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1.
Cells ; 11(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36359814

RESUMO

Low back pain is a clinically highly relevant musculoskeletal burden and is associated with inflammatory as well as degenerative processes of the intervertebral disc. However, the pathophysiology and cellular pathways contributing to this devastating condition are still poorly understood. Based on previous evidence, we hypothesize that tissue renin-angiotensin system (tRAS) components, including the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2), are present in human nucleus pulposus (NP) cells and associated with inflammatory and degenerative processes. Experiments were performed with NP cells from four human donors. The existence of angiotensin II, angiotensin II type 1 receptor (AGTR1), AGTR2, MAS-receptor (MasR), and ACE2 in human NP cells was validated with immunofluorescent staining and gene expression analysis. Hereafter, the cell viability was assessed after adding agonists and antagonists of the target receptors as well as angiotensin II in different concentrations for up to 48 h of exposure. A TNF-α-induced inflammatory in vitro model was employed to assess the impact of angiotensin II addition and the stimulation or inhibition of the tRAS receptors on inflammation, tissue remodeling, expression of tRAS markers, and the release of nitric oxide (NO) into the medium. Furthermore, protein levels of IL-6, IL-8, IL-10, and intracellular as well as secreted angiotensin II were assessed after exposing the cells to the substances, and inducible nitric oxide synthase (iNOS) levels were evaluated by utilizing Western blot. The existence of tRAS receptors and angiotensin II were validated in human NP cells. The addition of angiotensin II only showed a mild impact on gene expression markers. However, there was a significant increase in NO secreted by the cells. The gene expression ratios of pro-inflammatory/anti-inflammatory cytokines IL-6/IL-10, IL-8/IL-10, and TNF-α/IL-10 were positively correlated with the AGTR1/AGTR2 and AGTR1/MAS1 ratios, respectively. The stimulation of the AGTR2 MAS-receptor and the inhibition of the AGTR1 receptor revealed beneficial effects on the gene expression of inflammatory and tissue remodeling markers. This finding was also present at the protein level. The current data showed that tRAS components are expressed in human NP cells and are associated with inflammatory and degenerative processes. Further characterization of the associated pathways is warranted. The findings indicate that tRAS modulation might be a novel therapeutic approach to intervertebral disc disease.


Assuntos
Núcleo Pulposo , Sistema Renina-Angiotensina , Humanos , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2 , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
JOR Spine ; 5(3): e1226, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36203861
3.
JOR Spine ; 5(3): e1215, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36203866

RESUMO

Background: During the intervertebral disc (IVD) degeneration process, initial degenerative events occur at the extracellular matrix level, with the appearance of neoepitope peptides formed by the cleavage of aggrecan and collagen. This study aims to elucidate the spatial and temporal alterations of aggrecan and collagen neoepitope level during IVD degeneration. Methods: Bovine caudal IVDs were cultured under four different conditions to mimic different degenerative situations. Samples cultured after 1- or 8-days were collected for analysis. Human IVD samples were obtained from patients diagnosed with lumbar disc herniation (LDH) or adolescent idiopathic scoliosis (AIS). After immunohistochemical (IHC) staining of Aggrecanase Cleaved C-terminus Aggrecan Neoepitope (NB100), MMP Cleaved C-terminus Aggrecan Neoepitope (MMPCC), Collagen Type 1α1 1/4 fragment (C1α1) and Collagenase Cleaved Type I and II Collagen Neoepitope (C1,2C), staining optical density (OD)/area in extracellular matrix (OECM) and pericellular zone (OPCZ) were analyzed. Conditioned media of the bovine IVD was collected to measure protein level of inflammatory cytokines and C1,2C. Results: For the bovine IVD sections, the aggrecan MMPCC neoepitope was accumulated in nucleus pulposus (NP) and cartilage endplate (EP) regions following mechanical overload in the one strike model after long-term culture; as for the TNF-α induced degeneration, the OECM and OPCZ of collagen C1,2C neoepitope was significantly increased in the outer AF region after long-term culture; moreover, the C1,2C was only detected in conditioned medium from TNF-α injection + Degenerative loading group after 8 days of culture. LDH patients showed higher MMPCC OECM in NP and higher C1,2C OECM in AF region compared with AIS patients. Conclusions: In summary, aggrecan and collagen neoepitope profiles showed degeneration induction trigger- and region-specific differences in the IVD organ culture models. Different IVD degeneration types are correlated with specific neoepitope expression profiles. These neoepitopes may be helpful as biomarkers of ECM degradation in early IVD degeneration and indicators of different degeneration phenotypes.

