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Structural and biochemical modification of a collagen scaffold to selectively enhance MSC tenogenic, chondrogenic, and osteogenic differentiation.

ABSTRACT: Biomaterial approaches for engineering orthopedic interfaces such as the tendon-bone junction (TBJ) are limited by a lack of understanding of how insoluble (microstructure, composition) and soluble regulators of stem cell fate work in concert to promote bioactivity and differentiation. One strategy for regenerating the interface is to design biomaterials containing spatially graded structural properties sufficient to induce divergent mesenchymal stem cell (MSC) differentiation into multiple interface-specific phenotypes. This work explores the hypothesis that selective structural modification to a 3D collagen-glycosaminoglycan (CG) scaffold combined with biochemical supplementation can drive human bone-marrow-derived MSC differentiation down tenogenic, osteogenic, and chondrogenic lineages. Tenogenic differentiation is enhanced in geometrically anisotropic scaffolds versus a standard isotropic control. Notably, blebbistatin treatment abrogates this microstructurally driven effect. Further, enhanced osteogenic differentiation and new mineral synthesis are achieved by incorporation of a calcium phosphate mineral phase within the CG scaffold along with the use of osteogenic induction media. Finally, chondrogenic differentiation is optimally driven by combining chondrogenic induction media with a reduced density scaffold that promotes increased cellular condensation, significantly higher expression of chondrogenic genes, and increased GAG deposition. Together these data provide critical insight regarding design rules for elements of an integrated biomaterial platform for orthopedic interface regeneration.

SUBMITTER: Caliari SR

PROVIDER: S-EPMC4107041 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Structural and biochemical modification of a collagen scaffold to selectively enhance MSC tenogenic, chondrogenic, and osteogenic differentiation.

Caliari Steven R SR Harley Brendan A C BA

Advanced healthcare materials 20140225 7

Biomaterial approaches for engineering orthopedic interfaces such as the tendon-bone junction (TBJ) are limited by a lack of understanding of how insoluble (microstructure, composition) and soluble regulators of stem cell fate work in concert to promote bioactivity and differentiation. One strategy for regenerating the interface is to design biomaterials containing spatially graded structural properties sufficient to induce divergent mesenchymal stem cell (MSC) differentiation into multiple inte ...[more]

PMID: 24574180

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Project description:BackgroundMesenchymal stem cells (MSCs) have prospective applications in regenerative medicine and tissue engineering but to what extent phenotype and differentiation capacity alter with ageing is uncertain. Consequently, any loss in functionality with age would have profound consequences for the maintenance of tissue viability and the quality of tissues. Proteomics enables the set of proteins responsible for a particular cell phenotype to be identified, as well as enabling insights into mechanisms responsible for age-related alterations in musculoskeletal tissues. Few proteomic studies have been undertaken regarding age-related effects on tissue engineered into cartilage and bone, and none for tendon. This study provides a proteome inventory for chondrogenic, osteogenic and tenogenic constructs synthesised from human MSCs, and elucidates proteomic alterations as a consequence of donor age.MethodsHuman bone-marrow derived MSCs from young (n = 4, 21.8 years ± 2.4SD) and old (n = 4, 65.5 years ± 8.3SD) donors were used to make chondrogenic, osteogenic and tenogenic tissue-engineered constructs. We utilised an analytical method relying on extracted peptide intensities as a label-free approach for peptide quantitation by liquid chromatography-mass spectrometry. Results were validated using western blotting.ResultsWe identified proteins that were differentially expressed with ageing; 128 proteins in chondrogenic constructs, 207 in tenogenic constructs and four in osteogenic constructs. Differentially regulated proteins were subjected to bioinformatic analysis to ascertain their molecular functions and the signalling pathways. For all construct types, age-affected proteins were involved in altered cell survival and death, and antioxidant and cytoskeletal changes. Energy and protein metabolism were the principle pathways affected in tenogenic constructs, whereas lipid metabolism was strongly affected in chondrogenic constructs and mitochondrial dysfunction in osteogenic constructs.ConclusionsOur results imply that further work on MSC-based therapeutics for the older population needs to focus on oxidative stress protection. The differentially regulated proteome characterised by this study can potentially guide translational research specifically aimed at effective clinical interventions.

