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1.
Plast Reconstr Surg ; 153(3): 637-646, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-37224290

RESUMO

BACKGROUND: The standard graft material for alveolar cleft repair (ACR) is autogenous iliac crest. A promising alternative potential graft adjunct-newborn human umbilical cord mesenchymal stem cells (h-UCMSCs)-has yet to be explored in vivo. Their capacity for self-renewal, multipotent differentiation, and proliferation allows h-UCMSCs to be harnessed for regenerative medicine. This study sought to evaluate the efficacy of using tissue-derived h-UCMSCs and their osteogenic capabilities to improve ACR in a murine model. METHODS: Foxn1 mice were separated into three groups with the following calvarial defects: no treatment (empty defect; n = 6), poly(D,L-lactide-co-glycolide) (PLGA) scaffold ( n = 6), or h-UCMSCs with PLGA ( n = 4). Bilateral 2-mm-diameter parietal bone critical-sized defects were created using a dental drill. Microcomputed tomography (microCT) imaging was performed 1, 2, 3, and 4 weeks postoperatively. The mice were euthanized 4 weeks postoperatively for RNAScope, immunohistochemical, and histological analysis. RESULTS: No mice experienced complications during the follow-up period. MicroCT imaging and histological analysis demonstrated that the no-treatment and PLGA-only defects remained patent without significant defect size differences across groups. In contrast, the h-UCMSCs with PLGA group had significantly greater bone fill on microCT and histological analysis. CONCLUSIONS: This study demonstrates a successful calvarial defect model for the investigation of h-UCMSC-mediated osteogenesis and bone repair. Evidence reveals that PLGA alone has neither short-term effects on bone formation nor any unwanted side effects, making it an attractive scaffold. Further investigation using h-UCMSCs with PLGA in larger animals is warranted to advance future translation to patients requiring ACR. CLINICAL RELEVANCE STATEMENT: The authors' results demonstrate a successful murine calvarial defect model for the investigation of h-UCMSC-mediated osteogenesis and bone repair, and they provide preliminary evidence for the safe and efficacious use of this graft adjunct in alveolar cleft repair.


Assuntos
Osteogênese , Alicerces Teciduais , Humanos , Camundongos , Animais , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Microtomografia por Raio-X , Regeneração Óssea , Células-Tronco , Diferenciação Celular , Cordão Umbilical , Crânio/cirurgia , Crânio/patologia
2.
Cytotherapy ; 22(10): 581-591, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32718875

RESUMO

BACKGROUND AIMS: Umbilical cord (UC) tissue is recognized as an advantageous source of mesenchymal stromal cells (MSCs), whose therapeutic properties are being actively evaluated in pre-clinical and clinical trials. In recognition of its potential value, storage of UC tissue or cells from UC tissue in newborn stem cell banks is now commonplace; however, strategies for isolating UC-derived MSCs (UCMSCs) from UC tissue have not been standardized. The majority of newborn stem cell banks take one of two approaches to cord tissue processing and cryopreservation: enzymatic digestion of the fresh tissue with cryopreservation of the subsequent cell suspension or cryopreservation of the tissue as a composite whole with later, post-thaw isolation of cells by explantation. Evaluation of UCMSCs derived by these two principal preparation and cryopreservation strategies is important to understanding whether the methods currently employed by newborn stem cell banks retain the desirable clinical attributes of UC cells. METHODS: UCMSCs were isolated from 10 UC tissue samples by both explantation and enzymatic digestion methods to allow for comparison of cells from the same donor. Cell isolates from both methods were compared pre- and post-cryopreservation as well as after serial passaging. Cell viability, morphology, growth kinetics, immunophenotype, cytokine secretion and differentiation capacity were evaluated. RESULTS: UCMSCs could be derived from fresh UC tissue by both explantation and digestion methods and from thawed UC tissue by explantation. Initial cell populations isolated by digestion were heterogeneous and took longer to enrich for UCMSCs in culture than populations obtained by explantation. However, once isolated and enriched, UCMSCs obtained by either method showed no significant difference in viability, morphology, rate of proliferation, surface marker expression, levels of cytokine secretion or differentiation capacity. CONCLUSIONS: Derivation of UCMSCs by explantation after thawing UC cryopreserved as a composite tissue may be favorable in terms of initial purity and number of cells achievable by a specific passage. However, we observed no evidence of functional difference between UCMSCs derived by explanation or digestion, suggesting that cells isolated from cryopreserved material obtained by either method maintain their therapeutic properties.