4.
ACS Biomater Sci Eng ; 8(9): 3969-3976, 2022 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-35977717

RESUMO

A new generation of bioreactors with integrated six degrees of freedom (6 DOF) aims to mimic more accurately the natural intervertebral disc (IVD) load. We developed and validated in a biological and mechanical study a specimen holder and corresponding ex vivo IVD organ model according to the bioreactor requirements for multiaxial loading and a long-term IVD culture. IVD height changes and cell viability were compared between the 6 DOF model and the standard 1 DOF model throughout the 3 weeks of cyclic compressive loading in the uniaxial bioreactor. Furthermore, the 6 DOF model and holder were loaded for 9 days in the multiaxial bioreactor under development using the same conditions, and the IVDs were evaluated for cell viability. The interface of the IVD model and specimen holder, enhanced with fixation screws onto the bone, was tested in compression, torsion, lateral bending, and tension. Additionally, critical motions such as tension and bending were assessed for a combination of side screws and top screws or side screws and adhesive. The 6 DOF model loaded in the uniaxial bioreactor maintained similar cell viability in the IVD regions as the 1 DOF model. The viability was high after 2 weeks throughout the whole IVD and reduced by more than 30% in the inner annulus fibrous after 3 weeks. Similarly, the IVDs remained highly viabile when cultured in the multiaxial bioreactor. In both models, IVD height changes after loading were in the range of typical physiological conditions. When differently directed motions were applied, the holder-IVD interface remained stable under hyper-physiological loading levels using a side screw approach in compression and torsion and the combination of side and top screws in tension and bending. We thus conclude that the developed holding system is mechanically reliable and biologically compatible for application in a new generation of multiaxial bioreactors.


Assuntos
Disco Intervertebral , Reatores Biológicos , Disco Intervertebral/fisiologia , Técnicas de Cultura de Órgãos
5.
Medicina (Kaunas) ; 58(8)2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35893113

RESUMO

Background and Objectives: Commonly being the first step in trauma routine imaging, up to 67% fractures are missed on plain radiographs of the thoracolumbar (TL) spine. The aim of this study was to develop a deep learning model that detects traumatic fractures on sagittal radiographs of the TL spine. Identifying vertebral fractures in simple radiographic projections would have a significant clinical and financial impact, especially for low- and middle-income countries where computed tomography (CT) and magnetic resonance imaging (MRI) are not readily available and could help select patients that need second level imaging, thus improving the cost-effectiveness. Materials and Methods: Imaging studies (radiographs, CT, and/or MRI) of 151 patients were used. An expert group of three spinal surgeons reviewed all available images to confirm presence and type of fractures. In total, 630 single vertebra images were extracted from the sagittal radiographs of the 151 patients-302 exhibiting a vertebral body fracture, and 328 exhibiting no fracture. Following augmentation, these single vertebra images were used to train, validate, and comparatively test two deep learning convolutional neural network models, namely ResNet18 and VGG16. A heatmap analysis was then conducted to better understand the predictions of each model. Results: ResNet18 demonstrated a better performance, achieving higher sensitivity (91%), specificity (89%), and accuracy (88%) compared to VGG16 (90%, 83%, 86%). In 81% of the cases, the "warm zone" in the heatmaps correlated with the findings, suggestive of fracture within the vertebral body seen in the imaging studies. Vertebras T12 to L2 were the most frequently involved, accounting for 48% of the fractures. A4, A3, and A1 were the most frequent fracture types according to the AO Spine Classification. Conclusions: ResNet18 could accurately identify the traumatic vertebral fractures on the TL sagittal radiographs. In most cases, the model based its prediction on the same areas that human expert classifiers used to determine the presence of a fracture.