Project description:BackgroundCollagen is one of the most commonly used natural biomaterials for tendon tissue engineering. One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment, proliferation, and tenogenic differentiation of cells. However, there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in a collagen sponge-based 3D culture system.AimTo identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.MethodsWe constructed a 3D culture system based on a type I collagen sponge scaffold. The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy. Primary BMSCs were isolated from Sprague-Dawley rats. Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay. The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot, respectively. The deposited collagen was assessed by Sirius Red staining.ResultsTransforming growth factor ?1 (TGF-?1) showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7 (GDF-7) and insulin-like growth factor 1 (IGF-1) in both the 2D and 3D cultures, and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-?1 treatment. In the 2D culture, the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-?1, IGF-1, or GDF-7 treatment. However, TGF-?1 and GDF-7 could increase the cell proliferation in the 3D culture. Strangely, we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-?1. Moreover, TGF-?1 promoted more collagen deposition in both the 2D and 3D cultures.ConclusionCollagen sponge-based 3D culture with TGF-?1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.

Project description:Biodegradable synthetic scaffolds hold great promise for oral and craniofacial guided tissue regeneration and bone regeneration. To overcome the limitations of current scaffold materials in terms of osteogenic and antimicrobial properties, we have developed a novel silver-modified/collagen-coated electrospun poly-lactic-co-glycolic acid/polycaprolactone (PLGA/PCL) scaffold (PP-pDA-Ag-COL) with improved antimicrobial and osteogenic properties. Our novel scaffold was generated by electrospinning a basic PLGA/PCL matrix, followed by silver nanoparticles (AgNPs) impregnation via in situ reduction, polydopamine coating, and then coating by collagen I. The three intermediate materials involved in the fabrication of our scaffolds, namely, PLGA/PCL (PP), PLGA/PCL-polydopamine (PP-pDA), and PLGA/PCL-polydopamine-Ag (PP-pDA-Ag), were used as control scaffolds. Scanning electron micrographs and mechanical testing indicated that the unique three-dimensional structures with randomly oriented nanofibrous electrospun scaffold architectures, the elasticity modulus, and the tensile strength were maintained after modifications. CCK-8 cell proliferation analysis demonstrated that the PP-pDA-Ag-COL scaffold was associated with higher MC3T3 proliferation rates than the three control scaffolds employed. Scanning electron and fluorescence light microscopy illustrated that PP-pDA-Ag-COL scaffolds significantly enhanced MC3T3 cell adhesion compared to the control scaffolds after 12 and 24 h culture, in tandem with the highest ?1 integrin expression levels, both at the mRNA level and the protein level. Alkaline phosphatase activity, BMP2, and RUNX2 expression levels of MC3T3 cells cultured on PP-pDA-Ag-COL scaffolds for 7 and 14 days were also significantly higher when compared to controls (P < 0.001). There was a wider antibacterial zone associated in PP-pDA-Ag-COL and PP-pDA-Ag scaffolds versus control scaffolds (P < 0.05), and bacterial fluorescence was reduced on the Ag-modified scaffolds after 24 h inoculation against Staphylococcus aureus and Streptococcus mutans. In a mouse periodontal disease model, the PP-pDA-Ag-COL scaffold enhanced alveolar bone regeneration (31.8%) and was effective for periodontitis treatment. These results demonstrate that our novel PP-pDA-Ag-COL scaffold enhanced biocompatibility and osteogenic and antibacterial properties and has therapeutic potential for alveolar/craniofacial bone regeneration.

Project description:Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-?1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-? may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia's influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167?kPa) or stiff (49.6?kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-?3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia's effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering strategies often aim to drive progenitor cell differentiation by replicating the local environment of the native tissue, including by regulating oxygen concentration and mechanical stiffness. However, the pathways that regulate cellular responses to mechanotransduction and hypoxia may not act independently, but rather also impact one another. Here, we show that on soft, but not stiff surfaces, hypoxia impacts human MSC (hMSC) morphology and colony formation, and inhibition of cytoskeletal tension negates the hypoxia-mediated upregulation of molecular markers of chondrogenesis. These observations suggest that hypoxia's effects during hMSC chondrogenesis are modulated, at least in part, by mechanosensitive pathways, and may impact strategies to develop scaffolds for cartilage tissue engineering, as hypoxia's chondrogenic effects may be enhanced on soft materials.

Project description:BackgroundThere is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects.MethodsEVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry.ResultsAlthough Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM.ConclusionsThis study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

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Structural and biochemical modification of a collagen scaffold to selectively enhance MSC tenogenic, chondrogenic, and osteogenic differentiation.

Publications

Structural and biochemical modification of a collagen scaffold to selectively enhance MSC tenogenic, chondrogenic, and osteogenic differentiation.

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