentration. For every 1-year increase in age, the odds of having at least an average CTP prevalence and CTP concentration decreased by 1.5% and 1.6%, respectively. For every 1 million cells/mL increase in cell concentration, the odds of having at least an average CTP prevalence and CTP concentration increased by 2.2% and 7.9%, respectively. Sex, race, body mass index (BMI), and the presence of osteoporosis did not influence CTP prevalence or CTP concentration. CONCLUSIONS: BMA-derived CTPs were obtained from all patient groups. CTP prevalence and CTP concentration decreased with age. Cell concentration decreased with age and positively correlated with total CTP prevalence and CTP concentration. The mean CTP concentration in patients >60 years of age was a third of the CTP concentration in patients <30 years of age. CLINICAL RELEVANCE: Proper BMA techniques are necessary to obtain a high-quality yield and composition of cells and CTPs. The reduced CTP concentration and CTP prevalence in the elderly may be mitigated by the use of cell processing methods that increase CTP concentration and CTP prevalence (e.g., by removing red blood cells, serum, and non-CTPs or by increasing aspirate volumes). Cell concentration in the BMA can be measured at the point of care and is an appropriate initial assessment of the quality of BMA.


Assuntos
Células da Medula Óssea/citologia , Células do Tecido Conjuntivo/citologia , Células-Tronco/citologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , Contagem de Células , Criança , Feminino , Humanos , Ílio/citologia , Masculino , Pessoa de Meia-Idade , Osteoporose/patologia , Fatores Sexuais , Adulto Jovem
3.
BMC Genomics ; 21(1): 417, 2020 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-32571211

RESUMO

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


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Ílio/citologia , Mandíbula/citologia , Maxila/citologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/genética , Humanos , Ílio/química , Mandíbula/química , Maxila/química , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/citologia , Especificidade de Órgãos , Análise de Sequência de RNA/métodos , Sequenciamento do Exoma
4.
Neurol Med Chir (Tokyo) ; 60(2): 83-93, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31956170

RESUMO

We analyzed the cell characteristics, neuroprotective, and transplantation effects of human cranial bone-derived mesenchymal stem cells (hcMSCs) in ischemic stroke model rats compared with human iliac bone-derived mesenchymal stem cells (hiMSCs). The expressions of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF ) as neurotrophic factors were analyzed in both MSCs. hiMSCs or hcMSCs were intravenously administered into ischemic stroke model rats at 3 or 24 h after middle cerebral artery occlusion (MCAO) and neurological function was evaluated. The survival rate of neuroblastoma × glioma hybrid cells (NG108-15) after 3 or 24 h oxidative or inflammatory stress and the neuroprotective effects of hiMSCs or hcMSCs-conditioned medium (CM) on 3 or 24 h oxidative or inflammatory stress-exposed NG108-15 cells were analyzed. The expressions of BDNF and VEGF were higher in hcMSCs than in hiMSCs. hcMSCs transplantation at 3 h after MCAO resulted in significant functional recovery compared with that in the hiMSCs or control group. The survival rate of stress-exposed NG108-15 was lower after 24 h stress than after 3 h stress. The survival rates of NG108-15 cells cultured with hcMSCs-CM after 3 h oxidative or inflammatory stress were significantly higher than in the control group. Our results suggest that hcMSCs transplantation in the early stage of ischemic stroke suppresses the damage of residual nerve cells and leads to functional recovery through the strong expressions of neurotrophic factors. This is the first report demonstrating a functional recovery effect after ischemic stroke following hcMSCs transplantation.


Assuntos
Modelos Animais de Doenças , Intervenção Médica Precoce , AVC Isquêmico/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Humanos , Ílio/citologia , Infarto da Artéria Cerebral Média/terapia , Infusões Intravenosas , Fatores de Crescimento Neural/metabolismo , Crânio/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Oral Dis ; 26(1): 131-144, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31541596

RESUMO

OBJECTIVE: Secondary alveolar bone grafting is an essential part in the treatment of alveolar cleft deformity. Autologous iliac bone is the most favorable grafting source. However, the factors regulating postoperative bone formation are unclear. Investigations are needed to found whether the alveolar bone niche and bone marrow mesenchymal stem cells (BMSCs) derived from the jaw bone (BMSCs-J) affected the osteogenesis of BMSCs from the ilium (BMSCs-I). MATERIALS AND METHODS: The effect of BMSCs-J on BMSCs-I was investigated using a co-culture model. The exosomes were purified by sequential centrifugation. The osteoblastic differentiation of BMSCs was analyzed in vitro and in vivo. RESULTS: Co-culture with BMSCs-J increased the alkaline phosphatase (ALP) activity, Alizarin Red S (ARS) staining, and osteogenic gene expression in BMSCs-I. Transmission electron microscopy and nanoparticle tracking analysis verified the presence of exosomes in the culture supernatants of BMSCs. Exosomes secreted by BMSCs-J enhanced the ALP activity, ARS staining, osteogenic gene expression of BMSCs-I in vitro, and new bone formation in vivo. Blocking the secretion of exosomes using siRNA for Rab27a inhibited the effect of BMSCs-J. CONCLUSION: Exosomes played a role in the interaction between BMSCs-J and BMSCs-I, thereby leading to the enhanced osteogenic capacity of BMSCs-I and bone formation.