Assuntos
Fraturas da Coluna Vertebral , Vértebras Torácicas , Inteligência Artificial , Humanos , Vértebras Lombares/lesões , Radiografia , Estudos Retrospectivos , Fraturas da Coluna Vertebral/diagnóstico por imagem , Fraturas da Coluna Vertebral/cirurgia , Vértebras Torácicas/diagnóstico por imagem , Vértebras Torácicas/lesões
6.
JOR Spine ; 5(2): e1211, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35783911
7.
Mater Today Bio ; 16: 100357, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35880098

RESUMO

The tumor microenvironment (TME), consisting of extracellular matrix, proteins, stromal cells, and a vascular system, is reported to have a key role in cancer progression and prognosis. Thereby, the interaction between the vascular network and tumor mass is an important feature of the TME since the anticancer agents which are delivered to the TME can trigger the vascular response and influence the therapeutic outcome of the treatment. To identify and develop new therapeutic strategies, 3D in vitro models that recapitulate the complexity of the TME are urgently needed. Among them, vascularized tumor models are a promising approach, allowing to target tumor angiogenesis and reduce tumor growth. By using sound patterning, cells can be condensed locally into highly reproducible patterns through the action of mild hydrodynamic forces. Here, we use a soundwave-driven cell assembly approach to create a ring-shaped microcapillary network in fibrin hydrogel. Then, we generate a 3D vascularized tumor model by combining a tumor heterotypic spheroid, consisting of fibroblasts and Malignant Pleural Mesothelioma (MPM) cells, with the surrounding vascular ring. Based on its shape, we name it Saturn-like vascularized Tumor Model (STM). The growth of the microcapillary network is monitored over time by fluorescence imaging. The area covered by the microcapillary network, and its continuous increase in presence of the heterotypic tumor spheroid was monitored. Interestingly, this effect is enhanced when treating the STM with the anticancer agent Cisplatin. Overall, we show the use of sound patterning as a fast and cell-friendly approach to spatially organize and condense cells, to generate a 3D in vitro platform from which simple readouts of drug tests can be extracted by image analysis, with the potential to provide a model system for tailored tumor therapy.

8.
Arthritis Res Ther ; 24(1): 105, 2022 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-35545776

RESUMO

Osteoarthritis (OA) is one of the most common musculoskeletal degenerative diseases and contributes to heavy socioeconomic burden. Current pharmacological and conventional non-pharmacological therapies aim at relieving the symptoms like pain and disability rather than modifying the underlying disease. Surgical treatment and ultimately joint replacement arthroplasty are indicated in advanced stages of OA. Since the underlying mechanisms of OA onset and progression have not been fully elucidated yet, the development of novel therapeutics to prevent, halt, or reverse the disease is laborious. Recently, small molecules of herbal origin have been reported to show potent anti-inflammatory, anti-catabolic, and anabolic effects, implying their potential for treatment of OA. Herein, the molecular mechanisms of these small molecules, their effect on physiological or pathological signaling pathways, the advancement of the extraction methods, and their potential clinical translation based on in vitro and in vivo evidence are comprehensively reviewed.


Assuntos
Artroplastia de Substituição , Osteoartrite , Anti-Inflamatórios/uso terapêutico , Humanos , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Dor/tratamento farmacológico , Transdução de Sinais
9.
JOR Spine ; 5(1): e1200, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35386758
10.
Stem Cell Res Ther ; 13(1): 129, 2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-35346367

RESUMO

Extracellular vesicles (EVs) are advanced therapeutic strategies that can be used to efficiently treat diseases. Promising features of EVs include their innate therapeutic properties and ability to be engineered as targeted drug delivery systems. However, regulation of EV uptake is one challenge of EV therapy that must be overcome to achieve an efficient therapeutic outcome. Numerous efforts to improve the factors that affect EV uptake include the selection of a cell source, cell cultivation procedure, extraction and purification methods, storage, and administration routes. Limitations of rapid clearance, targeted delivery, and off-targeting of EVs are current challenges that must be circumvented. EV engineering can potentially overcome these limitations and provide an ideal therapeutic use for EVs. In this paper, we intend to discuss traditional strategies and their limitations, and then review recent advances in EV engineering that can be used to customize and control EV uptake for future clinical applications.