Assuntos
Separação Celular/métodos , Criopreservação , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Forma Celular , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , Hematopoese , Humanos , Imunofenotipagem , Recém-Nascido , Cinética
3.
Regen Med ; 14(4): 295-307, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31074319

RESUMO

Aim: Traumatic brain injury is a complex condition consisting of a mechanical injury with neurovascular disruption and inflammation with limited clinical interventions available. A growing number of studies report systemic delivery of human umbilical cord blood (HUCB) as a therapy for neural injuries. Materials & methods: HUCB cells from five donors were tested to improve blood-brain barrier integrity in a traumatic brain injury rat model at a dose of 2.5 × 107 cells/kg at 24 or 72 h postinjury and for immunomodulatory activity in vitro. Results & Conclusion: We observed that cells delivered 72 h postinjury significantly restored blood-brain barrier integrity. HUCB cells reduced the amount of TNF-α and IFN-γ released by activated primary rat splenocytes, which correlated with the expression of COX2 and IDO1.


Assuntos
Lesões Encefálicas/terapia , Encéfalo/irrigação sanguínea , Sangue Fetal/transplante , Inflamação/terapia , Cordão Umbilical/citologia , Animais , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Extravasamento de Materiais Terapêuticos e Diagnósticos/patologia , Humanos , Imunomodulação , Inflamação/complicações , Inflamação/patologia , Masculino , Ratos Sprague-Dawley , Baço/patologia , Fator de Necrose Tumoral alfa/metabolismo
4.
Cytotherapy ; 20(4): 564-575, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29429941

RESUMO

BACKGROUND: Umbilical cord (UC) tissue can be collected in a noninvasive procedure and is enriched in progenitor cells with potential therapeutic value. Mesenchymal stromal cells (MSCs) can be reliably harvested from fresh or cryopreserved UC tissue by explant outgrowth with no apparent impact on functionality. A number of stem cell banks offer cryopreservation of UC tissue, alongside cord blood, for future cell-based applications. In this setting, measuring and monitoring UC quality is critical. MATERIALS AND METHODS: UC explants were evaluated using a plating and scoring system accounting for cell attachment and proliferation. Explant scores for fresh and cryopreserved-then-thawed tissue from the same UC were compared. Metabolic activity of composite UC tissue was also assayed after exposure of the tissue to conditions anticipated to affect UC quality and compared with explant scores within the same UC. RESULTS: All fresh and cryopreserved tissues yielded MSC-like cells, and cryopreservation of the tissue did not prevent the ability to isolate MSCs by the explant method. Thawed UC tissue scores were 91% (±0.6%; P = 0.0009) that of the fresh, biologically identical tissue. Within the same UC, explant scores correlated well to both cell yield (R2 = 0.85) and tissue metabolic activity (R2 = 0.69). DISCUSSION: A uniform explant scoring assay can provide information about the quality of composite UC tissue. Such quantitative measurement is useful for analysis of tissue variability and process monitoring. Additionally, a metabolic assay of UC tissue health provides results that correlate well to explant scoring results.


Assuntos
Bioensaio , Coleta de Tecidos e Órgãos , Cordão Umbilical , Bioensaio/métodos , Bioensaio/normas , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Criopreservação/métodos , Criopreservação/normas , Estudos de Avaliação como Assunto , Sangue Fetal/química , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Saúde , Humanos , Recém-Nascido , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Metaboloma , Controle de Qualidade , Padrões de Referência , Bancos de Tecidos/normas , Coleta de Tecidos e Órgãos/métodos , Coleta de Tecidos e Órgãos/normas , Cordão Umbilical/química , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo , Cordão Umbilical/cirurgia
5.
Cell Transplant ; 24(11): 2323-35, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25197983