Assuntos
Células da Medula Óssea/citologia , Exossomos/fisiologia , Ílio/citologia , Células-Tronco Mesenquimais/citologia , Osteogênese , Fosfatase Alcalina/metabolismo , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Humanos , Arcada Osseodentária/citologia
6.
Cells ; 8(8)2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31434236

RESUMO

Osteoarthritis (OA) is a degenerative condition that involves the production of inflammatory cytokines (e.g., interleukin-1ß (IL-1ß), tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)) that stimulate degradative enzymes, matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS) resulting in articular cartilage breakdown. The presence of interleukin-1ß (IL-1ß) is one reason for poor clinical outcomes in current cell-based tissue engineering strategies for treating focal early osteoarthritic defects. Mesenchymal stem cells (MSCs) are a potential cell source for articular cartilage regeneration, although IL-1ß has been shown to inhibit in vitro chondrogenesis. In vivo, articular chondrocytes reside under a low oxygen environment between 2-5% oxygen (physioxia) and have been shown to enhance in vitro MSC chondrogenic matrix content with reduced hypertrophic marker expression under these conditions. The present investigation sought to understand the effect of physioxia on IL-1ß inhibited MSC chondrogenesis. MSCs expanded under physioxic (2% oxygen) and hyperoxic (20%) conditions, then chondrogenically differentiated as pellets in the presence of TGF-ß1 and either 0.1 or 0.5 ng/mL IL-1ß. Results showed that there were donor variations in response to physioxic culture based on intrinsic GAG content under hyperoxia. In physioxia responsive donors, MSC chondrogenesis significantly increased GAG and collagen II content, whilst hypertrophic markers were reduced compared with hyperoxia. In the presence of IL-1ß, these donors showed a significant increase in cartilage matrix gene expression and GAG content relative to hyperoxic conditions. In contrast, a set of MSC donors were unresponsive to physioxia and showed no significant increase in matrix production independent of IL-1ß presence. Thus, physioxia has a beneficial effect on MSC cartilage matrix production in responsive donors with or without IL-1ß application. The mechanisms controlling the MSC chondrogenic response in both physioxia responsive and unresponsive donors are to be elucidated in future investigations.


Assuntos
Cartilagem Articular/citologia , Condrogênese/fisiologia , Ílio/citologia , Interleucina-1beta/metabolismo , Células-Tronco Mesenquimais/citologia , Oxigênio/metabolismo , Adulto , Células Cultivadas , Humanos , Masculino , Osteoartrite/terapia , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta1/metabolismo , Adulto Jovem
7.
J Pediatr Orthop ; 39(7): 382-386, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31305383

RESUMO

BACKGROUND: Autologous bone marrow aspirates are utilized to treat various conditions in children. The biological value of bone marrow aspirate depends on the concentration of competent osteoblastic progenitors present in the aspirate. It has been shown in adults that increasing bone marrow aspiration volume beyond 2 mL decreases the concentration of osteoblast progenitor cells because of dilution of the sample with peripheral blood. The effect of varying bone marrow aspiration volumes on the osteoblast cell content has not been determined in children. METHODS: In total, 21 children (3 male and 18 female patients, age range 8 mo to 14 y) scheduled for pelvic osteotomy were included in the study. Three separate bone marrow aspirates of 1, 5, and 10 mL were obtained from the anterior superior iliac crest. Total number of nucleated cells was counted per aspirate and the prevalence of alkaline phosphatase-positive colony-forming units was determined per million nucleated cells. RESULTS: We measured a significant, proportional increase in the total number of nucleated bone marrow precursor cells between the 1 and 5 mL samples (mean±SD, 27±13 and 152±78 million nucleated cells, respectively; P<0.0001). When the aspiration volume doubled from 5 to 10 mL the total number of nucleated cells was 178±76 million (P=0.17). A proportional increase from 2214 alkaline phosphatase-positive colony-forming units in the 1 mL sample to 14,100 alkaline phosphatase-positive colony-forming units in the 5 mL sample was observed. However, the number of colony-forming units per aspirate decreased to 11,880 in the 10 mL sample. CONCLUSIONS: These data demonstrate that in children aspiration up to 5 mL bone marrow from the iliac crest yields a proportional increase in osteoblastic progenitor cells per aspirate. Increasing the aspiration volume beyond 5 mL results in hemodilution, rather than further selection of osteoblastic material. CLINICAL RELEVANCE: These data provide clinicians with a guideline for optimizing aspiration volume of bone marrow in children. LEVEL OF EVIDENCE: Level II-development of diagnostic criteria on basis of consecutive patients.