Assuntos
Vesículas Extracelulares , Transporte Biológico , Sistemas de Liberação de Medicamentos/métodos , Vesículas Extracelulares/metabolismo
11.
J Orthop Res ; 40(7): 1661-1671, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-34662464

RESUMO

The mouse outer annulus fibrosus (AF) was previously shown to contain CD146+ AF cells, while in vitro culture and exposure to transforming growth factor-beta (TGF-ß) further increased the expression of CD146. However, neither the specific function of CD146 nor the underlying mechanism of TGF-ß upregulation of CD146+ AF cells have been elucidated yet. In the current study, CD146 expression and its role in cultured human AF cells was investigated studying the cells' capacity for matrix contraction and gene expression of functional AF markers. In addition, TGF-ß pathways were blocked by several pathway inhibitors and short hairpin RNAs (shRNAs) targeting SMAD and non-SMAD pathways to investigate their involvement in TGF-ß-induced CD146 upregulation. Results showed that knockdown of CD146 led to reduction in AF cell-mediated collagen gel contraction, downregulation of versican and smooth muscle protein 22α (SM22α), and upregulation of scleraxis. TGF-ß-induced CD146 upregulation was significantly blocked by inhibition of TGF-ß receptor ALK5, and partially inhibited by shRNA against SMAD2 and SMAD4 and by an Protein Kinase B (AKT) inhibitor. Interestingly, the inhibition of extracellular signal-regulated kinases (ERK) pathway induced CD146 upregulation. In conclusion, CD146 was shown to be crucial to maintain the cell contractility of human AF cells in vitro. Furthermore, TGF-ß upregulated CD146 via ALK5 signaling cascade, partially through SMAD2, SMAD4, and AKT pathway, whereas, ERK was shown to be a potential negative modulator. Our findings suggest that CD146 can potentially be used as a functional marker in AF repair strategies.


Assuntos
Anel Fibroso , Antígeno CD146 , Fator de Crescimento Transformador beta , Anel Fibroso/metabolismo , Antígeno CD146/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt , Receptor do Fator de Crescimento Transformador beta Tipo I , Proteína Smad2 , Proteína Smad4 , Fator de Crescimento Transformador beta/metabolismo
12.
Tissue Eng Part B Rev ; 28(5): 949-965, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-34579558

RESUMO

Musculoskeletal disorders are the most common reason of chronic pain and disability, representing an enormous socioeconomic burden worldwide. In this review, new biomedical application fields for Raman spectroscopy (RS) technique related to skeletal tissues are discussed, showing that it can provide a comprehensive profile of tissue composition in situ, in a rapid, label-free, and nondestructive manner. RS can be used as a tool to study tissue alterations associated to aging, pathologies, and disease treatments. The main advantage with respect to currently applied methods in clinics is its ability to provide specific information on molecular composition, which goes beyond other diagnostic tools. Being compatible with water, RS can be performed without pretreatment on unfixed, hydrated tissue samples, without any labeling and chemical fixation used in histochemical methods. This review first provides the description of the basic principles of RS as a biotechnology tool and is introduced into the field of currently available RS-based techniques, developed to enhance Raman signals. The main spectral processing, statistical tools, fingerprint identification, and available databases are mentioned. The recent literature has been analyzed for such applications of RS as tendon and ligaments, cartilage, bone, and tissue engineered constructs for regenerative medicine. Several cases of proof-of-concept preclinical studies have been described. Finally, advantages, limitations, future perspectives, and challenges for the translation of RS into clinical practice have been also discussed. Impact statement Raman spectroscopy (RS) is a powerful noninvasive tool giving access to molecular vibrations and characteristics of samples in a wavelength window of 600 to 3200 cm-1, thus giving access to a molecular fingerprint of biological samples in a nondestructive way. RS could not only be used in clinical diagnostics, but also be used for quality control of tissues and tissue-engineered constructs, reducing number of samples, time, and the variety of analysis required in the quality control chain before implantation.