RESUMO

Cell-based angiogenic therapies offer potential for the repair of ischemic injuries, while avoiding several of the limitations associated with material-based growth factor delivery strategies. Evidence supports that applying MSCs as spheroids rather than dispersed cells can improve retention and enhance therapeutic effect through increased secretion of angiogenic factors due to hypoxia. However, while spheroid culture appears to modulate MSC behavior, there has been little investigation of how major culture parameters that affect cellular oxygen tension, such as external oxygenation and culture size, impact the angiogenic potential of spheroids. We cultured equal numbers of adipose-derived stem cells (ASCs) as spheroids containing 10,000 (10k) or 60,000 (60k) cells each, in 20% and 2% oxygen. VEGF secretion varied among the sample groups, with 10k, 2% O2 spheroids exhibiting the highest production. Spheroid-conditioned media was applied to HUVEC monolayers, and proliferation was assessed. Spheroids of either size in 2% oxygen induced comparable proliferation compared to a 2 ng/ml VEGF control sample, while spheroids in 20% oxygen induced less proliferation. Spheroids were also applied in coculture with HUVEC monolayers, and induction of migration through a Transwell membrane was evaluated. Sixty thousand, 2% O2 spheroids induced similar levels of migration as VEGF controls, while 10k, 2% O2 spheroids induced significantly more. Ten thousand, 20% spheroids performed no better than VEGF-free controls. We conclude that the therapeutic ability of ASC spheroids to stimulate angiogenesis in endothelial cells is affected by both culture size and oxygenation parameters, suggesting that, while ASC spheroids offer potential in the treatment of injured and ischemic tissues, careful consideration of culture size in respect to in vivo local oxygen tension will be necessary for optimal results.


Assuntos
Células Endoteliais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Esferoides Celulares/transplante , Fator A de Crescimento do Endotélio Vascular/metabolismo , Tecido Adiposo/metabolismo , Técnicas de Cultura de Células , Hipóxia Celular , Meios de Cultivo Condicionados , Humanos , Veias Umbilicais/citologia
6.
Tissue Eng Part A ; 19(21-22): 2330-8, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23668629

RESUMO

Adipose-derived stem cells (ADSCs) possess potent angiogenic properties and represent a source for cell-based approaches to delivery of bioactive factors to drive vascularization of tissues. Hypoxic signaling appears to be largely responsible for triggering release of these angiogenic cytokines, including vascular endothelial growth factor (VEGF). Three-dimensional (3D) culture may promote activation of hypoxia-induced pathways, and has furthermore been shown to enhance cell survival by promoting cell-cell interactions while increasing angiogenic potential. However, the development of hypoxia within ADSC spheroids is difficult to characterize. In the present study, we investigated the impact of spheroid size on hypoxia-inducible transcription factor (HIF)-1 activity in spheroid cultures under atmospheric and physiological oxygen conditions using a fluorescent marker. Hypoxia could be induced and modulated by controlling the size of the spheroid; HIF-1 activity increased with spheroid size and with decreasing external oxygen concentration. Furthermore, VEGF secretion was impacted by the hypoxic status of the culture, increasing with elevated HIF-1 activity, up to the point at which viability was compromised. Together, these results suggest the ability to use 3D culture geometry as a means to control output of angiogenic factors from ADSCs, and imply that at a particular environmental oxygen concentration an optimal culture size for cytokine production exists. Consideration of culture geometry and microenvironmental conditions at the implantation site will be important for successful realization of ADSCs as a pro-angiogenic therapy.


Assuntos
Adipócitos/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo
7.
Tissue Eng Part A ; 19(13-14): 1583-91, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23394201

RESUMO

Tissue engineering is a promising approach to repair critical-size defects in bone. Damage to vasculature at the defect site can create a lower O2 environment compared with healthy bone. Local O2 levels influence stem cell behavior, as O2 is not only a nutrient, but also a signaling molecule. The hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates a wide range of O2-related genes and its contribution in bone repair/formation is an important area that can be exploited. In this study, we examined the effect of low O2 environments (1% and 2% O2) on the osteogenic differentiation of adipose-derived stem cells in both two-dimensional (2-D) and three-dimensional (3-D) culture systems. To determine the role of HIF-1 in the differentiation process, an inhibitor was used to block the HIF-1 activity. The samples were examined for osteogenesis markers as measured by quantification of the alkaline phosphatase (ALP) activity, mineral deposition, and expression of osteonectin (ON) and osteopontin (OPN). Results show a downregulation of the osteogenic markers (ALP activity, mineralization, ON, OPN) in both 1% and 2% O2 when compared to 20% O2 in both 2-D and 3-D culture. Vascular endothelial growth factor secretion over 28 days was significantly higher in low O2 environments and HIF-1 inhibition reduced this effect. The inhibition of the HIF-1 activity did not have a significant impact on the expression of the osteogenic markers, suggesting HIF-1-independent inhibition of osteogenic differentiation in hypoxic conditions.