Assuntos
Células da Medula Óssea , Ílio/citologia , Células-Tronco , Adolescente , Fosfatase Alcalina/análise , Biópsia por Agulha , Medula Óssea , Transplante de Medula Óssea , Contagem de Células , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Osteoblastos , Sucção
8.
Cytotherapy ; 21(2): 189-199, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30700393

RESUMO

BACKGROUND: Chronic venous leg ulcers (VLUs) are a common problem in clinical practice and available treatments are not satisfactory. The use of adjuvant therapies in combination with lower limb compression may lead to improved healing rates. Chronic wounds are candidates for new strategies in the emergent field of regenerative medicine. Bone marrow-derived cells (BMDCs) contain cells and secrete cytokines known to participate in wound healing. Thus, BMDC therapy seems a logical strategy for the treatment of chronic wounds. Our objective was to evaluate feasibility, safety and initial clinical outcome of autologous BMDC therapy associated with standard treatment in patients with VLUs. METHODS: We conducted an open-label, single-arm, prospective pilot clinical trial in four patients with six chronic VLUs. The study protocol was approved by the institutional and national review boards and ethics committees. Bone marrow was harvest, processed and then administered by multiple injections into the ulcers. All patients received standard treatment and non-healing characteristics of the VLUs were confirmed at study entry. RESULTS: Ulcer size and wound pain evaluated 12 months after BMDC treatment were significantly reduced (P < 0.05). BMDC treatment was safe and well tolerated in long-term follow-up. DISCUSSION: Despite the low number of patients studied, our results showed that autologous BMDC treatment could be a useful, feasible and safe procedure to enhance ulcer healing. However, randomized controlled trials with more patients are needed to address this question and translate this approach into clinical practice.


Assuntos
Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/métodos , Medicina Regenerativa/métodos , Transplante Autólogo/métodos , Úlcera Varicosa/terapia , Idoso , Estudos de Viabilidade , Feminino , Seguimentos , Humanos , Ílio/citologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Resultado do Tratamento , Cicatrização
9.
Vet Pathol ; 55(6): 853-860, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29940832

RESUMO

Bone marrow (BM) cytology and histopathology are complementary tools used to investigate hematological diseases. The purpose of this study was to determine if there are site-dependent differences in the diagnostic quality, myeloid to erythroid ratio (MER), and discordant findings in samples from different sites in the same dog. Eighteen apparently healthy dogs were used in the study. The sequence of sample acquisition was randomized according to a Latin square, and samples for BM cytology and histology were collected from both humeri and both ilial crests immediately after death. Board-certified clinical and anatomical pathologists read the cytology and histology, respectively. The data were analyzed using a mixed-effect model. The site of BM acquisition did not affect BM sample quality. The rate of discordant clinical findings between sites was 0.05 (95% confidence interval, 0.01-0.13). In general, by cytology, the MERs were slightly but significantly greater in samples from the ilial crests than from the humeri ( P = .01). The measured MER for histology was nearly twice that for cytology for all sites ( P < .001). In conclusion, there was a low-rate, site-dependent discordance in diagnostic findings in BM samples and differences in MER between the ilial crest and the humerus. A similar study is justified in sick dogs with hematological disease to determine the effect of sampling site on discordant findings between sites.


Assuntos
Células da Medula Óssea/citologia , Medula Óssea/anatomia & histologia , Cães/anatomia & histologia , Células Eritroides/citologia , Células Mieloides/citologia , Manejo de Espécimes/veterinária , Animais , Biópsia por Agulha Fina/métodos , Biópsia por Agulha Fina/veterinária , Doenças do Cão/patologia , Feminino , Úmero/citologia , Ílio/citologia , Masculino , Manejo de Espécimes/métodos
10.
PLoS One ; 13(5): e0197969, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29795650