Assuntos
Análise Espectral Raman , Engenharia Tecidual , Humanos , Análise Espectral Raman/métodos , Estudos Prospectivos , Cartilagem , Água
13.
Carbohydr Polym ; 277: 118828, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34893245

RESUMO

Hyaluronic acid (HA) is a key component of the intervertebral disc (IVD) that is widely investigated as an IVD biomaterial. One persisting challenge is introducing materials capable of supporting cell encapsulation and function, yet with sufficient mechanical stability. In this study, a hybrid interpenetrating polymer network (IPN) was produced as a non-covalent hydrogel, based on a covalently cross-linked HA (HA-BDDE) and HA-poly(N-isopropylacrylamide) (HA-pNIPAM). The hybrid IPN was investigated for its physicochemical properties, with histology and gene expression analysis to determine matrix deposition in vitro and in an ex vivo model. The IPN hydrogel displayed cohesiveness for at least one week and rheological properties resembling native nucleus pulposus (NP) tissue. When implanted in an ex vivo IVD organ culture model, the IPN supported cell viability, phenotype expression of encapsulated NP cells and IVD matrix production over four weeks under physiological loading. Overall, our results indicate the therapeutic potential of this HA-based IPN hydrogel for IVD regeneration.


Assuntos
Resinas Acrílicas/farmacologia , Ácido Hialurônico/química , Hidrogéis/química , Disco Intervertebral/efeitos dos fármacos , Núcleo Pulposo/efeitos dos fármacos , Resinas Acrílicas/química , Animais , Bovinos , Portadores de Fármacos/química , Disco Intervertebral/patologia , Núcleo Pulposo/patologia
14.
Bioact Mater ; 9: 281-298, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34820571

RESUMO

The osteogenic microenvironment of bone-repairing materials plays a key role in accelerating bone regeneration but remains incompletely defined, which significantly limits the application of such bioactive materials. Here, the transcriptional landscapes of different osteogenic microenvironments, including three-dimensional (3D) hydroxyapatite (HA) scaffolds and osteogenic medium (OM), for mesenchymal stromal cells (MSCs) in vitro were mapped at single-cell resolution. Our findings suggested that an osteogenic process reminiscent of endochondral ossification occurred in HA scaffolds through sequential activation of osteogenic-related signaling pathways, along with inflammation and angiogenesis, but inhibition of adipogenesis and fibrosis. Moreover, we revealed the mechanism during OM-mediated osteogenesis involves the ZBTB16 and WNT signaling pathways. Heterogeneity of MSCs was also demonstrated. In vitro ossification of LRRC75A+ MSCs was shown to have better utilization of WNT-related ossification process, and PCDH10+ MSCs with superiority in hydroxyapatite-related osteogenic process. These findings provided further understanding of the cellular activity modulated by OM conditions and HA scaffolds, providing new insights for the improvement of osteogenic biomaterials. This atlas provides a blueprint for research on MSC heterogeneity and the osteogenic microenvironment of HA scaffolds and a database reference for the application of bioactive materials for bone regeneration.

15.
JOR Spine ; 5(4): e1239, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36601374
16.
Global Spine J ; : 21925682211058158, 2021 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-34870488

RESUMO

OBJECTIVE: Resource allocation to research activities is challenging and there is limited evidence to justify decisions. Members of AO Spine were surveyed to understand the research practices and needs of spine surgeons worldwide. METHODS: An 84-item survey was distributed to the AO Spine community in September of 2020. Respondent demographics and insights regarding research registries, training and education, mentorship, grants and financial support, and future directions were collected. Responses were anonymous and compared among regions. RESULTS: A total of 333 spine surgeons representing all geographic regions responded; 52.3% were affiliated with an academic/university hospital, 91.0% conducted clinical research, and 60.9% had 5+ years of research experience. There was heterogeneity among research practices and needs across regions. North American respondents had more research experience (P = .023), began conducting research early on (P < .001), had an undergraduate science degree (P < .001), and were more likely to have access to a research coordinator or support staff (P = .042) compared to other regions. While all regions expressed having the same challenges in conducting research, Latin America, and Middle East/Northern Africa respondents were less encouraged to do research (P < .001). Despite regional differences, there was global support for research registries and research training and education. CONCLUSION: To advance spine care worldwide, spine societies should establish guidelines, conduct studies on pain management, and support predictive analytic modeling. Tailoring local/regional programs according to regional needs is advised. These results can assist spine societies in developing long-term research strategies and provide justified rationale to governments and funding agencies.