Assuntos
Tecido Adiposo/citologia , Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Osteogênese/fisiologia , Osteonectina/metabolismo , Osteopontina/metabolismo , Engenharia Tecidual/métodos
8.
Int J Artif Organs ; 36(3): 175-83, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23404645

RESUMO

PURPOSE: Hypoxia is a major cause for failure of encapsulated islet grafts. Three-dimensional (3D) re-aggregation and hypoxic preconditioning are used to help overcome this obstacle. However, it is still difficult to identify hypoxic cells in a 3D system. We evaluate the efficacy of a fluorescent system for detecting HIF-1 activity in live ß-cells. Identification of HIF-1 activity and correlation with insulin secretion and viability will allow for more informed implant construction and better prediction of post-transplantational function.
 METHODS: MIN6 cells were infected with the marker virus and rotationally cultured to form clusters. Clusters were encapsulated in PEG hydrogels and incubated in 20%, 2%, or 1% O2. Gels were imaged daily for hypoxia marker signaling and for morphological observation. Daily GSIS was quantified by insulin ELSIA and cell viability was assessed by LIVE/DEAD staining.
 RESULTS: Clusters cultured in 2% and 1% O2 displayed high levels of HIF activity compared to 20% O2 clusters. 20% O2 clusters maintained viability and achieved a smooth, islet-like morphology by Day 14. Clusters in 2% and 1% O2 failed to associate cohesively and showed reduced viability. As a whole, constructs cultured in 20% O2 exhibited 10-fold higher GSIS than constructs in 2% and 1% O2.
 CONCLUSIONS: Our marker is an effective approach for identifying cellular hypoxia in 3D cultures. ß-cell clusters in 2% and 1% O2 are similarly affected by reduced oxygen tension, with HIF-1 activity correlating to reduced GSIS and impaired cell/cluster morphology. Simultaneous aggregative culture and hypoxic conditioning may not be beneficial to ß-cell transplantation.


Assuntos
Hipóxia Celular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Linhagem Celular Tumoral , Forma Celular , Células Secretoras de Insulina/citologia , Insulinoma/metabolismo , Ilhotas Pancreáticas/citologia , Camundongos , Neoplasias Pancreáticas/metabolismo
9.
J Biomed Mater Res B Appl Biomater ; 97(1): 148-55, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21290593

RESUMO

A common obstacle to the survival of encapsulated tissue is oxygen insufficiency. This appears particularly true of encapsulated pancreatic ß-cells. Our work investigates a fluorescent hypoxia detection system for early recognition of hypoxic stress in encapsulated pancreatic tissue. Murine insulinoma (MIN6) cells were engineered to produce a red fluorescent protein under the control of hypoxia-inducible-factor-1. Aggregates of these cells were encapsulated in poly(ethylene glycol) hydrogels at densities of 200,000, 600,000, and 1 million cells per capsule then incubated in either a 1% or 20% oxygen environment. Cell function was evaluated by daily measurement of glucose-stimulated insulin secretion. Encapsulated cells were also fluorescently imaged periodically over 72 h for expression of the marker signal. Results indicate that oxygen insufficiency severely impacts insulin release from MIN6 cells, and that large aggregates are especially vulnerable to oxygen limitations. Our marker was found to be successfully indicative of hypoxia and could be used as a predictor of subsequent insulin release. Further work will be required to fully characterize signal dynamics and to evaluate in vivo efficacy. The method presented here represents a unique and valuable approach to detecting hypoxic stress in living tissues which may prove useful to a variety of fields of biological research.


Assuntos
Bioensaio/métodos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Luminescentes/biossíntese , Consumo de Oxigênio , Animais , Biomarcadores/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Células Imobilizadas/citologia , Células Imobilizadas/metabolismo , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Proteínas Luminescentes/genética , Camundongos , Proteína Vermelha Fluorescente
10.
J Vis Exp ; (58)2011 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-22215075

RESUMO

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic ß-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim. Biologically-derived materials, such as alginate and agarose, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma. An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact ß-cells whose insulin secretory function is highly dependent on oxygen. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously. The virus is stored in 10% glycerol at -80° C as many 150 µL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 10(10) pfu/mL. Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1α for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.


Assuntos
Agregação Celular/fisiologia , Hipóxia Celular/fisiologia , Células Secretoras de Insulina/citologia , Animais , Materiais Biocompatíveis/química , Linhagem Celular , Hidrogéis/química , Células Secretoras de Insulina/química , Células Secretoras de Insulina/transplante , Camundongos , Polietilenoglicóis/química , Propano/análogos & derivados , Propano/química , Transdução de Sinais
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