RESUMO

The potential use of bone progenitors, multipotential stromal cells (MSCs) helping spine fusion is increasing, but convenient MSC sources and effective processing methods are critical factors yet to be optimised. The aim of this study was to test the effect of bone marrow processing on the MSC abundance and to compare the differentiation capabilities of vertebral body-bone marrow (VB-BM) MSCs versus iliac crest-bone marrow (IC-BM) MSCs. We assessed the effect of the red blood cell lysis (ammonium chloride, AC) and density-gradient centrifugation (Lymphoprep™, LMP), on the extracted VB-BM and IC-BM MSC numbers. The MSC abundance (indicated by colony counts and CD45lowCD271high cell numbers), phenotype, proliferation and tri-lineage differentiation of VB-BM MSCs were compared with donor-matched IC-BM MSCs. Importantly, the MSC attachment and osteogenesis were examined when VB-BM and IC-BM samples were loaded on a beta-tricalcium phosphate scaffold. In contrast to LMP, using AC yielded more colonies from IC-BM and VB-BM aspirates (p = 0.0019 & p = 0.0201 respectively). For IC-BM and VB-BM, the colony counts and CD45lowCD271high cell numbers were comparable (p = 0.5186, p = 0.2640 respectively). Furthermore, cultured VB-BM MSCs exhibited the same phenotype, proliferative and adipogenic potential, but a higher osteogenic and chondrogenic capabilities than IC-BM MSCs (p = 0.0010 and p = 0.0005 for calcium and glycosaminoglycan (GAG) levels, respectively). The gene expression data confirmed higher chondrogenesis for VB-BM MSCs than IC-BM MSCs, but osteogenic gene expression levels were comparable. When loaded on Vitoss™, both MSCs showed a similar degree of attachment and survival, but a better osteogenic ability was detected for VB-BM MSCs as measured by alkaline phosphatase activity (p = 0.0386). Collectively, the BM processing using AC had more MSC yield than using LMP. VB-BM MSCs have a comparable phenotype and proliferative capacity, but higher chondrogenesis and osteogenesis with or without using scaffold than donor-matched IC-BM MSCs. Given better accessibility, VB-BM could be an ideal MSC source for spinal bone fusion.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Linhagem da Célula , Ílio/citologia , Doenças da Coluna Vertebral/terapia , Fusão Vertebral/métodos , Coluna Vertebral/citologia , Células Estromais/citologia , Adolescente , Adulto , Idoso , Células da Medula Óssea/fisiologia , Proliferação de Células , Células Cultivadas , Condrogênese , Feminino , Humanos , Ílio/fisiologia , Masculino , Pessoa de Meia-Idade , Osteogênese , Doenças da Coluna Vertebral/patologia , Coluna Vertebral/fisiologia , Transplante de Células-Tronco , Células Estromais/fisiologia , Adulto Jovem
11.
J Dent Res ; 97(11): 1229-1235, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29772189

RESUMO

This study compared the osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) of iliac and alveolar origins (I-BMSCs and Al-BMSCs, respectively), which were transplanted in combination with ß tricalcium phosphate (ß-TCP) in peri-implant bone defects to investigate the osseointegration between dental implants and tissue-engineered bone in dogs. Specifically, I-BMSCs and Al-BMSCs were cultured, characterized, and seeded on ß-TCP and subjected to immunoblotting analyses and alkaline phosphatase activity assays. Subsequently, these cell-seeded scaffolds were implanted into defects that were freshly generated in the mandibular premolar areas of 4 dogs. The defects were covered with ß-TCP + Al-BMSCs ( n = 6), ß-TCP + I-BMSCs ( n = 6), or ß-TCP ( n = 6) or served as the blank control ( n = 6). After healing for 12 wk, the formation and mineralization of new bones were assessed through micro-computed tomographic, histologic, and histomorphometric analyses, and bone-to-implant contacts were measured in the specimens. It was evident that in this large animal model, I-BMSCs and Al-BMSCs manifested similarly strong osteogenic potential, as significantly more new bone was formed in the Al-BMSC and I-BMSC groups than otherwise ( P < 0.01). Therefore, Al-BMSCs are emerging as an efficient alternative for autologous mesenchymal stem cells in regenerative dental and maxillofacial therapies. I-BMSCs, if not restricted in their bioavailability, can also be of great utility in bone tissue-engineering applications.


Assuntos
Processo Alveolar/citologia , Regeneração Óssea , Ílio/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Processo Alveolar/diagnóstico por imagem , Processo Alveolar/fisiologia , Animais , Fosfatos de Cálcio/uso terapêutico , Cães , Transplante de Células-Tronco Mesenquimais/métodos , Engenharia Tecidual/métodos , Alicerces Teciduais , Microtomografia por Raio-X
12.
Mol Med Rep ; 17(5): 6621-6631, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29512721