18.
Bone Res ; 9(1): 46, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34707086

RESUMO

Tissue engineering is rapidly progressing toward clinical application. In the musculoskeletal field, there has been an increasing necessity for bone and cartilage replacement. Despite the promising translational potential of tissue engineering approaches, careful attention should be given to the quality of developed constructs to increase the real applicability to patients. After a general introduction to musculoskeletal tissue engineering, this narrative review aims to offer an overview of methods, starting from classical techniques, such as gene expression analysis and histology, to less common methods, such as Raman spectroscopy, microcomputed tomography, and biosensors, that can be employed to assess the quality of constructs in terms of viability, morphology, or matrix deposition. A particular emphasis is given to standards and good practices (GXP), which can be applicable in different sectors. Moreover, a classification of the methods into destructive, noninvasive, or conservative based on the possible further development of a preimplant quality monitoring system is proposed. Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages, including minimal invasiveness, making them suitable for the development of preimplant quality control systems.

19.
Biomedicines ; 9(7)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34356857

RESUMO

Biodegradable and bioresponsive polymer-based nanoparticles (NPs) can be used for oligonucleotide delivery, making them a promising candidate for mRNA-based therapeutics. In this study, we evaluated and optimized the efficiency of a cationic, hyperbranched poly(amidoamine)s-based nanoparticle system to deliver tdTomato mRNA to primary human bone marrow stromal cells (hBMSC), human synovial derived stem cells (hSDSC), bovine chondrocytes (bCH), and rat tendon derived stem/progenitor cells (rTDSPC). Transfection efficiencies varied among the cell types tested (bCH 28.4% ± 22.87, rTDSPC 18.13% ± 12.07, hBMSC 18.23% ± 14.80, hSDSC 26.63% ± 8.81) and while an increase of NPs with a constant amount of mRNA generally improved the transfection efficiency, an increase of the mRNA loading ratio (2:50, 4:50, or 6:50 w/w mRNA:NPs) had no impact. However, metabolic activity of bCHs and rTDSPCs was significantly reduced when using higher amounts of NPs, indicating a dose-dependent cytotoxic response. Finally, we demonstrate the feasibility of transfecting extracellular matrix-rich 3D cell culture constructs using the nanoparticle system, making it a promising transfection strategy for musculoskeletal tissues that exhibit a complex, dense extracellular matrix.

20.
Cells ; 10(8)2021 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-34440934

RESUMO

In the field of tissue engineering, progress has been made towards the development of new treatments for cartilage and bone defects. However, in vitro culture conditions for human bone marrow mesenchymal stromal cells (hBMSCs) have not yet been fully defined. To improve our understanding of cartilage and bone in vitro differentiation, we investigated the effect of culture conditions on hBMSC differentiation. We hypothesized that the use of two different culture media including specific growth factors, TGFß1 or BMP2, as well as low (2% O2) or high (20% O2) oxygen tension, would improve the chondrogenic and osteogenic potential, respectively. Chondrogenic and osteogenic differentiation of hBMSCs isolated from multiple donors and expanded under the same conditions were directly compared. Chondrogenic groups showed a notable upregulation of chondrogenic markers compared with osteogenic groups. Greater sGAG production and deposition, and collagen type II and I accumulation occurred for chondrogenic groups. Chondrogenesis at 2% O2 significantly reduced ALP gene expression and reduced type I collagen deposition, producing a more stable and less hypertrophic chondrogenic phenotype. An O2 tension of 2% did not inhibit osteogenic differentiation at the protein level but reduced ALP and OC gene expression. An upregulation of ALP and OC occurred during osteogenesis in BMP2 containing media under 20% O2; BMP2 free osteogenic media downregulated ALP and also led to higher sGAG release. A higher mineralization was observed in the presence of BMP2 during osteogenesis. This study demonstrates how the modulation of O2 tension, combined with tissue-specific growth factors and media composition can be tailored in vitro to promote chondral or endochondral differentiation while using the same donor cell population.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Condrogênese/fisiologia , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Imuno-Histoquímica , Osteogênese/fisiologia , Engenharia Tecidual
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