RESUMO

Activation of the transcription factor hypoxia inducible factor­1α (HIF-1α) is considered critical for the stimulation of osteogenic markers including runt­related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin, which are closely associated with forkhead boxclass O1 (Foxo1) levels in osteoblasts. The present study explored the associations between HIF­1α and Foxo1 in the regulation of cell viability, proliferation and apoptosis of osteoblasts. Osteoblasts obtained from children's iliac cancellous bone were used in the present study, which were confirmed by immunofluorescence staining for the osteoblast marker osteocalcin. The results revealed that the levels of reactive oxygen species and apoptosis were markedly increased in cells with knockdown of HIF­1α. By contrast, these were reduced in response to overexpressed HIF­1α. In addition, HIF­1α overexpression significantly stimulated cell viability, which was suppressed by silencing HIF­1α. HIF­1α overexpression also significantly increased the transcriptional and translational levels of Foxo1. Conversely, silencing HIF­1α markedly suppressed the expression levels of Foxo1. Furthermore, silencing HIF­1α reduced the expression of osteogenic markers, including Runx2, ALP and osteocalcin. Runx2 and ALP expression induced by HIF1α were markedly reversed by Foxo1 small interfering (si)RNA, whereas osteocalcin was not significantly affected by Foxo1 siRNA. Therefore, the cooperation of and interactions between HIF­1α and Foxo1 may be involved in the regulation of osteoblast markers, and serve a pivotal role in the proliferation and apoptosis of osteoblast. The HIF1α­induced expression of Runx2 and ALP may be completely dependent on the expression levels of Foxo1, and in turn, osteocalcin may be partially dependent on Foxo1 expression.


Assuntos
Apoptose/fisiologia , Osso Esponjoso/metabolismo , Proliferação de Células/fisiologia , Proteína Forkhead Box O1/biossíntese , Regulação da Expressão Gênica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ílio/metabolismo , Osteoblastos/metabolismo , Antígenos de Diferenciação/biossíntese , Osso Esponjoso/citologia , Pré-Escolar , Feminino , Humanos , Ílio/citologia , Masculino , Osteoblastos/citologia
13.
Eur J Orthop Surg Traumatol ; 28(3): 457-461, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29094213

RESUMO

BACKGROUND: Different cell populations from bone marrow were used in various clinical trials for cardiac diseases during last decade. Four clinical studies are ongoing in our institution and enroll patients with cardiac diseases, coronary disease, type 2 diabetes, and osteoarthritis. The density gradient is used to separate bone marrow mononuclear cells. Joint replacement procedures were associated with significant loss of tissue. Usually, excess tissue as bone marrow, peripheral blood and fat are removed to clean operation site. The aim of this study is to prove whether removed tissue during joint replacement procedure can be considered as a significant source of mononuclear cells. METHODS: Excised tissue obtained during joint replacement procedure was collected by AutoLog system. Bone marrow tissue was collected by iliac crest puncture. Mononuclear cells from both sources were isolated by using Ficoll density gradient centrifugation. Flow cytometry was used to detect mononuclear cell, CD34+ population counts and cell viability. Tissue processing yields between the group of joint replacement and iliac crest puncture group were compared. RESULTS: Together, 34 bone marrow tissue processings were performed. On average, samples contained 46.31 ± 9.35 ml of bone marrow solution. Average cell yield in final product was 28.64 ± 9.35 × 106 MNCs and 0.77 ± 1.51 × 106 CD34+ population. In case of tissue removed during joint replacement nine processings were performed. On average samples contained 450 ± 157.69 ml of tissue solution. Average cell yield in final product was 76.67 ± 35.42 × 106 MNCs and 1.33 ± 0.97 × 106 CD34+ population. CONCLUSIONS: Tissue processing analysis shows that tissue removed during joint replacement procedure can be assumed as a significant source of mononuclear cells. Methods used for bone marrow-derived mononuclear cell extraction can be applied to the excess tissue.


Assuntos
Monócitos/transplante , Coleta de Tecidos e Órgãos/métodos , Antígenos CD34 , Artroplastia de Quadril/métodos , Artroplastia do Joelho/métodos , Estudos de Casos e Controles , Centrifugação com Gradiente de Concentração/métodos , Feminino , Citometria de Fluxo , Humanos , Ílio/citologia , Ílio/cirurgia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Quadril/cirurgia , Osteoartrite do Joelho/cirurgia , Punções/métodos , Transplante Autólogo/métodos
14.
Morphologie ; 102(336): 48-53, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29102553

RESUMO

Primary hyperoxaluria is a rare genetic disorder characterized by oxalate overproduction, leading to kidney failure due to nephrocalcinosis, and is eventually responsible for systemic oxalosis. Bone impairment, secondary to oxalate deposits, is one of the many complications that may occur. Skeletal involvement can be difficult to diagnose because of lack of clinical symptoms and therefore needs to be confirmed by invasive testing, such as transiliac bone biopsy. If confirmed, bone oxalosis is the proof of disease severity and that combined liver-kidney transplantation should be performed.


Assuntos
Oxalato de Cálcio/metabolismo , Hiperoxalúria Primária/metabolismo , Ílio/patologia , Nefrocalcinose/metabolismo , Adulto , Biópsia , Densidade Óssea , Oxalato de Cálcio/urina , Humanos , Hiperoxalúria Primária/tratamento farmacológico , Hiperoxalúria Primária/genética , Hiperoxalúria Primária/urina , Ílio/citologia , Ílio/diagnóstico por imagem , Falência Renal Crônica/metabolismo , Falência Renal Crônica/terapia , Transplante de Rim , Masculino , Microrradiografia , Nefrocalcinose/diagnóstico por imagem , Nefrocalcinose/genética , Nefrocalcinose/urina , Osteoblastos/patologia , Piridoxina/uso terapêutico , Diálise Renal , Transaminases/genética
15.
Growth Factors ; 35(4-5): 179-188, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-29228886

RESUMO

Bone defect treatment belongs to the most challenging fields in orthopedic surgery and requires the well-coordinated application of mesenchymal stem cells (MSC) and differentiation factors. MSC isolated from reaming material (RMSC) and iliac crest (BMSC) in combination with bone morphogenetic protein-7 (BMP-7) and insulin-like growth factor-1 (IGF-1) have been used. The short half-life of both factors limit their applications: a burst release of the factor can probably not induce sustainable differentiation. We stimulated MSC in osteogenic differentiation medium with three different concentrations of BMP-7 or IGF-1: Group A was stimulated continuously, group B for 24 h and group C remained without any stimulation. Osteogenic differentiation was measured after seven and 14 days by alizarin red staining and alkaline phosphatase (ALP) activity. Continuous stimulation led to higher levels of osteogenic differentiation than short-term stimulation. This could lead to a reconsideration of established application forms for differentiation factors, aiming to provide a more sustained release.


Assuntos
Proteína Morfogenética Óssea 7/farmacologia , Diferenciação Celular , Fator de Crescimento Insulin-Like I/farmacologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Idoso , Células Cultivadas , Feminino , Humanos , Ílio/citologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade
16.
J Bone Joint Surg Am ; 99(19): 1673-1682, 2017 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-28976432

RESUMO

BACKGROUND: The rational design and optimization of tissue engineering strategies for cell-based therapy requires a baseline understanding of the concentration and prevalence of osteogenic progenitor cell populations in the source tissues. The aim of this study was to (1) define the efficiency of, and variation among individuals in, bone marrow aspiration as a means of osteogenic connective tissue progenitor (CTP-O) harvest compared with harvest from iliac cancellous bone, and (2) determine the location of CTP-Os within native cancellous bone and their distribution between the marrow-space and trabecular-surface tissue compartments. METHODS: Eight 2-mL bone marrow aspiration (BMA) samples and one 7-mm transcortical biopsy sample were obtained from the anterior iliac crest of 33 human subjects. Two cell populations were obtained from the iliac cancellous bone (ICB) sample. The ICB sample was placed into αMEM (alpha-minimal essential medium) with antibiotic-antimycotic and minced into small pieces (1 to 2 mm in diameter) with a sharp osteotome. Cells that could be mechanically disassociated from the ICB sample were defined as marrow-space (IC-MS) cells, and cells that were disassociated only after enzymatic digestion were defined as trabecular-surface (IC-TS) cells. The 3 sources of bone and marrow-derived cells were compared on the basis of cellularity and the concentration and prevalence of CTP-Os through colony-forming unit (CFU) analysis. RESULTS: Large variation was seen among patients with respect to cell and CTP-O yield from the IC-MS, IC-TS, and BMA samples and in the relative distribution of CTP-Os between the IC-MS and IC-TS fractions. The CTP-O prevalence was highest in the IC-TS fraction, which was 11.4-fold greater than in the IC-MS fraction (p < 0.0001) and 1.7-fold greater than in the BMA fraction. However, the median concentration of CTP-Os in the ICB (combining MS and TS fractions) was only 3.04 ± 1.1-fold greater than that in BMA (4,265 compared with 1,402 CTP/mL; p = 0.00004). CONCLUSIONS: Bone marrow aspiration of a 2-mL volume at a given needle site is an effective means of harvesting CTP-Os, albeit diluted with peripheral blood. However, the median concentration of CTP-Os is 3-fold less than from native iliac cancellous bone. The distribution of CTP-Os between the IC-MS and IC-TS fractions varies widely among patients. CLINICAL RELEVANCE: Bone marrow aspiration is an effective means of harvesting CTP-Os but is associated with dilution with peripheral blood. Overall, we found that 63.5% of all CTP-Os within iliac cancellous bone resided on the trabecular surface; however, 48% of the patients had more CTP-Os contributed by the IC-MS than the IC-TS fraction.


Assuntos
Células da Medula Óssea , Ílio/citologia , Sucção , Coleta de Tecidos e Órgãos/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Artroplastia de Quadril , Transplante de Medula Óssea , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
17.
Regen Med ; 12(8): 917-928, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28891421

RESUMO

AIM: We review relevant anatomy of the iliac crest, and describe an interventional technique to maximize harvesting of desired progenitor cells with ultrasound to guide safe trochar placement. MATERIALS & METHODS: We validated the technique on both sides of the pelvis in four human cadavers. RESULTS: Using ultrasound guidance, 32 BMA needles were placed in a safe zone along various portions of the iliac crest. CONCLUSION: Ultrasound guidance can improve accuracy of bone marrow aspirations form the iliac crest. Mastery of this procedure will facilitate cell harvest and aid in patient safety when procuring mesenchymal stem cells from a bone marrow source.


Assuntos
Doenças Ósseas/terapia , Células da Medula Óssea/citologia , Ílio/cirurgia , Células-Tronco Multipotentes/citologia , Procedimentos Ortopédicos/instrumentação , Regeneração , Ultrassonografia/métodos , Cadáver , Feminino , Humanos , Ílio/citologia , Ílio/diagnóstico por imagem , Masculino , Agulhas , Sucção
18.
Actual. osteol ; 13(2): 104-115, Mayo - Ago. 2017. ilus, graf, tab
Artigo em Espanhol | LILACS | ID: biblio-1117988

RESUMO

La osteonecrosis de maxilar asociada a aminobisfosfonatos (BRONJ) constituye un efecto secundario del tratamiento crónico con los más potentes. Un modelo experimental permitiría determinar la patogenia de dicha alteración. La oveja presenta características orales y del metabolismo óseo similar al humano y permite realizar manipulaciones bucales. Se evaluaron cambios clínicos, remodelación ósea y masa ósea maxilar en ovejas hembras adultas tratadas con zolendronato (ZOL), durante 22 meses y utilizando dosis equivalente al tratamiento de neoplasias. Seis ovariectomizadas (OVX) recibieron ZOL; 5 OVX y 4 SHAM (control) recibieron solución fisiológica. Al inicio, 4 y 22 meses se evaluó calcemia, fosfatemia, crosslaps (CTX) y fosfatasa alcalina ósea. Al final, se evaluó contenido mineral óseo de la hemimandíbula superior (CMO: mg/cm2). Al final del estudio, CTX disminuyó significativamente en ZOL (p<0,05) sin diferencias entre SHAM y OVX. En maxilar, los contenidos de Ca y P (g/g tejido) y CMO (g/cm2 ) disminuyeron en OVX vs. SHAM (p<0,05) y solo Ca y CMO respecto de ZOL (p<0,05). ZOL incrementó el contenido de Ca y CMO, mientras que el de P permaneció significativamente disminuido respecto de SHAM. La sobrevida en SHAM y OVX fue del 100% y en ZOL 77% (2 muertes); 2 ovejas del grupo ZOL presentaron necrosis de maxilar. Conclusiones: fue posible obtener desarrollo de BRONJ por tratamiento crónico con ZOL, el cual redujo notablemente la resorción y, según la relación Ca/P, posiblemente haya afectado la mineralización ósea. (AU)


Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a complication of chronic treatment with the most powerful aminobisphosphonates (BPs). An experimental animal model would allow to determine the pathogenesis of this complication. Ewes exhibit similar oral cavity characteristics and bone metabolism as humans, and they are suitable for oral cavity interventions. We examined herein the clinical manifestations, bone remodeling status, and maxillary bone mass in adult female ewes treated with zoledronate (ZOL) for 22 months. Six ovariectomized (OVX) ewes received ZOL; and 5 OVX and 4 SHAM animals received saline solution. At the start of the experiment, and at the 4 and 22 month-time points serum Ca, P, crosslaps (CTX), and bone alkaline phosphatase were measured. Bone mineral content (BMC) of the superior hemimandible was measured at the end of the experiment. At this time point, CTX was significantly decreased only in the ZOL group (p<0.05). Ca and P content (g/g tissue) and BMC in the mandible were significantly decreased in the OVX group compared to SHAM animals (p<0.05) and only Ca content and BMC were decreased when compared to ZOL (p<0.05). ZOL treatment increased the Ca content and BMC, whereas the P content remained low compared to the SHAM group (p<0.05). All ewes from the SHAM and OVX groups and 77% of the animals from the ZOL group survived until the end of the experiment, whereas two ewes of ZOL group exhibited BRONJ. Conclusion: under our experimental conditions, it was possible to induce BRONJ by the chronic ZOL administration, which in turn induced a high reduction in bone resorption as well as possibly impaired bone mineralization, based on the Ca/P ratio in the mandible. (AU)


Assuntos
Animais , Difosfonatos/efeitos adversos , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/patologia , Ácido Zoledrônico/efeitos adversos , Extração Dentária , Doenças Ósseas Metabólicas/induzido quimicamente , Ovinos/metabolismo , Ovinos/sangue , Biomarcadores/sangue , Densidade Óssea/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Densitometria , Desenvolvimento Experimental , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/etiologia , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/imunologia , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/prevenção & controle , Ácido Zoledrônico/administração & dosagem , Glucocorticoides/uso terapêutico , Analgésicos/uso terapêutico , Ílio/citologia , Anestésicos Dissociativos/uso terapêutico , Lidocaína/uso terapêutico , Maxila/citologia , Maxila/efeitos dos fármacos , Maxila/metabolismo , Maxila/diagnóstico por imagem , Antibacterianos/uso terapêutico
19.
J Dent Res ; 96(9): 984-991, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28521114

RESUMO

Transplantation of mesenchymal stem cells (MSCs), which possess self-renewing properties and multipotency, into a periodontal defect is thought to be a useful option for periodontal tissue regeneration. However, developing more reliable and predictable implantation techniques is still needed. Recently, we generated clumps of an MSC/extracellular matrix (ECM) complex (C-MSC), which consisted of cells and self-produced ECM. C-MSCs can regulate their cellular functions in vitro and can be grafted into a defect site, without any artificial scaffold, to induce bone regeneration. Accordingly, this study aimed to evaluate the effect of C-MSC transplantation on periodontal tissue regeneration in beagle dogs. Seven beagle dogs were employed to generate a premolar class III furcation defect model. MSCs isolated from dog ilium were seeded at a density of 7.0 × 104 cells/well into 24-well plates and cultured in growth medium supplemented with 50 µg/mL ascorbic acid for 4 d. To obtain C-MSCs, confluent cells were scratched using a micropipette tip and were then torn off as a cellular sheet. The sheet was rolled up to make round clumps of cells. C-MSCs were maintained in growth medium or osteoinductive medium (OIM) for 5 or 10 d. The biological properties of C-MSCs were evaluated in vitro, and their periodontal tissue regenerative activity was tested by using a dog class III furcation defect model. Immunofluorescence analysis revealed that type I collagen fabricated the form of C-MSCs. OIM markedly elevated calcium deposition in C-MSCs at day 10, suggesting its osteogenic differentiation capacity. Both C-MSCs and C-MSCs cultured with OIM transplantation without an artificial scaffold into the dog furcation defect induced periodontal tissue regeneration successfully compared with no graft, whereas osteogenic-differentiated C-MSCs led to rapid alveolar bone regeneration. These findings suggested that the use of C-MSCs refined by self-produced ECM may represent a novel predictable periodontal tissue regenerative therapy.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Matriz Extracelular/metabolismo , Regeneração Tecidual Guiada Periodontal/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Doenças Periodontais/terapia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Cães , Ílio/citologia , Células-Tronco Mesenquimais/citologia , Microtomografia por Raio-X
20.
BMC Nephrol ; 18(1): 134, 2017 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-28407760

RESUMO

BACKGROUND: Patients with an impaired renal function show a high incidence of bone and mineral disturbances. These 'chronic kidney disease - mineral and bone disorders' (CKD-MBD) range from high turnover osteoporosis to adynamic bone disease. Currently, the histomorphometric analysis of a bone biopsy taken from the iliac crest is viewed as the gold standard for CKD-MBD subtype differentiation. However, the clinical relevance of such a biopsy is questionable since iliac crest fractures are an extremely rare finding. Therefore, we aimed to elucidate if the histomorphometric parameter 'trabecular bone volume (BV/TV)' from the iliac crest is representative for other biopsy locations. We chose two skeletal sites of higher fracture risk for testing, namely, the tibial bone and the lumbar spine, to examine if the current gold standard of bone biopsy is indeed golden. METHODS: Bone biopsies were taken from 12 embalmed body donors at the iliac crest, the proximal tibia, and the lumbar vertebral body, respectively. Masson-Goldner stained sections of methyl methacrylate embedded biopsies were used for trabecular bone volume calculation. Furthermore, exemplary µ-computed tomography (XtremeCT) scans with subsequent analysis were performed. RESULTS: Median values of trabecular bone volume were comparable between all body donors with median (interquartile range, IQR) 18.3% (10.9-22.9%) at the iliac crest, 21.5% (9.5-40.1%) at the proximal tibia, and 16.3% (11.4-25.0%) at the lumbar spine. However, single values showed extensive intra-individual variation, which were also confirmed by XtremeCT imaging. CONCLUSIONS: Distinct intra-individual heterogeneity of trabecular bone volume elucidate why a bone biopsy from one site does not necessarily predict patient relevant endpoints like hip or spine fractures. Physicians interpreting bone biopsy results should know this limitation of the current gold standard for CKD-MBD diagnostic, especially, when systemic therapeutic decisions should be based on it.


Assuntos
Biópsia/métodos , Ílio/citologia , Vértebras Lombares/citologia , Tíbia/citologia , Idoso , Cadáver , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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